Archive for the ‘Bone Marrow Stem Cells’ Category

by Jos Domen*, Amy Wagers** and Irving L. Weissman***

Blood and the system that forms it, known as the hematopoietic system, consist of many cell types with specialized functions (see Figure 2.1). Red blood cells (erythrocytes) carry oxygen to the tissues. Platelets (derived from megakaryocytes) help prevent bleeding. Granulocytes (neutrophils, basophils and eosinophils) and macrophages (collectively known as myeloid cells) fight infections from bacteria, fungi, and other parasites such as nematodes (ubiquitous small worms). Some of these cells are also involved in tissue and bone remodeling and removal of dead cells. B-lymphocytes produce antibodies, while T-lymphocytes can directly kill or isolate by inflammation cells recognized as foreign to the body, including many virus-infected cells and cancer cells. Many blood cells are short-lived and need to be replenished continuously; the average human requires approximately one hundred billion new hematopoietic cells each day. The continued production of these cells depends directly on the presence of Hematopoietic Stem Cells (HSCs), the ultimate, and only, source of all these cells.

Figure 2.1. Hematopoietic and stromal cell differentiation.

2001 Terese Winslow (assisted by Lydia Kibiuk)

The search for stem cells began in the aftermath of the bombings in Hiroshima and Nagasaki in 1945. Those who died over a prolonged period from lower doses of radiation had compromised hematopoietic systems that could not regenerate either sufficient white blood cells to protect against otherwise nonpathogenic infections or enough platelets to clot their blood. Higher doses of radiation also killed the stem cells of the intestinal tract, resulting in more rapid death. Later, it was demonstrated that mice that were given doses of whole body X-irradiation developed the same radiation syndromes; at the minimal lethal dose, the mice died from hematopoietic failure approximately two weeks after radiation exposure.1 Significantly, however, shielding a single bone or the spleen from radiation prevented this irradiation syndrome. Soon thereafter, using inbred strains of mice, scientists showed that whole-body-irradiated mice could be rescued from otherwise fatal hematopoietic failure by injection of suspensions of cells from blood-forming organs such as the bone marrow.2 In 1956, three laboratories demonstrated that the injected bone marrow cells directly regenerated the blood-forming system, rather than releasing factors that caused the recipients' cells to repair irradiation damage.35 To date, the only known treatment for hematopoietic failure following whole body irradiation is transplantation of bone marrow cells or HSCs to regenerate the blood-forming system in the host organisms.6,7

The hematopoietic system is not only destroyed by the lowest doses of lethal X-irradiation (it is the most sensitive of the affected vital organs), but also by chemotherapeutic agents that kill dividing cells. By the 1960s, physicians who sought to treat cancer that had spread (metastasized) beyond the primary cancer site attempted to take advantage of the fact that a large fraction of cancer cells are undergoing cell division at any given point in time. They began using agents (e.g., chemical and X-irradiation) that kill dividing cells to attempt to kill the cancer cells. This required the development of a quantitative assessment of damage to the cancer cells compared that inflicted on normal cells. Till and McCulloch began to assess quantitatively the radiation sensitivity of one normal cell type, the bone marrow cells used in transplantation, as it exists in the body. They found that, at sub-radioprotective doses of bone marrow cells, mice that died 1015 days after irradiation developed colonies of myeloid and erythroid cells (see Figure 2.1 for an example) in their spleens. These colonies correlated directly in number with the number of bone marrow cells originally injected (approximately 1 colony per 7,000 bone marrow cells injected).8 To test whether these colonies of blood cells derived from single precursor cells, they pre-irradiated the bone marrow donors with low doses of irradiation that would induce unique chromosome breaks in most hematopoietic cells but allow some cells to survive. Surviving cells displayed radiation-induced and repaired chromosomal breaks that marked each clonogenic (colony-initiating) hematopoietic cell.9 The researchers discovered that all dividing cells within a single spleen colony, which contained different types of blood cells, contained the same unique chromosomal marker. Each colony displayed its own unique chromosomal marker, seen in its dividing cells.9 Furthermore, when cells from a single spleen colony were re-injected into a second set of lethally-irradiated mice, donor-derived spleen colonies that contained the same unique chromosomal marker were often observed, indicating that these colonies had been regenerated from the same, single cell that had generated the first colony. Rarely, these colonies contained sufficient numbers of regenerative cells both to radioprotect secondary recipients (e.g., to prevent their deaths from radiation-induced blood cell loss) and to give rise to lymphocytes and myeloerythroid cells that bore markers of the donor-injected cells.10,11 These genetic marking experiments established the fact that cells that can both self-renew and generate most (if not all) of the cell populations in the blood must exist in bone marrow. At the time, such cells were called pluripotent HSCs, a term later modified to multipotent HSCs.12,13 However, identifying stem cells in retrospect by analysis of randomly chromosome-marked cells is not the same as being able to isolate pure populations of HSCs for study or clinical use.

Achieving this goal requires markers that uniquely define HSCs. Interestingly, the development of these markers, discussed below, has revealed that most of the early spleen colonies visible 8 to 10 days after injection, as well as many of the later colonies, visible at least 12 days after injection, are actually derived from progenitors rather than from HSCs. Spleen colonies formed by HSCs are relatively rare and tend to be present among the later colonies.14,15 However, these findings do not detract from Till and McCulloch's seminal experiments to identify HSCs and define these unique cells by their capacities for self-renewal and multilineage differentiation.

While much of the original work was, and continues to be, performed in murine model systems, strides have been made to develop assays to study human HSCs. The development of Fluorescence Activated Cell Sorting (FACS) has been crucial for this field (see Figure 2.2). This technique enables the recognition and quantification of small numbers of cells in large mixed populations. More importantly, FACS-based cell sorting allows these rare cells (1 in 2000 to less than 1 in 10,000) to be purified, resulting in preparations of near 100% purity. This capability enables the testing of these cells in various assays.

Figure 2.2. Enrichment and purification methods for hematopoietic stem cells. Upper panels illustrate column-based magnetic enrichment. In this method, the cells of interest are labeled with very small iron particles (A). These particles are bound to antibodies that only recognize specific cells. The cell suspension is then passed over a column through a strong magnetic field which retains the cells with the iron particles (B). Other cells flow through and are collected as the depleted negative fraction. The magnet is removed, and the retained cells are collected in a separate tube as the positive or enriched fraction (C). Magnetic enrichment devices exist both as small research instruments and large closed-system clinical instruments.

Lower panels illustrate Fluorescence Activated Cell Sorting (FACS). In this setting, the cell mixture is labeled with fluorescent markers that emit light of different colors after being activated by light from a laser. Each of these fluorescent markers is attached to a different monoclonal antibody that recognizes specific sets of cells (D). The cells are then passed one by one in a very tight stream through a laser beam (blue in the figure) in front of detectors (E) that determine which colors fluoresce in response to the laser. The results can be displayed in a FACS-plot (F). FACS-plots (see figures 3 and 4 for examples) typically show fluorescence levels per cell as dots or probability fields. In the example, four groups can be distinguished: Unstained, red-only, green-only, and red-green double labeling. Each of these groups, e.g., green fluorescence-only, can be sorted to very high purity. The actual sorting happens by breaking the stream shown in (E) into tiny droplets, each containing 1 cell, that then can be sorted using electric charges to move the drops. Modern FACS machines use three different lasers (that can activate different set of fluorochromes), to distinguish up to 8 to 12 different fluorescence colors and sort 4 separate populations, all simultaneously.

Magnetic enrichment can process very large samples (billions of cells) in one run, but the resulting cell preparation is enriched for only one parameter (e.g., CD34) and is not pure. Significant levels of contaminants (such as T-cells or tumor cells) remain present. FACS results in very pure cell populations that can be selected for several parameters simultaneously (e.g., Linneg, CD34pos, CD90pos), but it is more time consuming (10,000 to 50,000 cells can be sorted per second) and requires expensive instrumentation.

2001 Terese Winslow (assisted by Lydia Kibiuk)

Assays have been developed to characterize hematopoietic stem and progenitor cells in vitro and in vivo (Figure 2.3).16,17In vivo assays that are used to study HSCs include Till and McCulloch's classical spleen colony forming (CFU-S) assay,8 which measures the ability of HSC (as well as blood-forming progenitor cells) to form large colonies in the spleens of lethally irradiated mice. Its main advantage (and limitation) is the short-term nature of the assay (now typically 12 days). However, the assays that truly define HSCs are reconstitution assays.16,18 Mice that have been quot;preconditionedquot; by lethal irradiation to accept new HSCs are injected with purified HSCs or mixed populations containing HSCs, which will repopulate the hematopoietic systems of the host mice for the life of the animal. These assays typically use different types of markers to distinguish host and donor-derived cells.

For example, allelic assays distinguish different versions of a particular gene, either by direct analysis of dna or of the proteins expressed by these alleles. These proteins may be cell-surface proteins that are recognized by specific monoclonal antibodies that can distinguish between the variants (e.g., CD45 in Figure 2.3) or cellular proteins that may be recognized through methods such as gel-based analysis. Other assays take advantage of the fact that male cells can be detected in a female host by detecting the male-cell-specific Y-chromosome by molecular assays (e.g., polymerase chain reaction, or PCR).

Figure 2.3. Assays used to detect hematopoietic stem cells. The tissue culture assays, which are used frequently to test human cells, include the ability of the cells to be tested to grow as quot;cobblestonesquot; (the dark cells in the picture) for 5 to 7 weeks in culture. The Long Term Culture-Initiating Cell assay measures whether hematopoietic progenitor cells (capable of forming colonies in secondary assays, as shown in the picture) are still present after 5 to 7 weeks of culture.

In vivo assays in mice include the CFU-S assay, the original stem cell assay discussed in the introduction. The most stringent hematopoietic stem cell assay involves looking for the long-term presence of donor-derived cells in a reconstituted host. The example shows host-donor recognition by antibodies that recognize two different mouse alleles of CD45, a marker present on nearly all blood cells. CD45 is also a good marker for distinguishing human blood cells from mouse blood cells when testing human cells in immunocompromised mice such as NOD/SCID. Other methods such as pcr-markers, chromosomal markers, and enzyme markers can also be used to distinguish host and donor cells.

Small numbers of HSCs (as few as one cell in mouse experiments) can be assayed using competitive reconstitutions, in which a small amount of host-type bone marrow cells (enough to radioprotect the host and thus ensure survival) is mixed in with the donor-HSC population. To establish long-term reconstitutions in mouse models, the mice are followed for at least 4 months after receiving the HSCs. Serial reconstitution, in which the bone marrow from a previously-irradiated and reconstituted mouse becomes the HSC source for a second irradiated mouse, extends the potential of this assay to test lifespan and expansion limits of HSCs. Unfortunately, the serial transfer assay measures both the lifespan and the transplantability of the stem cells. The transplantability may be altered under various conditions, so this assay is not the sine qua non of HSC function. Testing the in vivo activity of human cells is obviously more problematic.

Several experimental models have been developed that allow the testing of human cells in mice. These assays employ immunologically-incompetent mice (mutant mice that cannot mount an immune response against foreign cells) such as SCID1921 or NOD-SCID mice.22,23 Reconstitution can be performed in either the presence or absence of human fetal bone or thymus implants to provide a more natural environment in which the human cells can grow in the mice. Recently NOD/SCID/c-/- mice have been used as improved recipients for human HSCs, capable of complete reconstitution with human lymphocytes, even in the absence of additional human tissues.24 Even more promising has been the use of newborn mice with an impaired immune system (Rag-2-/-C-/-), which results in reproducible production of human B- and T-lymphoid and myeloerythroid cells.25 These assays are clearly more stringent, and thus more informative, but also more difficult than the in vitro HSC assays discussed below. However, they can only assay a fraction of the lifespan under which the cells would usually have to function. Information on the long-term functioning of cells can only be derived from clinical HSC transplantations.

A number of assays have been developed to recognize HSCs in vitro (e.g., in tissue culture). These are especially important when assaying human cells. Since transplantation assays for human cells are limited, cell culture assays often represent the only viable option. In vitro assays for HSCs include Long-Term Culture-Initializing Cell (LTC-IC) assays2628 and Cobble-stone Area Forming Cell (CAFC) assays.29 LTC-IC assays are based on the ability of HSCs, but not more mature progenitor cells, to maintain progenitor cells with clonogenic potential over at least a five-week culture period. CAFC assays measure the ability of HSCs to maintain a specific and easily recognizable way of growing under stromal cells for five to seven weeks after the initial plating. Progenitor cells can only grow in culture in this manner for shorter periods of time.

While initial experiments studied HSC activity in mixed populations, much progress has been made in specifically describing the cells that have HSC activity. A variety of markers have been discovered to help recognize and isolate HSCs. Initial marker efforts focused on cell size, density, and recognition by lectins (carbohydrate-binding proteins derived largely from plants),30 but more recent efforts have focused mainly on cell surface protein markers, as defined by monoclonal antibodies. For mouse HSCs, these markers include panels of 8 to 14 different monoclonal antibodies that recognize cell surface proteins present on differentiated hematopoietic lineages, such as the red blood cell and macrophage lineages (thus, these markers are collectively referred to as quot;Linquot;),13,31 as well as the proteins Sca-1,13,31 CD27,32 CD34,33 CD38,34 CD43,35 CD90.1(Thy-1.1),13,31 CD117(c-Kit),36 AA4.1,37 and MHC class I,30 and CD150.38 Human HSCs have been defined with respect to staining for Lin,39 CD34,40 CD38,41 CD43,35 CD45RO,42 CD45RA,42 CD59,43 CD90,39 CD109,44 CD117,45 CD133,46,47CD166,48 and HLA DR(human).49,50 In addition, metabolic markers/dyes such as rhodamine123 (which stains mitochondria),51 Hoechst33342 (which identifies MDR-type drug efflux activity),52 Pyronin-Y (which stains RNA),53 and BAAA (indicative of aldehyde dehydrogenase enzyme activity)54 have been described. While none of these markers recognizes functional stem cell activity, combinations (typically with 3 to 5 different markers, see examples below) allow for the purification of near-homogenous populations of HSCs. The ability to obtain pure preparations of HSCs, albeit in limited numbers, has greatly facilitated the functional and biochemical characterization of these important cells. However, to date there has been limited impact of these discoveries on clinical practice, as highly purified HSCs have only rarely been used to treat patients (discussed below). The undeniable advantages of using purified cells (e.g., the absence of contaminating tumor cells in autologous transplantations) have been offset by practical difficulties and increased purification costs.

Figure 2.4. Examples of Hematopoietic Stem Cell staining patterns in mouse bone marrow (top) and human mobilized peripheral blood (bottom). The plots on the right show only the cells present in the left blue box. The cells in the right blue box represent HSCs. Stem cells form a rare fraction of the cells present in both cases.

HSC assays, when combined with the ability to purify HSCs, have provided increasingly detailed insight into the cells and the early steps involved in the differentiation process. Several marker combinations have been developed that describe murine HSCs, including [CD117high, CD90.1low, Linneg/low, Sca-1pos],15 [CD90.1low, Linneg, Sca-1pos Rhodamine123low],55 [CD34neg/low, CD117pos, Sca-1pos, Linneg],33 [CD150 pos, CD48neg, CD244neg],38 and quot;side-populationquot; cells using Hoechst-dye.52 Each of these combinations allows purification of HSCs to near-homogeneity. Figure 2.4 shows an example of an antibody combination that can recognize mouse HSCs. Similar strategies have been developed to purify human HSCs, employing markers such as CD34, CD38, Lin, CD90, CD133 and fluorescent substrates for the enzyme, aldehyde dehydrogenase. The use of highly purified human HSCs has been mainly experimental, and clinical use typically employs enrichment for one marker, usually CD34. CD34 enrichment yields a population of cells enriched for HSC and blood progenitor cells but still contains many other cell types. However, limited trials in which highly FACS-purified CD34pos CD90pos HSCs (see Figure 2.4) were used as a source of reconstituting cells have demonstrated that rapid reconstitution of the blood system can reliably be obtained using only HSCs.5658

The purification strategies described above recognize a rare subset of cells. Exact numbers depend on the assay used as well as on the genetic background studied.16 In mouse bone marrow, 1 in 10,000 cells is a hematopoietic stem cell with the ability to support long-term hematopoiesis following transplantation into a suitable host. When short-term stem cells, which have a limited self-renewal capacity, are included in the estimation, the frequency of stem cells in bone marrow increases to 1 in 1,000 to 1 in 2,000 cells in humans and mice. The numbers present in normal blood are at least ten-fold lower than in marrow.

None of the HSC markers currently used is directly linked to an essential HSC function, and consequently, even within a species, markers can differ depending on genetic alleles,59 mouse strains,60 developmental stages,61 and cell activation stages.62,63 Despite this, there is a clear correlation in HSC markers between divergent species such as humans and mice. However, unless the ongoing attempts at defining the complete HSC gene expression patterns will yield usable markers that are linked to essential functions for maintaining the quot;stemnessquot; of the cells,64,65 functional assays will remain necessary to identify HSCs unequivocally.16

More recently, efforts at defining hematopoietic populations by cell surface or other FACS-based markers have been extended to several of the progenitor populations that are derived from HSCs (see Figure 2.5). Progenitors differ from stem cells in that they have a reduced differentiation capacity (they can generate only a subset of the possible lineages) but even more importantly, progenitors lack the ability to self-renew. Thus, they have to be constantly regenerated from the HSC population. However, progenitors do have extensive proliferative potential and can typically generate large numbers of mature cells. Among the progenitors defined in mice and humans are the Common Lymphoid Progenitor (CLP),66,67 which in adults has the potential to generate all of the lymphoid but not myeloerythroid cells, and a Common Myeloid Progenitor (CMP), which has the potential to generate all of the mature myeloerythroid, but not lymphoid, cells.68,69 While beyond the scope of this overview, hematopoietic progenitors have clinical potential and will likely see clinical use.70,71

Figure 2.5. Relationship between several of the characterized hematopoietic stem cells and early progenitor cells. Differentiation is indicated by colors; the more intense the color, the more mature the cells. Surface marker distinctions are subtle between these early cell populations, yet they have clearly distinct potentials. Stem cells can choose between self-renewal and differentiation. Progenitors can expand temporarily but always continue to differentiate (other than in certain leukemias). The mature lymphoid (T-cells, B-cells, and Natural Killer cells) and myeloerythroid cells (granulocytes, macrophages, red blood cells, and platelets) that are produced by these stem and progenitor cells are shown in more detail in Figure 2.1.

HSCs have a number of unique properties, the combination of which defines them as such.16 Among the core properties are the ability to choose between self-renewal (remain a stem cell after cell division) or differentiation (start the path towards becoming a mature hematopoietic cell). In addition, HSCs migrate in regulated fashion and are subject to regulation by apoptosis (programmed cell death). The balance between these activities determines the number of stem cells that are present in the body.

One essential feature of HSCs is the ability to self-renew, that is, to make copies with the same or very similar potential. This is an essential property because more differentiated cells, such as hematopoietic progenitors, cannot do this, even though most progenitors can expand significantly during a limited period of time after being generated. However, for continued production of the many (and often short-lived) mature blood cells, the continued presence of stem cells is essential. While it has not been established that adult HSCs can self-renew indefinitely (this would be difficult to prove experimentally), it is clear from serial transplantation experiments that they can produce enough cells to last several (at least four to five) lifetimes in mice. It is still unclear which key signals allow self-renewal. One link that has been noted is telomerase, the enzyme necessary for maintaining telomeres, the DNA regions at the end of chromosomes that protect them from accumulating damage due to DNA replication. Expression of telomerase is associated with self-renewal activity.72 However, while absence of telomerase reduces the self-renewal capacity of mouse HSCs, forced expression is not sufficient to enable HSCs to be transplanted indefinitely; other barriers must exist.73,74

It has proven surprisingly difficult to grow HSCs in culture despite their ability to self-renew. Expansion in culture is routine with many other cells, including neural stem cells and ES cells. The lack of this capacity for HSCs severely limits their application, because the number of HSCs that can be isolated from mobilized blood, umbilical cord blood, or bone marrow restricts the full application of HSC transplantation in man (whether in the treatment of nuclear radiation exposure or transplantation in the treatment of blood cell cancers or genetic diseases of the blood or blood-forming system). Engraftment periods of 50 days or more were standard when limited numbers of bone marrow or umbilical cord blood cells were used in a transplant setting, reflecting the low level of HSCs found in these native tissues. Attempts to expand HSCs in tissue culture with known stem-cell stimulators, such as the cytokines stem cell factor/steel factor (KitL), thrombopoietin (TPO), interleukins 1, 3, 6, 11, plus or minus the myeloerythroid cytokines GM-CSF, G-CSF, M-CSF, and erythropoietin have never resulted in a significant expansion of HSCs.16,75 Rather, these compounds induce many HSCs into cell divisions that are always accompanied by cellular differentiation.76 Yet many experiments demonstrate that the transplantation of a single or a few HSCs into an animal results in a 100,000-fold or greater expansion in the number of HSCs at the steady state while simultaneously generating daughter cells that permitted the regeneration of the full blood-forming system.7780 Thus, we do not know the factors necessary to regenerate HSCs by self-renewing cell divisions. By investigating genes transcribed in purified mouse LT-HSCs, investigators have found that these cells contain expressed elements of the Wnt/fzd/beta-catenin signaling pathway, which enables mouse HSCs to undergo self-renewing cell divisions.81,82 Overexpression of several other proteins, including HoxB48386 and HoxA987 has also been reported to achieve this. Other signaling pathways that are under investigation include Notch and Sonic hedgehog.75 Among the intracellular proteins thought to be essential for maintaining the quot;stem cellquot; state are Polycomb group genes, including Bmi-1.88 Other genes, such as c-Myc and JunB have also been shown to play a role in this process.89,90Much remains to be discovered, including the identity of the stimuli that govern self-renewal in vivo, as well as the composition of the environment (the stem cell quot;nichequot;) that provides these stimuli.91 The recent identification of osteoblasts, a cell type known to be involved in bone formation, as a critical component of this environment92,93 will help to focus this search. For instance, signaling by Angiopoietin-1 on osteoblasts to Tie-2 receptors on HSCs has recently been suggested to regulate stem cell quiescence (the lack of cell division).94 It is critical to discover which pathways operate in the expansion of human HSCs to take advantage of these pathways to improve hematopoietic transplantation.

Differentiation into progenitors and mature cells that fulfill the functions performed by the hematopoietic system is not a unique HSC property, but, together with the option to self-renew, defines the core function of HSCs. Differentiation is driven and guided by an intricate network of growth factors and cytokines. As discussed earlier, differentiation, rather than self-renewal, seems to be the default outcome for HSCs when stimulated by many of the factors to which they have been shown to respond. It appears that, once they commit to differentiation, HSCs cannot revert to a self-renewing state. Thus, specific signals, provided by specific factors, seem to be needed to maintain HSCs. This strict regulation may reflect the proliferative potential present in HSCs, deregulation of which could easily result in malignant diseases such as leukemia or lymphoma.

Migration of HSCs occurs at specific times during development (i.e., seeding of fetal liver, spleen and eventually, bone marrow) and under certain conditions (e.g., cytokine-induced mobilization) later in life. The latter has proven clinically useful as a strategy to enhance normal HSC proliferation and migration, and the optimal mobilization regimen for HSCs currently used in the clinic is to treat the stem cell donor with a drug such as cytoxan, which kills most of his or her dividing cells. Normally, only about 8% of LT-HSCs enter the cell cycle per day,95,96 so HSCs are not significantly affected by a short treatment with cytoxan. However, most of the downstream blood progenitors are actively dividing,66,68 and their numbers are therefore greatly depleted by this dose, creating a demand for a regenerated blood-forming system. Empirically, cytokines or growth factors such as G-CSF and KitL can increase the number of HSCs in the blood, especially if administered for several days following a cytoxan pulse. The optimized protocol of cytoxan plus G-CSF results in several self-renewing cell divisions for each resident LT-HSC in mouse bone marrow, expanding the number of HSCs 12- to 15-fold within two to three days.97 Then, up to one-half of the daughter cells of self-renewing dividing LT-HSCs (estimated to be up to 105 per mouse per day98) leave the bone marrow, enter the blood, and within minutes engraft other hematopoietic sites, including bone marrow, spleen, and liver.98 These migrating cells can and do enter empty hematopoietic niches elsewhere in the bone marrow and provide sustained hematopoietic stem cell self-renewal and hematopoiesis.98,99 It is assumed that this property of mobilization of HSCs is highly conserved in evolution (it has been shown in mouse, dog and humans) and presumably results from contact with natural cell-killing agents in the environment, after which regeneration of hematopoiesis requires restoring empty HSC niches. This means that functional, transplantable HSCs course through every tissue of the body in large numbers every day in normal individuals.

Apoptosis, or programmed cell death, is a mechanism that results in cells actively self-destructing without causing inflammation. Apoptosis is an essential feature in multicellular organisms, necessary during development and normal maintenance of tissues. Apoptosis can be triggered by specific signals, by cells failing to receive the required signals to avoid apoptosis, and by exposure to infectious agents such as viruses. HSCs are not exempt; apoptosis is one mechanism to regulate their numbers. This was demonstrated in transgenic mouse experiments in which HSC numbers doubled when the apoptosis threshold was increased.76 This study also showed that HSCs are particularly sensitive and require two signals to avoid undergoing apoptosis.

The best-known location for HSCs is bone marrow, and bone marrow transplantation has become synonymous with hematopoietic cell transplantation, even though bone marrow itself is increasingly infrequently used as a source due to an invasive harvesting procedure that requires general anesthesia. In adults, under steady-state conditions, the majority of HSCs reside in bone marrow. However, cytokine mobilization can result in the release of large numbers of HSCs into the blood. As a clinical source of HSCs, mobilized peripheral blood (MPB) is now replacing bone marrow, as harvesting peripheral blood is easier for the donors than harvesting bone marrow. As with bone marrow, mobilized peripheral blood contains a mixture of hematopoietic stem and progenitor cells. MPB is normally passed through a device that enriches cells that express CD34, a marker on both stem and progenitor cells. Consequently, the resulting cell preparation that is infused back into patients is not a pure HSC preparation, but a mixture of HSCs, hematopoietic progenitors (the major component), and various contaminants, including T cells and, in the case of autologous grafts from cancer patients, quite possibly tumor cells. It is important to distinguish these kinds of grafts, which are the grafts routinely given, from highly purified HSC preparations, which essentially lack other cell types.

In the late 1980s, umbilical cord blood (UCB) was recognized as an important clinical source of HSCs.100,101 Blood from the placenta and umbilical cord is a rich source of hematopoietic stem cells, and these cells are typically discarded with the afterbirth. Increasingly, UCB is harvested, frozen, and stored in cord blood banks, as an individual resource (donor-specific source) or as a general resource, directly available when needed. Cord blood has been used successfully to transplant children and (far less frequently) adults. Specific limitations of UCB include the limited number of cells that can be harvested and the delayed immune reconstitution observed following UCB transplant, which leaves patients vulnerable to infections for a longer period of time. Advantages of cord blood include its availability, ease of harvest, and the reduced risk of graft-versus-host-disease (GVHD). In addition, cord blood HSCs have been noted to have a greater proliferative capacity than adult HSCs. Several approaches have been tested to overcome the cell dose issue, including, with some success, pooling of cord blood samples.101,102 Ex vivo expansion in tissue culture, to which cord blood cells are more amenable than adult cells, is another approach under active investigation.103

The use of cord blood has opened a controversial treatment strategyembryo selection to create a related UCB donor.104 In this procedure, embryos are conceived by in vitro fertilization. The embryos are tested by pre-implantation genetic diagnosis, and embryos with transplantation antigens matching those of the affected sibling are implanted. Cord blood from the resulting newborn is then used to treat this sibling. This approach, successfully pioneered at the University of Minnesota, can in principle be applied to a wide variety of hematopoietic disorders. However, the ethical questions involved argue for clear regulatory guidelines.105

Embryonic stem (ES) cells form a potential future source of HSCs. Both mouse and human ES cells have yielded hematopoietic cells in tissue culture, and they do so relatively readily.106 However, recognizing the actual HSCs in these cultures has proven problematic, which may reflect the variability in HSC markers or the altered reconstitution behavior of these HSCs, which are expected to mimic fetal HSC. This, combined with the potential risks of including undifferentiated cells in an ES-cell-derived graft means that, based on the current science, clinical use of ES cell-derived HSCs remains only a theoretical possibility for now.

An ongoing set of investigations has led to claims that HSCs, as well as other stem cells, have the capacity to differentiate into a much wider range of tissues than previously thought possible. It has been claimed that, following reconstitution, bone marrow cells can differentiate not only into blood cells but also muscle cells (both skeletal myocytes and cardiomyocytes),107111 brain cells,112,113 liver cells,114,115 skin cells, lung cells, kidney cells, intestinal cells,116 and pancreatic cells.117 Bone marrow is a complex mixture that contains numerous cell types. In addition to HSCs, at least one other type of stem cell, the mesenchymal stem cell (MSC), is present in bone marrow. MSCs, which have become the subject of increasingly intense investigation, seem to retain a wide range of differentiation capabilities in vitro that is not restricted to mesodermal tissues, but includes tissues normally derived from other embryonic germ layers (e.g., neurons).118120MSCs are discussed in detail in Dr. Catherine Verfaillie's testimony to the President's Council on Bioethics at this website: refer to Appendix J (page 295) and will not be discussed further here. However, similar claims of differentiation into multiple diverse cell types, including muscle,111 liver,114 and different types of epithelium116 have been made in experiments that assayed partially- or fully-purified HSCs. These experiments have spawned the idea that HSCs may not be entirely or irreversibly committed to forming the blood, but under the proper circumstances, HSCs may also function in the regeneration or repair of non-blood tissues. This concept has in turn given rise to the hypothesis that the fate of stem cells is quot;plastic,quot; or changeable, allowing these cells to adopt alternate fates if needed in response to tissue-derived regenerative signals (a phenomenon sometimes referred to as quot;transdifferentiationquot;). This in turn seems to bolster the argument that the full clinical potential of stem cells can be realized by studying only adult stem cells, foregoing research into defining the conditions necessary for the clinical use of the extensive differentiation potential of embryonic stem cells. However, as discussed below, such quot;transdifferentiationquot; claims for specialized adult stem cells are controversial, and alternative explanations for these observations remain possible, and, in several cases, have been documented directly.

While a full discussion of this issue is beyond the scope of this overview, several investigators have formulated criteria that must be fulfilled to demonstrate stem cell plasticity.121,122 These include (i) clonal analysis, which requires the transfer and analysis of single, highly-purified cells or individually marked cells and the subsequent demonstration of both quot;normalquot; and quot;plasticquot; differentiation outcomes, (ii) robust levels of quot;plasticquot; differentiation outcome, as extremely rare events are difficult to analyze and may be induced by artefact, and (iii) demonstration of tissue-specific function of the quot;transdifferentiatedquot; cell type. Few of the current reports fulfill these criteria, and careful analysis of individually transplanted KTLS HSCs has failed to show significant levels of non-hematopoietic engraftment.123,124In addition, several reported trans-differentiation events that employed highly purified HSCs, and in some cases a very strong selection pressure for trans-differentiation, now have been shown to result from fusion of a blood cell with a non-blood cell, rather than from a change in fate of blood stem cells.125127 Finally, in the vast majority of cases, reported contributions of adult stem cells to cell types outside their tissue of origin are exceedingly rare, far too rare to be considered therapeutically useful. These findings have raised significant doubts about the biological importance and immediate clinical utility of adult hematopoietic stem cell plasticity. Instead, these results suggest that normal tissue regeneration relies predominantly on the function of cell type-specific stem or progenitor cells, and that the identification, isolation, and characterization of these cells may be more useful in designing novel approaches to regenerative medicine. Nonetheless, it is possible that a rigorous and concerted effort to identify, purify, and potentially expand the appropriate cell populations responsible for apparent quot;plasticityquot; events, characterize the tissue-specific and injury-related signals that recruit, stimulate, or regulate plasticity, and determine the mechanism(s) underlying cell fusion or transdifferentiation, may eventually enhance tissue regeneration via this mechanism to clinically useful levels.

Recent progress in genomic sequencing and genome-wide expression analysis at the RNA and protein levels has greatly increased our ability to study cells such as HSCs as quot;systems,quot; that is, as combinations of defined components with defined interactions. This goal has yet to be realized fully, as computational biology and system-wide protein biochemistry and proteomics still must catch up with the wealth of data currently generated at the genomic and transcriptional levels. Recent landmark events have included the sequencing of the human and mouse genomes and the development of techniques such as array-based analysis. Several research groups have combined cDNA cloning and sequencing with array-based analysis to begin to define the full transcriptional profile of HSCs from different species and developmental stages and compare these to other stem cells.64,65,128131 Many of the data are available in online databases, such as the NIH/NIDDK Stem Cell Genome Anatomy Projects. While transcriptional profiling is clearly a work in progress, comparisons among various types of stem cells may eventually identify sets of genes that are involved in defining the general quot;stemnessquot; of a cell, as well as sets of genes that define their exit from the stem cell pool (e.g., the beginning of their path toward becoming mature differentiated cells, also referred to as commitment). In addition, these datasets will reveal sets of genes that are associated with specific stem cell populations, such as HSCs and MSCs, and thus define their unique properties. Assembly of these datasets into pathways will greatly help to understand and to predict the responses of HSCs (and other stem cells) to various stimuli.

The clinical use of stem cells holds great promise, although the application of most classes of adult stem cells is either currently untested or is in the earliest phases of clinical testing.132,133 The only exception is HSCs, which have been used clinically since 1959 and are used increasingly routinely for transplantations, albeit almost exclusively in a non-pure form. By 1995, more than 40,000 transplants were performed annually world-wide.134,135 Currently the main indications for bone marrow transplantation are either hematopoietic cancers (leukemias and lymphomas), or the use of high-dose chemotherapy for non-hematopoietic malignancies (cancers in other organs). Other indications include diseases that involve genetic or acquired bone marrow failure, such as aplastic anemia, thalassemia sickle cell anemia, and increasingly, autoimmune diseases.

Transplantation of bone marrow and HSCs are carried out in two rather different settings, autologous and allogeneic. Autologous transplantations employ a patient's own bone marrow tissue and thus present no tissue incompatibility between the donor and the host. Allogeneic transplantations occur between two individuals who are not genetically identical (with the rare exceptions of transplantations between identical twins, often referred to as syngeneic transplantations). Non-identical individuals differ in their human leukocyte antigens (HLAs), proteins that are expressed by their white blood cells. The immune system uses these HLAs to distinguish between quot;selfquot; and quot;nonself.quot; For successful transplantation, allogeneic grafts must match most, if not all, of the six to ten major HLA antigens between host and donor. Even if they do, however, enough differences remain in mostly uncharacterized minor antigens to enable immune cells from the donor and the host to recognize the other as quot;nonself.quot; This is an important issue, as virtually all HSC transplants are carried out with either non-purified, mixed cell populations (mobilized peripheral blood, cord blood, or bone marrow) or cell populations that have been enriched for HSCs (e.g., by column selection for CD34+ cells) but have not been fully purified. These mixed population grafts contain sufficient lymphoid cells to mount an immune response against host cells if they are recognized as quot;non-self.quot; The clinical syndrome that results from this quot;non-selfquot; response is known as graft-versus-host disease (GVHD).136

In contrast, autologous grafts use cells harvested from the patient and offer the advantage of not causing GVHD. The main disadvantage of an autologous graft in the treatment of cancer is the absence of a graft-versusleukemia (GVL) or graft-versus-tumor (GVT) response, the specific immunological recognition of host tumor cells by donor-immune effector cells present in the transplant. Moreover, the possibility exists for contamination with cancerous or pre-cancerous cells.

Allogeneic grafts also have disadvantages. They are limited by the availability of immunologically-matched donors and the possibility of developing potentially lethal GVHD. The main advantage of allogeneic grafts is the potential for a GVL response, which can be an important contribution to achieving and maintaining complete remission.137,138

Today, most grafts used in the treatment of patients consist of either whole or CD34+-enriched bone marrow or, more likely, mobilized peripheral blood. The use of highly purified hematopoietic stem cells as grafts is rare.5658 However, the latter have the advantage of containing no detectable contaminating tumor cells in the case of autologous grafts, therefore not inducing GVHD, or presumably GVL,139141in allogeneic grafts. While they do so less efficiently than lymphocyte-containing cell mixtures, HSCs alone can engraft across full allogeneic barriers (i.e., when transplanted from a donor who is a complete mismatch for both major and minor transplantation antigens).139141The use of donor lymphocyte infusions (DLI) in the context of HSC transplantation allows for the controlled addition of lymphocytes, if necessary, to obtain or maintain high levels of donor cells and/or to induce a potentially curative GVL-response.142,143 The main problems associated with clinical use of highly purified HSCs are the additional labor and costs144 involved in obtaining highly purified cells in sufficient quantities.

While the possibilities of GVL and other immune responses to malignancies remain the focus of intense interest, it is also clear that in many cases, less-directed approaches such as chemotherapy or irradiation offer promise. However, while high-dose chemotherapy combined with autologous bone marrow transplantation has been reported to improve outcome (usually measured as the increase in time to progression, or increase in survival time),145154 this has not been observed by other researchers and remains controversial.155161 The tumor cells present in autologous grafts may be an important limitation in achieving long-term disease-free survival. Only further purification/ purging of the grafts, with rigorous separation of HSCs from cancer cells, can overcome this limitation. Initial small scale trials with HSCs purified by flow cytometry suggest that this is both possible and beneficial to the clinical outcome.56 In summary, purification of HSCs from cancer/lymphoma/leukemia patients offers the only possibility of using these cells post-chemotherapy to regenerate the host with cancer-free grafts. Purification of HSCs in allotransplantation allows transplantation with cells that regenerate the blood-forming system but cannot induce GVHD.

An important recent advance in the clinical use of HSCs is the development of non-myeloablative preconditioning regimens, sometimes referred to as quot;mini transplants.quot;162164 Traditionally, bone marrow or stem cell transplantation has been preceded by a preconditioning regimen consisting of chemotherapeutic agents, often combined with irradiation, that completely destroys host blood and bone marrow tissues (a process called myeloablation). This creates quot;spacequot; for the incoming cells by freeing stem cell niches and prevents an undesired immune response of the host cells against the graft cells, which could result in graft failure. However, myeloablation immunocompromises the patient severely and necessitates a prolonged hospital stay under sterile conditions. Many protocols have been developed that use a more limited and targeted approach to preconditioning. These nonmyeloablative preconditioning protocols, which combine excellent engraftment results with the ability to perform hematopoietic cell transplantation on an outpatient basis, have greatly changed the clinical practice of bone marrow transplantation.

FACS purification of HSCs in mouse and man completely eliminates contaminating T cells, and thus GVHD (which is caused by T-lymphocytes) in allogeneic transplants. Many HSC transplants have been carried out in different combinations of mouse strains. Some of these were matched at the major transplantation antigens but otherwise different (Matched Unrelated Donors or MUD); in others, no match at the major or minor transplantation antigens was expected. To achieve rapid and sustained engraftment, higher doses of HSCs were required in these mismatched allogeneic transplants than in syngeneic transplants.139141,165167 In these experiments, hosts whose immune and blood-forming systems were generated from genetically distinct donors were permanently capable of accepting organ transplants (such as the heart) from either donor or host, but not from mice unrelated to the donor or host. This phenomenon is known as transplant-induced tolerance and was observed whether the organ transplants were given the same day as the HSCs or up to one year later.139,166Hematopoietic cell transplant-related complications have limited the clinical application of such tolerance induction for solid organ grafts, but the use of non-myeloablative regimens to prepare the host, as discussed above, should significantly reduce the risk associated with combined HSC and organ transplants. Translation of these findings to human patients should enable a switch from chronic immunosuppression to prevent rejection to protocols wherein a single conditioning dose allows permanent engraftment of both the transplanted blood system and solid organ(s) or other tissue stem cells from the same donor. This should eliminate both GVHD and chronic host transplant immunosuppression, which lead to many complications, including life-threatening opportunistic infections and the development of malignant neoplasms.

We now know that several autoimmune diseasesdiseases in which immune cells attack normal body tissuesinvolve the inheritance of high risk-factor genes.168 Many of these genes are expressed only in blood cells. Researchers have recently tested whether HSCs could be used in mice with autoimmune disease (e.g., type 1 diabetes) to replace an autoimmune blood system with one that lacks the autoimmune risk genes. The HSC transplants cured mice that were in the process of disease development when nonmyeloablative conditioning was used for transplant.169 It has been observed that transplant-induced tolerance allows co-transplantation of pancreatic islet cells to replace destroyed islets.170 If these results using nonmyeloablative conditioning can be translated to humans, type 1 diabetes and several other autoimmune diseases may be treatable with pure HSC grafts. However, the reader should be cautioned that the translation of treatments from mice to humans is often complicated and time-consuming.

Banking is currently a routine procedure for UCB samples. If expansion of fully functional HSCs in tissue culture becomes a reality, HSC transplants may be possible by starting with small collections of HSCs rather than massive numbers acquired through mobilization and apheresis. With such a capability, collections of HSCs from volunteer donors or umbilical cords could be theoretically converted into storable, expandable stem cell banks useful on demand for clinical transplantation and/or for protection against radiation accidents. In mice, successful HSC transplants that regenerate fully normal immune and blood-forming systems can be accomplished when there is only a partial transplantation antigen match. Thus, the establishment of useful human HSC banks may require a match between as few as three out of six transplantation antigens (HLA). This might be accomplished with stem cell banks of as few as 4,00010,000 independent samples.

Leukemias are proliferative diseases of the hematopoietic system that fail to obey normal regulatory signals. They derive from stem cells or progenitors of the hematopoietic system and almost certainly include several stages of progression. During this progression, genetic and/or epigenetic changes occur, either in the DNA sequence itself (genetic) or other heritable modifications that affect the genome (epigenetic). These (epi)genetic changes alter cells from the normal hematopoietic system into cells capable of robust leukemic growth. There are a variety of leukemias, usually classified by the predominant pathologic cell types and/or the clinical course of the disease. It has been proposed that these are diseases in which self-renewing but poorly regulated cells, so-called "leukemia stem cells" (LSCs), are the populations that harbor all the genetic and epigenetic changes that allow leukemic progression.171176 While their progeny may be the characteristic cells observed with the leukemia, these progeny cells are not the self-renewing "malignant" cells of the disease. In this view, the events contributing to tumorigenic transformation, such as interrupted or decreased expression of "tumor suppressor" genes, loss of programmed death pathways, evasion of immune cells and macrophage surveillance mechanisms, retention of telomeres, and activation or amplification of self-renewal pathways, occur as single, rare events in the clonal progression to blast-crisis leukemia. As LT HSCs are the only selfrenewing cells in the myeloid pathway, it has been proposed that most, if not all, progression events occur at this level of differentiation, creating clonal cohorts of HSCs with increasing malignancy (see Figure 2.6). In this disease model, the final event, explosive selfrenewal, could occur at the level of HSC or at any of the known progenitors (see Figures 2.5 and 2.6). Activation of the -catenin/lef-tcf signal transduction and transcription pathway has been implicated in leukemic stem cell self-renewal in mouse AML and human CML.177 In both cases, the granulocyte-macrophage progenitors, not the HSCs or progeny blast cells, are the malignant self-renewing entities. In other models, such as the JunB-deficient tumors in mice and in chronic-phase CML in humans, the leukemic stem cell is the HSC itself.90,177 However, these HSCs still respond to regulatory signals, thus representing steps in the clonal progression toward blast crisis (see Figure 2.6).

Figure 2.6. Leukemic progression at the hematopoietic stem cell level. Self-renewing HSCs are the cells present long enough to accumulate the many activating events necessary for full transformation into tumorigenic cells. Under normal conditions, half of the offspring of HSC cell divisions would be expected to undergo differentiation, leaving the HSC pool stable in size. (A) (Pre) leukemic progression results in cohorts of HSCs with increasing malignant potential. The cells with the additional event (two events are illustrated, although more would be expected to occur) can outcompete less-transformed cells in the HSC pool if they divide faster (as suggested in the figure) or are more resistant to differentiation or apoptosis (cell death), two major exit routes from the HSC pool. (B) Normal HSCs differentiate into progenitors and mature cells; this is linked with limited proliferation (left). Partially transformed HSCs can still differentiate into progenitors and mature cells, but more cells are produced. Also, the types of mature cells that are produced may be skewed from the normal ratio. Fully transformed cells may be completely blocked in terminal differentiation, and large numbers of primitive blast cells, representing either HSCs or self-renewing, transformed progenitor cells, can be produced. While this sequence of events is true for some leukemias (e.g., AML), not all of the events occur in every leukemia. As with non-transformed cells, most leukemia cells (other than the leukemia stem cells) can retain the potential for (limited) differentiation.

Many methods have revealed contributing protooncogenes and lost tumor suppressors in myeloid leukemias. Now that LSCs can be isolated, researchers should eventually be able to assess the full sequence of events in HSC clones undergoing leukemic transformation. For example, early events, such as the AML/ETO translocation in AML or the BCR/ABL translocation in CML can remain present in normal HSCs in patients who are in remission (e.g., without detectable cancer).177,178 The isolation of LSCs should enable a much more focused attack on these cells, drawing on their known gene expression patterns, the mutant genes they possess, and the proteomic analysis of the pathways altered by the proto-oncogenic events.173,176,179 Thus, immune therapies for leukemia would become more realistic, and approaches to classify and isolate LSCs in blood could be applied to search for cancer stem cells in other tissues.180

After more than 50 years of research and clinical use, hematopoietic stem cells have become the best-studied stem cells and, more importantly, hematopoietic stem cells have seen widespread clinical use. Yet the study of HSCs remains active and continues to advance very rapidly. Fueled by new basic research and clinical discoveries, HSCs hold promise for such indications as treating autoimmunity, generating tolerance for solid organ transplants, and directing cancer therapy. However, many challenges remain. The availability of (matched) HSCs for all of the potential applications continues to be a major hurdle. Efficient expansion of HSCs in culture remains one of the major research goals. Future developments in genomics and proteomics, as well as in gene therapy, have the potential to widen the horizon for clinical application of hematopoietic stem cells even further.

Notes:

* Cellerant Therapeutics, 1531 Industrial Road, San Carlos, CA 94070. Current address: Department of Surgery, Arizona Health Sciences Center, 1501 N. Campbell Avenue, P.O. Box 245071, Tucson, AZ 857245071,e-mail: jdomen@surgery.arizona.edu.

** Section on Developmental and Stem Cell Biology, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215, E-mail: Amy_Wagers@harvard.edu

*** Director, Institute for Cancer/Stem Cell Biology and Medicine, Professor of Pathology and Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, Irv@stanford.edu.

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Bone Marrow (Hematopoietic) Stem Cells | stemcells.nih.gov

a, Hindlimb bone marrow cellularity (n=9 mice for -catulin+/+,n=4 mice for -catulinGFP/+ and n=9 mice for -catulinGFP/GFP genotype) and spleen cellularity (n=6 mice for -catulin+/+,n=4 mice for -catulinGFP/+ and n=6 mice for -catulinGFP/GFP genotype), spleen mass (7 mice for -catulin+/+,n=4 mice for -catulinGFP/+ and n=7 mice for -catulinGFP/GFP genotype). b, White blood cell (WBC), red blood cell (RBC) and platelet (PLT) counts per microliter of peripheral blood from 812 week old -catulin+/+, -catulinGFP/+, and -catulinGFP/GFP mice (n=9 mice/genotype). c,d, Frequencies of mature hematopoietic cells and progenitors in the bone marrow of 812 week old -catulin+/+ and -catulinGFP/GFP mice (Pre-ProB cells were B220+sIgMCD43+CD24; ProB cells were B220+sIgMCD43+CD24+; Pre-B cells were B220+sIgMCD43; common lymphoid progenitors (CLPs) were Linc-kitlowSca1lowCD127+CD135+; common myeloid progenitors (CMPs) were Linc-kit+Sca1CD34+CD16/32; granulocyte-macrophage progenitors (GMPs) were Linc-kit+Sca1CD34+CD16/32+; and megakaryocyte-erythroid progenitors (MEPs) were Linc-kit+Sca1CD34CD16/32 (n=3 mice/genotype). e, Bone marrow CD150+CD48LSK HSC frequency, bone marrow CD150CD48LSK MPP frequency (n=12 mice/genotype in 12 independent experiments), and spleen HSC frequency (n=3 mice/genotype in 3 experiments). f, Percentage of HSCs and whole bone marrow cells that incorporated a 3 day pulse of BrdU in vivo (n=6 -catulin+/+, 9 -catulinGFP/+, and 7 -catulinGFP/GFP 812 week old mice in 3 independent experiments). g, Colony formation by HSCs in methylcellulose cultures (GM means granulocyte-macrophage colonies, GEMM means granulocyte-erythroid-macrophage-megakaryocyte colonies, Mk means megakaryocyte colonies; (n=5 mice/genotype in 5 independent experiments). h, Reconstitution of irradiated mice by 300,000 donor bone marrow cells from 812 week old -catulin+/+, -catulinGFP/+, or -catulinGFP/GFP mice competed against 300,000 recipient bone marrow cells (n=4 donor mice and 16 recipient mice for -catulin+/+, n=3 donor mice and 9 recipient mice for -catulinGFP/+, and n=4 donor mice and 18 recipients for -catulinGFP/GFP in 3 independent experiments). i, Serial transplantation of 3 million WBM cells from primary recipient mice shown in panel d into irradiated secondary recipient mice (n=4 primary -catulin+/+ recipients were transplanted into 17 secondary recipients and n=6 primary -catulinGFP/GFP recipients were transplanted into 20 secondary recipients). All data represent means.d. The statistical significance of differences between genotypes was assessed using Students t-tests or ANOVAs. None were significant.

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Human peripheral blood cells are isolated from adult whole blood or from a leukapheresis preparation produced using state-of-the-art apheresis systems. These apheresis collections, known as "Leuko Paks", contain very high concentrations of mononuclear cells and are available in the following Pak sizes: full, half and quarter Pak. Specific cell subsets are purified using STEMCELL's cell isolation products. Fresh whole blood products are collected directly into blood bags using acid-citrate-dextrose solution A (ACDA) or citrate-phosphate-dextrose (CPD) as the anticoagulant.

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See comment in PubMed Commons below N Engl J Med. 2012 Oct 18;367(16):1487-96. doi: 10.1056/NEJMoa1203517. Anasetti C, Logan BR, Lee SJ, Waller EK, Weisdorf DJ, Wingard JR, Cutler CS, Westervelt P, Woolfrey A, Couban S, Ehninger G, Johnston L, Maziarz RT, Pulsipher MA, Porter DL, Mineishi S, McCarty JM, Khan SP, Anderlini P, Bensinger WI, Leitman SF, Rowley SD, Bredeson C, Carter SL, Horowitz MM, Confer DL; Blood and Marrow Transplant Clinical Trials Network. Collaborators (182)

Horowitz MM, Carter SL, Confer DL, DiFronzo N, Wagner E, Merritt W, Wu R, Anasetti C, Logan BR, Lee SJ, Waller EK, Weisdorf DJ, Wingard JR, Couban S, Anderlini P, Bensinger WI, Leitman SF, Rowley SD, Carter SL, Karanes C, Horowitz MM, Confer DL, Allen C, Colby C, Gurgol C, Knust K, Foley A, King R, Mitchell P, Couban S, Pulsipher MA, Ehninger G, Johnston L, Khan SP, Maziarz RT, McCarty JM, Mineishi S, Porter DL, Bredeson C, Anasetti C, Lee S, Waller EK, Wingard JR, Cutler CS, Westervelt P, Woolfrey A, Logan BR, Carter SL, Lee SJ, Waller EK, Anasetti C, Logan BR, Lee SJ, Stadtmauer E, Wingard J, Vose J, Lazarus H, Cowan M, Wingard J, Westervelt P, Litzow M, Wu R, Geller N, Carter S, Confer D, Horowitz M, Poland N, Krance R, Carrum G, Agura E, Nademanee A, Sahdev I, Cutler C, Horwitz ME, Kurtzberg J, Waller EK, Woolfrey A, Rowley S, Brochstein J, Leber B, Wasi P, Roy J, Jansen J, Stiff PJ, Khan S, Devine S, Maziarz R, Nemecek E, Huebsch L, Couban S, McCarthy P, Johnston L, Shaughnessy P, Savoie L, Ball E, Vaughan W, Cowan M, Horn B, Wingard J, Silverman M, Abhyankar S, McGuirk J, Yanovich S, Ferrara J, Weisdorf D, Faber E Jr, Selby G, Rooms LM, Porter D, Agha M, Anderlini P, Lipton J, Pulsipher MA, Pulsipher MA, Shepherd J, Toze C, Kassim A, Frangoul H, McCarty J, Hurd D, DiPersio J, Westervelt P, Shenoy S, Agura E, Culler E, Axelrod F, Chambers L, Senaldi E, Nguyen KA, Engelman E, Hartzman R, Sutor L, Dickson L, Nademanee A, Khalife G, Lenes BA, Eames G, Sibley D, Gale P, Antin J, Ehninger G, Newberg NR, Gammon R, Montgomery M, Mair B, Rossmann S, Wada R, Waxman D, Ranlett R, Silverman M, Herzig G, Fried M, Atkinson E, Weitekamp L, Bigelow C, Miller JP, Miller JP, Miller JP, Miller JP, Miller JP, Miller JP, Miller JP, Miller JP, Miller JP, Miller JP, Miller JP, Miller JP, Miller JP, Miller JP, Miller JP, Miller JP, Price T, Young C, Hilbert R, Oh D, Cable C, Smith JW, Kalmin ND, Schultheiss K, Beck T, Lankiewicz MW, Sharp D.

Randomized trials have shown that the transplantation of filgrastim-mobilized peripheral-blood stem cells from HLA-identical siblings accelerates engraftment but increases the risks of acute and chronic graft-versus-host disease (GVHD), as compared with the transplantation of bone marrow. Some studies have also shown that peripheral-blood stem cells are associated with a decreased rate of relapse and improved survival among recipients with high-risk leukemia.

We conducted a phase 3, multicenter, randomized trial of transplantation of peripheral-blood stem cells versus bone marrow from unrelated donors to compare 2-year survival probabilities with the use of an intention-to-treat analysis. Between March 2004 and September 2009, we enrolled 551 patients at 48 centers. Patients were randomly assigned in a 1:1 ratio to peripheral-blood stem-cell or bone marrow transplantation, stratified according to transplantation center and disease risk. The median follow-up of surviving patients was 36 months (interquartile range, 30 to 37).

The overall survival rate at 2 years in the peripheral-blood group was 51% (95% confidence interval [CI], 45 to 57), as compared with 46% (95% CI, 40 to 52) in the bone marrow group (P=0.29), with an absolute difference of 5 percentage points (95% CI, -3 to 14). The overall incidence of graft failure in the peripheral-blood group was 3% (95% CI, 1 to 5), versus 9% (95% CI, 6 to 13) in the bone marrow group (P=0.002). The incidence of chronic GVHD at 2 years in the peripheral-blood group was 53% (95% CI, 45 to 61), as compared with 41% (95% CI, 34 to 48) in the bone marrow group (P=0.01). There were no significant between-group differences in the incidence of acute GVHD or relapse.

We did not detect significant survival differences between peripheral-blood stem-cell and bone marrow transplantation from unrelated donors. Exploratory analyses of secondary end points indicated that peripheral-blood stem cells may reduce the risk of graft failure, whereas bone marrow may reduce the risk of chronic GVHD. (Funded by the National Heart, Lung, and Blood Institute-National Cancer Institute and others; ClinicalTrials.gov number, NCT00075816.).

Survival after Randomization in the Intention-to-Treat Analysis

The P value is from a stratified binomial comparison at the 2-year point. The P value from a stratified log-rank test was also not significant. A total of 75 patients in each group were still alive at 36 months.

N Engl J Med. ;367(16):10.1056/NEJMoa1203517.

Outcomes after Transplantation, According to Study Group

Panel A shows the rate of overall survival, and Panel B the rate of disease-free survival. Panel C shows the incidence of death unrelated to relapse. Panel D shows the incidence of relapse. Panel E shows the incidence of neutrophil engraftment (>500 neutrophils per cubic millimeter), and Panel F the incidence of platelet engraftment (>20,000 platelets per cubic millimeter, without platelet transfusion during the prior 7 days). Panel G shows the incidence of acute graft-versus-host disease (GVHD) of grades II to IV, and Panel H the incidence of chronic GVHD. P values for the between-group differences in overall survival (Panel A) and disease-free survival (Panel B) are from a stratified binomial comparison at the 2-year point; P values from stratified log-rank tests for survival and disease-free survival were also not significant. All other P values shown are from stratified log-rank tests.

N Engl J Med. ;367(16):10.1056/NEJMoa1203517.

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Peripheral-blood stem cells versus bone marrow from ...

Abstract

Mesenchymal stem cells (MSCs) represent a promising tool for new clinical concepts in supporting cellular therapy. Bone marrow (BM) was the first source reported to contain MSCs. However, for clinical use, BM may be detrimental due to the highly invasive donation procedure and the decline in MSC number and differentiation potential with increasing age. More recently, umbilical cord blood (UCB), attainable by a less invasive method, was introduced as an alternative source for MSCs. Another promising source is adipose tissue (AT). We compared MSCs derived from these sources regarding morphology, the success rate of isolating MSCs, colony frequency, expansion potential, multiple differentiation capacity, and immune phenotype. No significant differences concerning the morphology and immune phenotype of the MSCs derived from these sources were obvious. Differences could be observed concerning the success rate of isolating MSCs, which was 100% for BM and AT, but only 63% for UCB. The colony frequency was lowest in UCB, whereas it was highest in AT. However, UCB-MSCs could be cultured longest and showed the highest proliferation capacity, whereas BM-MSCs possessed the shortest culture period and the lowest proliferation capacity. Most strikingly, UCB-MSCs showed no adipogenic differentiation capacity, in contrast to BM- and AT-MSCs. Both UCB and AT are attractive alternatives to BM in isolating MSC: AT as it contains MSCs at the highest frequency and UCB as it seems to be expandable to higher numbers.

Mesenchymal stem cells (MSCs) found in many adult tissues are an attractive stem cell source for the regeneration of damaged tissues in clinical applications because they are characterized as undifferentiated cells, able to self-renew with a high proliferative capacity, and possess a mesodermal differentiation potential [1].

Although bone marrow (BM) has been the main source for the isolation of multipotent MSCs, the harvest of BM is a highly invasive procedure and the number, differentiation potential, and maximal life span of MSCs from BM decline with increasing age [24]. Therefore, alternative sources from which to isolate MSCs are subject to intensive investigation.

One alternative source is umbilical cord blood (UCB), which can be obtained by a less invasive method, without harm for the mother or the infant [5]. However, controversy still exists whether full-term UCB can serve as a source for isolating multipotent MSCs: although some groups did not succeed in isolating MSCs [6, 7], we and other groups succeeded in isolating MSCs from full-term UCB [812].

Adipose tissue (AT) is another alternative source that can be obtained by a less invasive method and in larger quantities than BM. It has been demonstrated that AT contains stem cells similar to BM-MSCs, which are termed processed lipoaspirate (PLA) cells [13]. These cells can be isolated from cosmetic liposuctions in large numbers and grown easily under standard tissue culture conditions [13]. The multilineage differentiation capacity of PLA cells has been confirmed [13].

As BM-MSCs are best characterized, we asked whether MSCs derived from other sources share the characteristics of BM-MSCs. The aim of our study was to compare MSCs isolated from the three sources under identical in vitro conditions with respect to their morphology, frequency of colonies, expansion characteristics, multilineage differentiation capacity, immunophenotype, and success rate of isolating the cells.

We compared MSCs from BM and two alternative sources, namely UCB and AT, concerning basic MSC characteristics. All cells isolated from these three sources exhibited typical MSC characteristics: a fibroblastoid morphology, the formation of CFU-F, a multipotential differentiation capability, and the expression of a typical set of surface proteins. Whereas MSCs derived from the three sources expressed classic MSC marker proteins, but lacked hematopoietic and endothelial markers, we observed significant differences concerning the expression of CD90, CD105, and CD106. These molecules are described to be associated with hematopoiesis and cell migration [18 20]. It needs to be further investigated whether these molecules are functionally important for stroma and homing capacities. In a first approach, we created a comprehensive protein expression profile of undifferentiated UCB-MSCs, which will be extended to BM- and AT-MSCs and then correlated to functional properties [21].

Since the relevance of the observed differences of marker expression has not been properly investigated yet, differences concerning differentiation capacity seem to be more relevant for MSC quality at present. We demonstrated a multilineage differentiation capacity for BM- and AT-MSCs. Interestingly, UCB-MSCs could not be differentiated toward the adipogenic lineage, which was not related to the CFU-F origin. Actually, there are conflicting data concerning the adipogenic differentiation capacity of UCB-MSCs [9 12, 22, 23]. Nevertheless, we assume that UCB-MSCs are less sensitive toward the adipogenic differentiation (supported by results of Chang et al. [22]) which might be related to the ontogenetic age of these cells. This is further supported by the fact that adipocytes reside in adult human BM and AT but are absent in fetal BM and by the observation of an increased adipogenesis correlated with age [24]. Further comparative genomic or proteomic approaches are needed to assess the susceptibility toward adipogenesis of MSCs.

None of our UCB-MSCs showed adipogenic differentiation capacity, but all differentiated into both the chondro- and osteogenic lineages. In contrast, a tripotential differentiation capacity was observed for most AT samples but only for a few BM samples. One sample each of BM and AT was observed to undergo only the chondrogenic pathway. In accordance with this, a hierarchical or even restricted differentiation potential of MSCs has been reported [1, 13, 25].

In our study, investigations were limited to the mesodermal differentiation capacity. Based on recent reports, however, the spectrum of differentiation of MSCs does not seem to be restricted to this lineage. MSCs derived from all three tissues have been shown to differentiate into further mesodermal lineages and into endo- and ectodermal lineages as well [10 13, 26 33]. Comparative experiments need to be performed to assess responsiveness toward cardiomyogenic, endothelial, hepatic, neuronal, and pancreatic differentiation.

A high impact on clinical exploitation might be related to the abundance and expansion capacity of MSCs. Based on our results, both BM and AT are reliable sources for isolating and expanding MSCs in autologous settings since all preparations gave rise to MSCs. UCB, in contrast, had an isolation efficacy of a maximum of 63% [8]. We attribute these differences to the fact that MSCs are circulating in the prenatal organism and are residing in tissues of the adult [9]. Despite the low frequency of UCB-MSCs, the expansion potential was highest compared with other cell sources. Considering clinical applications, the resulting cell numbers may be similar to both BM and AT, which can be obtained at higher frequencies. One argument against AT might be the limited availability in some patients. However, we believe that due to the high frequency of AT-MSCs, also small fat reservoirs might be sufficient for MSC isolation. BM has been the main source for clinical application of MSCs, such as the treatment of osteogenesis imperfecta, graft versus host disease, and acute myocardial infarction [3436]. As the number, frequency, and differentiation capacity of BM-MSCs correlate negatively with age, they could be clinically inefficient when derived from elderly patients. In that case, an allogeneic approach would be required. In case a matching donor is required, BM or AT from HLA identical siblings, haplo-identical relatives, or HLA-screened donors might be best choice. Speculating on a off-the-shelf product requiring mass production, AT might be a solid starting basis due to the abundance, relatively easy harvest, and high MSCs frequency.

Transplantation of MSCs is currently a highly experimental procedure, resembling the early beginnings of hematopoietic stem cell transplantation. In the latter, BM has been replaced gradually by peripheral blood progenitor cells and umbilical cord blood. Also, in the field of MSCs, alternative sources are intensely investigated, and one day these new sources may replace BM. Taking into account all the advantages and disadvantages of the three sources discussed above, depending on the therapeutic indication, the clinical applications may be based on differentiation capacity, but more likely on the abundance, frequency, and expansion potential of the cells.

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Comparative Analysis of Mesenchymal Stem Cells from Bone ...

A stem cell transplant is a treatment for some types of cancer. For example, you might have one if you have leukemia, multiple myeloma, or some types of lymphoma. Doctors also treat some blood diseases with stem cell transplants.

In the past, patients who needed a stem cell transplant received a bone marrow transplant because the stem cells were collected from the bone marrow. Today, stem cells are usually collected from the blood, instead of the bone marrow. For this reason, they are now more commonly called stem cell transplants.

A part of your bones called bone marrow makes blood cells. Marrow is the soft, spongy tissue inside bones. It contains cells called hematopoietic stem cells (pronounced he-mah-tuh-poy-ET-ick). These cells can turn into several other types of cells. They can turn into more bone marrow cells. Or they can turn into any type of blood cell.

Certain cancers and other diseases keep hematopoietic stem cells from developing normally. If they are not normal, neither are the blood cells that they make. A stem cell transplant gives you new stem cells. The new stem cells can make new, healthy blood cells.

The main types of stem cell transplants and other options are discussed below.

Autologous transplant. Doctors call this an AUTO transplant. This type of stem cell transplant may also be called high-dose chemotherapy with autologous stem cell rescue.

In an AUTO transplant, you get your own stem cells after doctors treat the cancer. First, your health care team collects stem cells from your blood and freezes them. Next, you have powerful chemotherapy, and rarely, radiation therapy. Then, your health care team thaws your frozen stem cells. They put them back in your blood through a tube placed in a vein (IV).

It takes about 24 hours for your stem cells to reach the bone marrow. Then they start to grow, multiply, and help the marrow make healthy blood cells again.

Allogeneic transplantation. Doctors call this an ALLO transplant.

In an ALLO transplant, you get another persons stem cells. It is important to find someone whose bone marrow matches yours. This is because you have certain proteins on your white blood cells called human leukocyte antigens (HLA). The best donor has HLA proteins as much like yours as possible.

Matching proteins make a serious condition called graft-versus-host disease (GVHD) less likely. In GVHD, healthy cells from the transplant attack your cells. A brother or sister may be the best match. But another family member or volunteer might work.

Once you find a donor, you receive chemotherapy with or without radiation therapy. Next, you get the other persons stem cells through a tube placed in a vein (IV). The cells in an ALLO transplant are not typically frozen. So, doctors can give you the cells as soon after chemotherapy or radiation therapy as possible.

There are 2 types of ALLO transplants. The best type for each patient depends his or her age and health and the type of disease being treated.

Ablative, which uses high-dose chemotherapy

Reduced intensity, which uses milder doses of chemotherapy

If your health care team cannot find a matched adult donor, there are other options. Research is ongoing to determine which type of transplant will work best for different patients.

Umbilical cord blood transplant. This may be an option if you cannot find a donor match. Cancer centers around the world use cord blood.

Parent-child transplant and haplotype mismatched transplant. These types of transplants are being used more commonly. The match is 50%, instead of near 100%. Your donor might be a parent, child, brother, or sister.

Your doctor will recommend an AUTO or ALLO transplant based mostly on the disease you have. Other factors include the health of your bone marrow and your age and general health. For example, if you have cancer or other disease in your bone marrow, you will probably have an ALLO transplant. In this situation, doctors do not recommend using your own stem cells.

Choosing a transplant is complicated. You will need help from a doctor who specializes in transplants. So you might need to travel to a center that does many stem cell transplants. Your donor might need to go, too. At the center, you talk with a transplant specialist and have an examination and tests. Before a transplant, you should also think about non-medical factors. These include:

Who can care for you during treatment

How long you will be away from work and family responsibilities

If your insurance pays for the transplant

Who can take you to transplant appointments

Your health care team can help you find answers to these questions.

The information below tells you the main parts of AUTO and ALLO transplants. Your health care team usually does the steps in order. But sometimes certain steps happen in advance, such as collecting stem cells. Ask your doctor what to expect before, during, and after a transplant.

A doctor puts a thin tube called a transplant catheter in a large vein. The tube stays in until after the transplant. Your health care team will collect stem cells through this tube and give chemotherapy and other medications through the tube.

You get injections of a medication to raise your number of white blood cells. White blood cells help your body fight infections.

Your health care team collects stem cells, usually from your blood.

Time: 1 to 2 weeks

Where its done: Clinic or hospital building. You do not need to stay in the hospital overnight.

Time: 5 to 10 days

Where its done: Clinic or hospital. At many transplant centers, patients need to stay in the hospital for the duration of the transplant, usually about 3 weeks. At some centers, patients receive treatment in the clinic and can come in every day.

Time: Each infusion usually takes less than 30 minutes. You may receive more than 1 infusion.

Where its done: Clinic or hospital.

Time: approximately 2 weeks

Where its done: Clinic or hospital. You might be staying in the hospital or you might not.

Time: Varies based on how the stem cells are collected

Where its done: Clinic or hospital

Time: 5 to 7 days

Where its done: Many ALLO transplants are done in the hospital.

Time: 1 day

Where its done: Clinic or hospital.

You take antibiotics and other drugs. This includes medications to prevent graft-versus-host disease. You get blood transfusions through your catheter if needed. Your health care team takes care of any side effects from the transplant.

After the transplant, patients visit the clinic frequently at first and less often over time.

Time: Varies

For an ablative transplant, patients are usually in the hospital for about 4 weeks in total.

For a reduced intensity transplant, patients are in the hospital or visit the clinic daily for about 1 week.

The words successful transplant might mean different things to you, your family, and your doctor. Below are 2 ways to measure transplant success.

Your blood counts are back to safe levels. A blood count is the number of red cells, white cells, and platelets in your blood. A transplant makes these numbers very low for 1 to 2 weeks. This causes risks of:

Infection from low numbers of white cells, which fight infections

Bleeding from low numbers of platelets, which stop bleeding

Tiredness from low numbers of red cells, which carry oxygen

Doctors lower these risks by giving blood and platelet transfusions after a transplant. You also take antibiotics to help prevent infections. When the new stem cells multiply, they make more blood cells. Then your blood counts improve. This is one way to know if a transplant is a success.

It controls your cancer. Doctors do stem cell transplants with the goal of curing disease. A cure may be possible for some cancers, such as some types of leukemia and lymphoma. For other patients, remission is the best result. Remission is having no signs or symptoms of cancer. After a transplant, you need to see your doctor and have tests to watch for any signs of cancer or complications from the transplant.

Talking often with the doctor is important. It gives you information to make health care decisions. The questions below may help you learn more about stem cell transplant. You can also ask other questions that are important to you.

Which type of stem cell transplant would you recommend? Why?

If I will have an ALLO transplant, how will we find a donor? What is the chance of a good match?

What type of treatment will I have before the transplant? Will radiation therapy be used?

How long will my treatment take? How long will I stay in the hospital?

How will a transplant affect my life? Can I work? Can I exercise and do regular activities?

How will we know if the transplant works?

What if the transplant doesnt work? What if the cancer comes back?

What are the side effects? This includes short-term, such as during treatment and shortly after. It also includes long-term, such as years later.

What tests will I need later? How often will I need them?

If I am worried about managing the costs of treatment, who can help me with these concerns?

Bone Marrow Aspiration and Biopsy

Making Decisions About Cancer Treatment

Donating Blood and Platelets

Donating Umbilical Cord Blood

Explore BMT

Be the Match: National Marrow Donor Program

Blood & Marrow Transplant Information Network

U.S. Department of Health and Human Services: Understanding Transplantation as a Treatment Option

National Bone Marrow Transplant Link

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What is a Stem Cell Transplant (Bone Marrow Transplant ...

Mesenchymal stem cells (MSCs) are multipotent adult stem cells that are present in practically all tissues as a specialized population of mural cells/pericytes that lie on the abluminal side of blood vessels. Originally identified within the bone marrow (BM) stroma, not only do they provide microenvironmental support for hematopoietic stem cells (HSCs), but can also differentiate into various mesodermal lineages. MSCs can easily be isolated from the BM and subsequently expand in vitro and in addition they exhibit intriguing immunomodulatory properties, thereby emerging as attractive candidates for various therapeutic applications. This review addresses the concept of BM MSCs via a hematologist's point of view. In this context it discusses the stem cell properties that have been attributed to BM MSCs, as compared to those of the prototypic hematopoietic stem cell model and then gives a brief overview of the in vitro and vivo features of the former, emphasizing on their immunoregulatory properties and their hematopoiesis-supporting role. In addition, the qualitative and quantitative characteristics of BM MSCs within the context of a defective microenvironment, such as the one characterizing Myelodysplastic Syndromes are described and the potential involvement of these cells in the pathophysiology of the disease is discussed. Finally, emerging clinical applications of BM MSCs in the field of hematopoietic stem cell transplantation are reviewed and potential hazards from MSC use are outlined.

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Bone marrow mesenchymal stem cells: biological properties ...

ai, In vivo self-renewal of adult bone marrow CD45Nes-GFP+ cells in secondary transplants. a, Scheme showing the experimental paradigm. be, Primary ossicles showing numerous -galactosidase+ osteoblasts derived from CD45Nes-GFP+ cells (b, blue; d, e, dark deposits, arrowheads) but none from CD45Nes-GFP cells (c); haematopoietic areas (b, e; circled by dashed line) were detected only in the former group and frequently associated with GFP+ cells (e, green). f, Secondary ossicle showing numerous -galactosidase+ osteoblasts derived from CD45Nes-GFP+ cells (blue) and also haematopoietic areas (circled by dashed line). g, CD45+ haematopoietic cells (red) localized near Nes-GFP+ (green) cells in the ossicles; cell nuclei have been stained with DAPI (blue); grid, 50 m per square. h, i, Secondary ossicles yielded 8,557 537 GFP+ spheres (h) that generated Col2.3+ osteoblasts (i; blue precipitates). jm, Adult nestin+ MSCs contribute to endochondral lineages. jl, Femoral sections from 11-month-old Nes-creERT2/RCE:loxP double-transgenic mice 8 months after tamoxifen treatment showing the contribution of adult nestin+ cells to bone-lining osteoblasts (j), osteocytes (k; asterisks indicate GFP+ cells, arrowheads indicate GFP osteocytes) and collagen 1 type 2+ (red) chondrocytes (l). m, GFP+ (green) perivascular cells (asterisk) identical in frequency, morphology and distribution to Nes-GFP+ cells and osteoblasts (arrowhead) co-stained with anti-GFP antibodies (red). n, o, Bone marrow section of Nes-Gfp/Col2.3-cre/R26R triple-transgenic mouse showing X-gal+ osteoblasts (dark precipitates), GFP+ (green) and CD31+ vascular endothelial cells (red); Col2.3+ osteoblasts localized near Nes-GFP+ perivascular cells are indicated with arrowheads. p, q, Immunostaining for osterix (red) in trabecular bone section of a 5-week-old Nes-Gfp (green) mouse. g, jm, oq, Nuclei have been stained with DAPI (blue). j, l,m, p, q, Bone (b) margins are indicated with dashed lines. b, c, f, i, n, Bright field; g, j, k, p, q, fluorescence; d, e, h, l, m, o, overlapped fluorescence and bright field. Scale bars: 100 m (cf, h, i); 20 m (jq).

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Mesenchymal and haematopoietic stem cells form a unique ...

a. HSCs are found mainly adjacent to sinusoids throughout the bone marrow,,,, where endothelial cells and mesenchymal stromal cells promote HSC maintenance by producing SCF, CXCL12,,, and likely other factors. Similar cells may also promote HSC maintenance around other types of blood vessels, such as arterioles. The mesenchymal stromal cells can be identified based on their expression of Lepr-Cre, Prx1-Cre, Cxcl12-GFP, or Nestin-GFP transgene in mice and similar cells are likely to be identified by CD146 expression in humans. These perivascular stromal cells, which likely include Cxcl12-abundant Reticular (CAR) cells, are fated to form bone in vivo, express Mx-1-Cre and overlap with CD45/Ter119PDGFR +Sca-1+ stromal cells that are highly enriched for MSCs in culture. b. It is likely that other cells also contribute to this niche, likely including cells near bone surfaces in trabecular rich areas. Other cell types that regulate HSC niches include sympathetic nerves,, non-myelinating Schwann cells (which are also Nestin+), macrophages, osteoclasts, extracellular matrix ,, and calcium. Osteoblasts do not directly promote HSC maintenance but do promote the maintenance and perhaps the differentiation of certain lymphoid progenitors by secreting Cxcl12 and likely other factors,,,. Early lineage committed progenitors thus reside in an endosteal niche that is spatially and cellularly distinct from HSCs.

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The Bone Marrow Niche for Haematopoietic Stem Cells

This week I treated a patient with adipose SVF stem cells to augment a low stem cell yield from bone marrow. I dont do this often, as the quality of fat stem cells for orthopedic applications like arthritis is much less. We do use fat for an occasional structural graft in various procedures. Today I wanted to give you a clinicians eye view of the harvest procedures for both stem cell types that you wont see elsewhere, so let Fat vs Bone Marrow Stem Cells begin.

In summary, harvesting fat in a mini-liposuction is a violent affair, harvesting stem cells from a bone marrow aspirate is like an advanced blood draw. Let me explain.

In order to get fat through a mini-liposuction you need to first use a scalpel to open a small incision in the skin. This isnt at all required for a bone marrow aspiration as the needle is just inserted into the skin like any other needle. In the liposuction, the whole goal is disrupting large amounts of normal tissue. In fact, the stem cells live around the blood vessels, so you have to chew up as many blood vessels in the fat as possible to get a good stem cell yield. This involves placing a small wand like device under the skin and into the fat and moving it back and forth (through much resistance) to break apart large sections of tissue. The bone marrow aspiration simply involves directing the needle under the x-ray to the desired area of bone. The needle is then turned back and forth a few times to enter the bone (which is like hard plastic instead of cement). At this point in the liposuction the doctor must continue to break up large swaths of tissue with suction, sucking the broken tissue and blood vessels into a syringe. On the other hand, in the bone marrow aspiration the doctor simply draws the bone marrow aspirate (which looks like blood) into the syringe like a common blood draw.

The complication rates for these two procedures tell the rest of the story. Mini-liposuction procedures have surgical style complication rates of 3-10%, while bone marrow aspiration complication rates are so rare that only a handful occurred in more than 20,000 procedures in one U.K. registry. The upshot? It always makes me chuckle (in a bad way) when I hear fat stem cell advocates claim that a bone marrow aspiration procedure is so invasive. Youhavent seen invasive until youve seen a lipo-suction!

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Fat vs. Bone Marrow Stem Cells: A Clinicians Perspective

There are two different types of stem cell transplants:

To understand these treatments, it first helps to learn how the bone marrow and stem cells work.

Stem cells are blood cells at their earliest stage of development. All blood cells develop from stem cells. The full name for stem cells in the blood and bone marrow is haematopoietic stem cells, but in this booklet we call them stem cells.

Bone marrow is a spongy material inside the bones particularly the bones of the pelvis. The bone marrow is where stem cells are made.

Most of the time, almost all of your stem cells are in the bone marrow. There are usually only a very small number in the blood. Stem cells stay in the bone marrow while they develop into blood cells. Then, once they are fully mature, the blood cells are released into the bloodstream.

The three main types of blood cells are:

Illustration of bone marrow

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The levels of blood cells in your blood are measured in a blood test called a full blood count (FBC). Its often just called a blood count.

The figures below are a guide to the levels usually found in a healthy person.

These figures can vary from hospital to hospital. Your doctor or nurse can tell you what levels they use. They can also vary slightly between people from different ethnic groups.

The figures might look complicated when theyre written down, but in practice theyre used in a straightforward way. For example, youll hear doctors or nurses saying things like your haemoglobin is 140 or your neutrophils are 4.

Most people with cancer or leukaemia soon get used to these figures and what they mean. But you can always ask your medical team to explain if youre not sure.

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What are stem cells and bone marrow? - Information and ...

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Cryopreserved Leuko Pak and Whole Blood Products:

Donor Screening:Donors are screened for HIV (1 & 2), Hepatitis B, and Hepatitis C.

Cryopreserved products are shipped with negative test results from donor screening that is done within 90 days of collection.

Fresh Leuko Pak and Whole Blood Products:

Donor Screening:Donors are screened for HIV (1 & 2), Hepatitis B, and Hepatitis C.

If the donor was screened within 90 days of donation the product will be shipped with negative test results from donor screening.

If the donor was not screened within 90 days of collection, a test sample will be taken at the time of donation and the product will be shipped before the screening results are available. In the unlikely event that a test result is positive, the customer will be contacted as soon as possible (usually within 24-72 hours from the time of shipment).

Cryopreserved Cord Blood Products:

Donor Screening:Cord blood is only collected from mothers that have tested negative for HIV (1 & 2) and Hepatitis B during their pregnancy. Hepatitis C is tested for at the time of collection.

Cryopreserved products are shipped with negative test results from donor screening.

Fresh Cord Blood Products:

Donor Screening: Cord blood is only collected from mothers that have tested negative for HIV (1 & 2) and Hepatitis B during their pregnancy. Hepatitis C is tested for at the time of collection.

Fresh cord blood products are shipped with negative test results for HIV (1 & 2) and Hepatitis B donor screening. Hepatitis C test results are not available at the time of shipment. In the unlikely event that the Hepatitis C test result is positive, the customer will be contacted as soon as possible (usually within 24-72 hours from the time of shipment).

STEMCELL does not test for infectious diseases other than those listed above and the testing that is done cannot completely guarantee that the donor was virus-free. Therefore THESE PRODUCTS SHOULD BE TREATED AS POTENTIALLY INFECTIOUS and only used following appropriate handling precautions such as those described in biological safety level 2. When handling these products do not use sharps such as needles and syringes.

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Human Primary Cells - Stemcell Technologies

What are hematopoietic cell transplantation (HCT) and peripheral blood stem cell transplantation (PBSCT)?

HCT and PBSCT are procedures that use stem cells to treat a patient's malignancy or to repair diseased or defective bone marrow. A patient receives intensive chemotherapy with or without total body irradiation therapy in an attempt to kill all cancerous cells, but which also destroy his/her own bone marrow function. This therapy also causes immunosuppression, which prevents rejection of the newly transplanted stem cells from a related or unrelated donor.

There is little risk of rejection of a patient's own stem cells following autologous transplant. After transplantation, the new stem cells replace the damaged bone marrow and cells of the immune system.

How do HCT and PBSCT help patients?

HCT and PBSCT allow a patient to receive very high doses of chemotherapy and radiation designed to kill cancer cells. The high doses of therapy lead to the destruction of a patient's own marrow and immune system, which is then replaced by marrow from a donor or from peripheral blood stem cells that have been harvested before therapy.

How many HCTs and PBSCTs are performed at City of Hope?

City of Hope has performed more than 12,000 transplants for patients from virtually every state as well as from numerous countries. HCT and PBSCT patients at City of Hope have ranged in age from less than 1 year old to 79 years old. City of Hope's HCT program is one of America's largest, dedicated solely to the traditional and newer uses of this procedure.

Which diseases are HCT and PBSCT most frequently used to treat?

What is the difference between autologous and allogeneic HCT?

How are donors for allogeneic transplantations found?

About 30 percent of patients needing a transplant get one from a family member whose HLA testing has identified compatibility between a patient and donor. This matching of donor and recipient reduces the chance of marrow rejection and greatly increases the likelihood of a successful transplant. The remaining 70 percent of patients must find an unrelated donor whose marrow is compatible.

Currently, there are nearly 5 million volunteer donors in the National Marrow Donor Program (NMDP) Be The Match Registry. Almost 50 percent of patients searching the registry have at least one identically matched, unrelated donor. The NMDP is conducting a major effort at the 97 donor centers around the United States (of which City of Hope is one) to increase minority registration.

Because HLA types vary greatly between people of different ethnic backgrounds, increasing minority and ethnic representation will increase minority patients' chances of finding matches.

What is a mini-HCT?

Mini-HCT is a procedure that allows successful transplant of bone marrow without the use of high-dose chemo and radiation therapy. It is less intensive but allows transplant to be utilized in the treatment of older patients who may not be able to endure the intensity of traditional HCT transplant regimens.

Because many diseases, such as leukemia, lymphoma, myeloma and myelodysplasia, are more common in older patients, mini-HCTs allow these patients to potentially benefit from transplant.

What is bone marrow?

Bone marrow is the soft, spongy material found inside bones. Bone marrow contains stem cells that give rise to white blood cells (to fight infections), red blood cells (for oxygenation) and platelets (to prevent hemorrhaging). The chief function of bone marrow is to produce blood cells.

What are platelets?

Platelets are critical in the clotting process and to help control bleeding. Platelets are commonly used to treat leukemia and cancer patients undergoing chemotherapy and bone marrow transplants. Platelets are also used for trauma patients.

What are stem cells?

All blood cells develop from very immature cells called stem cells. Most stem cells are found in the bone marrow, although some, called peripheral blood stem cells, circulate in blood vessels throughout the body. Stem cells can divide to form more stem cells, or they can go through a series of cell divisions by which they become fully mature blood cells.

Who can donate bone marrow or peripheral blood stem cells?

Donating bone marrow or stem cell to someone suffering from a life-threatening disease is one of the greatest gifts you can provide, the gift of life. The first step is to join the National Marrow Donor Program (NMDP) Be The Match Registry. The NMDP maintains the registry of potential donors and searches this when people need a match. To join the registry, you need to complete a brief health questionnaire, sign a consent form, and provide a small blood sample to determine your tissue type.

At City of Hope, we ask that you donate a unit of blood or platelets to help offset the cost of a tissue-type test. Your tissue type will be compared to the tissue types of thousands of patients awaiting a bone marrow transplant. If you are ever a potential match, the City of Hope Donor Center will notify you to see if you are still interested in continuing with the process. If you are, a City of Hope staff member will request an additional blood sample. This sample will determine if the donor matches well enough to continue with the process.

Will patients need blood and platelet donations?

Blood donations from friends and family are a great source of encouragement and support for a patient needing transfusions. If your blood type is compatible with the patient, your donated blood can be given directly to your loved one. If your blood is not the same type, it is still important that you donate to help other City of Hope patients who are a blood type match and seriously in need of your help.

In most circumstances, platelet donations do not need be the same blood type. Therefore, most friends and family members can direct their platelet donations to their loved one. Because platelets can only be stored for 3-5 days, consistent support for our patients is crucial. You can help rally friends and family members by sponsoring blood drives for patients as well as arranging for group donations in our Donor Center.

Encourage friends and family members to call the CityofHopeBloodDonorCenter at 626- 471-7171 and schedule an appointment to donate blood and/or platelets or make arrangements for a blood drive in your community. To find a blood drive in your community, please call 626-301-8385.

Why do patients need platelets?

Before a patient receives a donor's marrow, his or her own marrow must be destroyed by a rigorous treatment of chemotherapy and/or radiation. Once the patient receives the donated marrow, it takes about 4 to 8 weeks for the new marrow to produce platelets. During that time period, the patient needs transfusions of platelets to help his/her blood to clot. City of Hope patients sometimes receive platelet transfusion on a daily basis.

What are the risks to marrow donors?

Virtually none. Bone marrow is extracted under general anesthesia in a procedure that takes less than an hour. Donors have commented that their buttocks felt sore for several days after aspiration. Contrary to organ donations, marrow is completely replenished by the body within a couple of weeks. There are no increased risks to the donor during this period. Historically, at HCT centers around the world, marrow has been donated by individuals less than 1 year old to 60 or 70 years old.

What are the possible complications associated with HCT and PBSCT?

Immediately following allogeneic transplantation, patients are immunosuppressed and unable to fight infection. Different drugs are administered during this critical period and isolation is sometimes necessary for the patient.

Another possible complication for patients receiving allogeneic transplantation is known as graft-versus-host disease (GvHD). Despite the close match between patient and donor, in GvHD, the donated marrow may recognize its new home as foreign and react against the host.

In addition, patients can acquire post-transplant cytomegalovirus (CMV) pneumonia. City of Hope has pioneered several outstanding advances for the prevention and treatment of this potentially fatal complication. Recurrent disease also is possible if the pre-transplant chemotherapy and irradiation therapy were not successful in killing all malignant cells.

In autologous transplantations there are few complications once the patient leaves the hospital, and the only risk is whether the disease will return, causing relapse.

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Bone Marrow and Blood Stem Cell Transplants - City of Hope

Stem Cell Research & Therapy20112:13

DOI: 10.1186/scrt54

BioMed Central Ltd.2011

Published: 14March2011

The vast majority of hematopoietic stem cells (HSCs) reside in specialized niches within the bone marrow during steady state, maintaining lifelong blood cell production. A small number of HSCs normally traffic throughout the body; however, exogenous stimuli can enhance their release from the niche and entry into the peripheral circulation. This process, termed mobilization, has become the primary means to acquire a stem cell graft for hematopoietic transplant at most transplant centers. Currently, the preferred method of HSC mobilization for subsequent transplantation is treatment of the donor with granulocyte colony-stimulating factor. The mobilizing effect of granulocyte colony-stimulating factor is not completely understood, but recent studies suggest that its capacity to mobilize HSCs, at least in part, is a consequence of alterations to the hematopoietic niche. The present article reviews some of the key mechanisms mediating HSC mobilization, highlighting recent advances and controversies in the field.

The online version of this article (doi:10.1186/scrt54) contains supplementary material, which is available to authorized users.

Higher organisms have the remarkable capacity to produce and maintain adequate numbers of blood cells throughout their entire lifespan to meet the normal physiological requirements of blood cell turnover, as well as to respond to needs for increased blood cell demand as a consequence of injury or infection. At the center of lifelong blood cell production is the hematopoietic stem cell (HSC), with the capacity to give rise to all mature circulating blood cell types. Regulation of HSC function is a highly complex process involving not only intrinsic cues within the HSC themselves, but signaling from the surrounding microenvironment in which they reside. It was first postulated by Schofield that defined local microenvironments created specialized stem cell niches that regulated HSCs [1]. Bone marrow is the primary HSC niche in mammals and is composed of stromal cells and an extracellular matrix of collagens, fibronectin, proteoglycans [2], and endosteal lining osteoblasts [36]. HSCs are thought to be tethered to osteoblasts, other stromal cells, and the extracellular matrix in this stem cell niche through a variety of adhesion molecule inter-actions, many of which are probably redundant systems.

Disruption of one or more of these niche interactions can result in release of HSCs from the niche and their trafficking from the bone marrow to the peripheral circulation, a process termed peripheral blood stem cell mobilization. Mobilization can be achieved through administration of chemotherapy [79], hematopoietic growth factors, chemokines and small-molecule chemokine receptor inhibitors or antibodies against HSC niche interactions [1012].

The process of mobilization has been exploited for collection of hematopoietic stem and progenitor cells (HSPCs) and is widely used for hematopoietic trans-plantation in both the autologous and allogeneic settings. Mobilized peripheral blood hematopoietic stem cell grafts are associated with more rapid engraftment, reduction in infectious complications and, in patients with advanced malignancies, lower regimen-related mor-tality [1315] compared with bone marrow grafts. In many transplantation centers, mobilized HSC grafts are now the preferred hematopoietic stem cell source used for human leukocyte antigen-identical sibling transplants as well as for matched related and unrelated donor transplants [16, 17]. Granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor and - more recently, for patients who fail to mobilize with a G-CSF or granulocyte-macrophage colony-stimulating factor - plerixafor (AMD3100) are the only US Food and Drug Administration-approved agents for mobilizing HSCs. Despite the clinical prevalence of peripheral blood stem and progenitor cell mobilization, the mechanisms orchestrating the release of these cells from the hematopoietic niche are still not completely understood. In the following sections, we highlight some of the key mechanistic findings concerning HSPC mobilization, with an emphasis on the effects of mobilizing agents on bone marrow niche interactions.

The most explored HSC niche interaction is between the CXC4 chemokine receptor (CXCR4) and its ligand, stromal cell-derived factor 1 (SDF-1). SDF-1 is produced by osteoblasts [18], a specialized set of reticular cells found in endosteal and vascular niches [19], endothelial cells and bone itself [20, 21], and high levels of SDF-1 were observed recently in nestin-positive mesenchymal stem cells [22]. HSPCs express CXCR4 and are chemoattracted to and retained within the bone marrow by SDF-1 [2325]. Genetic knockout of either CXCR4 [26] or SDF-1 [27] in mice is embryonically lethal, with a failure of HSPCs to tracffic to the bone marrow niche during development. In addition, conditional CXCR4 knockout in mice results in a substantial egress of hematopoietic cells from the bone marrow [28] and impaired ability of CXCR4 knockout HSPCs to be retained within the bone marrow after transplantation [29].

Many agents reported to mobilize HSCs have been shown to disrupt the CXCR4/SDF-1 axis. Most notably, the CXCR4 antagonist AMD3100 (Plerixafor; Mozobil, Genzyme Corporation, Cambridge, MA, USA) mobilizes HSPCs [3035]; and similarly, the CXCR4 antagonists T140 [36] and T134 [37] are both capable of mobilization. Partially agonizing CXCR4 with SDF-1 mimetics including (met)-SDF-1 [38], CTCE-0214 [39], and CTCE-0021 [35] also mobilizes HSCs through CXCR4 receptor desensitization and/or downregulation of surface CXCR4 expression. Intriguingly, these agents that directly disrupt the CXCR4/SDF-1 axis lead to rapid mobilization of HSPCs - that is, hours after treatment - in contrast to other mobilization agents like G-CSF, which take several days to maximally mobilize HSPCs.

Despite the abundance of evidence supporting a key role for the CXCR4/SDF-1 axis in HSPC retention/trafficking/mobilization, it is still not clear which population of cells within the bone marrow niche is the pre-dominate source of SDF-1. Some studies have demonstrated that SDF-1 production by osteoblasts is reduced after G-CSF treatment [21, 40, 41], and seminal work by Katayama and colleagues suggests that this reduction in osteoblast SDF-1 is at least partly mediated by the sympathetic nervous system [21]. Notwithstanding the fact that decreased levels of SDF-1 production by osteoblasts are routinely seen following G-CSF administration, however, other studies have questioned the relative importance of osteoblast-derived SDF-1 in HSC maintenance and mobilization [19, 22, 42]. A recent study by Christopher and colleagues indicated that reduction in osteoblast production of SDF-1 is a common mechanism of cytokine-induced HSC mobilization and showed a specific reduction in SDF-1 production in Col2.3-expressing osteoblasts with no reduction in Col2.3-negative stromal cells [43]. Mendez-Ferrer and colleagues, however, showed, using a similar approach, a substantial decrease in SDF-1 in a novel population of nestin-expressing mesenchymal stem cells [22], relative to a similar population of stromal cells described by Christopher and colleagues [43], although a direct comparison with defined osteoblasts was not made. Future studies are clearly required in order to define the specific niche cells responsible for SDF-1 production and HSC retention, and may identify specific targets for future HSC therapies.

Osteoblasts are important HSC regulators [36], and express numerous signaling molecules in addition to SDF-1 that regulate HSC function and retention in the bone marrow niche. Osteoblasts express vascular cell adhesion molecule 1 (VCAM-1), and targeting the inter-action between very late antigen 4 (VLA-4) and VCAM-1 with either antibodies against VLA-4 [44, 45], antibodies against VCAM-1 [46, 47], or a small molecule inhibitor of VLA-4 (BIO5192) [48] results in HPSC mobilization. In addition, the Eph-ephrin A3 signaling axis increases adhesion to fibronectin and VCAM-1, and disruption of this signaling axis in vivo with a soluble EphA3-Fc fusion protein mobilizes HSPCs [49].

Osteoblasts also express significant amounts of osteo-pontin, and HSPCs adhere to osteopontin via 1 integrins, such as VLA-4 [50]. Osteopontin is a negative regulator of HSC pool size within the bone marrow niche [50, 51], and knockout of osteopontin in mice results in endoge-nous HSPC mobilization and increases the mobilization response to G-CSF [52]. Future therapies that target osteopontin may not only increase the HSC pool size available for hematopoietic mobilization, but may also act to untether the expanded HSCs from the bone marrow niche, resulting in significantly enhanced HSC mobilization.

Mobilizing regimens of G-CSF are associated with suppression of niche osteoblasts [21, 41, 53], with increased osteoblast apoptosis [41] and osteoblast flattening [21], resulting in significant decreases in endosteal niche expression of many of the above-mentioned retention molecules. This suppression has been reported to be the result of altered sympathetic nervous system signaling to osteoblasts [21]. A recent report by Winkler and colleagues demonstrated that G-CSF treatment results in the reduction of endosteal-lining osteomacs, which results in suppression of osteoblasts [53]. This osteomac population of cells is F4/80+ Ly-6G+ CD11b+ and provides a yet to be determined positive supporting role for osteoblasts. When osteomacs are depleted using Mafia transgenic mice or by treatment of mice with clodronate-loaded liposomes, significant mobilization of HSPCs was observed. These findings support a mechanistic role for osteoblasts in mediating G-CSF-induced mobilization, independent of the sympathetic nervous system, and highlight that multiple mechanisms may be responsible for the mobilizing effects of G-CSF.

Osteoblasts and osteoclasts regulate/coordinate bone formation and bone resorption, respectively, within the bone marrow niche. A report from Kollet and colleagues suggested that osteoclasts can mediate HSPC mobilization [54], and proposed a model where the balance between osteoblasts and osteoclasts is required for homeostatic maintenance of the stem cell niche and HSPC pool size. In their model, increased osteoblasts - for example, after parathyroid hormone administration [3] - increase the stem cell pool size and adherence in the niche, whereas increased osteoclasts degrade the niche - facilitating release and egress of HSPCs.

A role for osteoclasts in mobilization was shown by treating mice with RANK ligand, which increased osteoclast activity that correlated with a moderate increase in hematopoietic progenitor cell (HPC) mobilization [54]. Similarly, bleeding mice or treating them with lipopoly-saccharide, two models of physiological stress, resulted in an increase in the number of bone marrow niche osteoclasts as well as HPC mobilization. Inhibition of osteoclasts, either by treatment with calcitonin or using a genetic knockout model of PTP in female mice, resulted in a reduced HPC mobilization response to G-CSF compared with controls, further suggesting that osteoclasts were involved in G-CSF-mediated mobilization. The authors proposed that osteoclast-derived proteolytic enzymes, such as cathepsin K, degraded important niche interaction components including SDF-1 and osteopontin, thereby facilitating mobilization [54]. A more recent study by the same laboratory demonstrated reduced osteoclast maturation and activity in CD45 knockout mice, which correlated with reduced mobilization to RANK ligand and G-CSF [55], providing an additional link between osteoclast activity and HSPC mobilization.

In contrast to studies showing that increased osteoclasts enhance HPC mobilization, an earlier report by Takamatsu and colleagues demonstrated that while G-CSF treatment increases osteoclast number and bone resorption in both BALB/c mice and humans, the increase in osteoclasts did not occur until 10 to 15 days or 6 to 8 days, respectively, after treatment with G-CSF [56] - a finding that has also been observed by other groups using similar systems [40, 57]. Since HSPC mobilization by G-CSF is typically evaluated after 4 to 5 days, the importance of osteoclasts to HSPC mobilization in response to G-CSF treatment remains unclear. Furthermore, treatment of mice with bisphosphonates, which inhibit osteoclast activity and/or number, prior to G-CSF administration does not result in an impaired HSPC mobilization response [53, 56]; in fact, in one case, bisphosphonate treatment increased mobilization by G-CSF [53]. These studies suggest that while osteoclasts elicit mechanisms that can induce hematopoietic stem and progenitor mobilization, their role in clinical HSC mobilization with G-CSF is not sufficiently defined and may not be a primary mechanism of mobilization.

The endosteal surface of bone, particularly at the site of resorbing osteoclasts, is a significant source of soluble extracellular calcium within the bone marrow niche. Studies by Adams and colleagues demonstrated that HSCs express calcium-sensing receptors and are chemo-attracted to soluble Ca2+ [58]. When the gene for the calcium-sensing receptor was knocked out, mice had reduced HSC content within the bone marrow niche and increased HSCs in peripheral blood. Moreover, calcium-sensing receptor-knockout HSCs failed to engraft in hematopoietic transplantation experiments. These results suggest that Ca2+ at the endosteal surface is an important retention signal within the hematopoietic niche and that pharmacologic antagonism of the HSC calcium-sensing receptor may represent a possible strategy for HSPC mobilization.

The bone marrow hematopoietic niche has been shown to be hypoxic [59, 60]. HSCs that reside in hypoxic niches have also been shown to have greater hematopoietic-repopulating ability than those that do not [61]. A known physiological response to hypoxia is stabilization of the transcription factor hypoxia inducible factor 1 (HIF-1). HIF-1 has been shown to upregulate erythropoietin production [62], numerous cell proliferation and survival genes [6365], the angiogenic vascular endothelial growth factor [66], and other genes. It has also been suggested that the hypoxic bone marrow niche maintains HIF-1 activity, thereby maintaining stem cells [67] - a hypothesis supported by the fact that hypoxic conditions expand human HSCs [68] and HPC populations [6971] in vitro. In response to G-CSF, both the hypoxic environment and HIF-1 expand within the bone marrow compartment [72] and increase production of vascular endothelial growth factor A; however, bone marrow vascular density and permeability are not increased [61]. HIF-1 also increases production of SDF-1 [73] and CXCR4 receptor expression [74], suggesting that hypoxia may be a physiological regulator of this important signaling axis within the hematopoietic niche.

HIF-1 has recently been reported to prevent hematopoietic cell damage caused by overproduction of reactive oxygen species [75], suggesting that the hypoxic niche helps maintain the long lifespan of HSCs. However, some small degree of reactive oxygen species signaling may be necessary for HSC mobilization. A recent report demonstrated that enhanced c-Met activity promotes HSPC mobilization by activating mTOR and increasing reactive oxygen species production in HSPCs [76], while inhibition of mTOR with rapamycin reduced HSC mobilization [76, 77]. Genetic knockout of the gene for thioredoxin-interacting protein also results in increased HSPC mobilization under stress conditions [78], suggesting a role for oxygen tension and reactive oxygen species in regulation of hematopoietic stem and progenitor mobilization. These findings clearly warrant additional exploration.

It has been known for some time that there is dynamic interaction between the bone marrow niche and the nervous system. Studies by Katayama and colleagues demonstrated that HSPC mobilization by G-CSF requires peripheral 2-adrenergic signals [21], showing that G-CSF mobilization was reduced in chemically sympathectomized mice treated with 6-hydroxydopamine, in mice treated with the -blocker propanolol, or in mice genetically deficient in the gene for dopamine -hydroxylase (Dbh), an enzyme that converts dopamine into norepinephrine. They also showed that treatment with the 2-adrenergic agonist clenbuterol reversed the phenotype of Dbh knockout mice [21]. Intriguingly, G-CSF attenuated osteoblast function via the sympathetic nervous system resulting in osteoblasts having a marked flattened appearance. The effects of nervous system signaling can also be mediated directly on HSCs, as human CD34+ hematopoietic cells express 2-adrenergic and dopamine receptors that are upregulated after G-CSF treatment [79]. Neurotransmitters serve as direct chemo-attractants to HSPCs, and treatment with norepinephrine results in HSC mobilization [79]. Norepinephrine treatment of mice has also been shown to increase CXCR4 receptor expression [80], perhaps suggesting that adrenergic signaling could directly affect CXCR4/SDF-1 signaling in HSPCs. Additional studies directly assessing effects of neurotransmitter signaling in HSPCs will help to further define the role of the nervous system in hematopoietic regulation.

Not only does the sympathetic nervous system affect HSC mobilization during stress situations, but it also regulates HSC trafficking via a circadian rhythm [81, 82]. 3-Adrenergic stimulations demonstrate regular oscillations controlling norepinephrine release, CXCR4 expression, and SDF-1 production, leading to rhythmic release of HSPCs from the bone marrow niche. Intriguingly, while optimal mobilization occurs in the morning in mice (Zeitgeber time 5), HSC mobilization circadian control is inverted in humans, with peak mobilization occurring later in the evening [81]. Mobilization by both G-CSF and AMD3100 is affected by circadian control of the CXCR4/SDF-1 axis. Recently, it was demonstrated that 2-adrenergic signaling upregulates the vitamin D receptor on osteoblasts; that expression of this receptor is necessary for the G-CSF-induced suppression of osteoblast function; and that vitamin D receptor knockout mice have reduced HSC mobilization [83]. Intriguingly, vitamin D receptor is an important regulator of extracellular calcium and HSPC localization [84] and the receptor is also regulated by circadian rhythms [85], possibly suggesting additional interconnected mobilization mechanisms. Further assessment of the role of nervous system signaling and vitamin D receptor signaling on other niche cells, particularly mesenchymal stem cells, should be performed.

There has been significant progress in understanding the mechanisms of action of G-CSF and other stimuli that increase HSPC trafficking/mobilization. As described in the present review, however, there is currently an abundance of proposed mechanisms that may be responsible for mobilization. This raises the question of whether the proposed mechanisms, be they HSPC intrinsic or manifested through the bone marrow niche, truly represent alternate and independent means to mobilize or enhance egress of HSPCs from bone marrow to the circulation, or whether we have not yet found the unifying mechanism.

Intriguingly, many of the proposed mechanisms of mobilization converge on the CXCR4/SDF-1 pathway (Figure

). Alterations of the osteoblast/osteoclast balance result in a reduction of SDF-1 production and/or degradation of SDF-1 by proteases. Signaling from the sympathetic nervous system, stimulated by G-CSF, can alter the osteoblast/osteoclast balance leading to reduced CXCR4/SDF-1 signaling and HSPC mobilization. Circadian rhythms act to reduce niche SDF-1 production and HSPC CXCR4 expression in an oscillating manner, suggesting that clinical mobilization should be performed at the trough of SDF-1 and CXCR4 expression (early night for humans) and perhaps suggesting that clinical transplantation should be performed at the peak of expression (early morning in humans). The hypoxic nature of the hematopoietic bone marrow niche may itself regulate the CXCR4/SDF-1 signaling axis, perhaps further identifying this axis as a unifying mobilization mechanism. The importance of CXCR4 signaling in HSPC retention and mobilization is certainly supported by the abundance of agents that directly antagonize, or compete with SDF-1 and partially agonize, the CXCR4 receptor and result in HSPC mobilization. Even a rapid mobilizing agent such as GRO (CXCR2 agonist) may function by increasing proteolytic cleavage of SDF-1 [

,

], or altering a homeostatic balance between the CXCR4 and CXCR2 signaling pathways [

].

Hematopoietic stem and progenitor mobilization converges on the CXCR4/SDF-1 signaling axis within the hematopoietic niche. Many of the proposed mechanisms for hematopoietic stem and progenitor mobilization function by altering the marrow microenvironmental CXC4 chemokine receptor (CXCR4)/stromal cell-derived factor 1 (SDF-1) signaling axis. Shown are representative mobilization mechanisms and their relationship to the CXCR4/SDF-1 axis. Question marks denote hypothetical linkage to the CXCR4/SDF-1 axis. G-CSF, granulocyte colony-stimulating factor; HSC, hematopoietic stem cell; HSPC, hematopoietic stem and progenitor cell; ROS, reactive oxygen species.

While perhaps connecting many of the proposed mechanistic pathways for HSPC mobilization, however, the CXCR4/SDF-1 pathway does not appear to be an exclusive target for HSPC mobilization. Continued investigation of the molecular mechanism(s) for action of G-CSF and other HSPC mobilizers is warranted and may define new molecular targets that can be used to enhance the magnitude and/or ease of HSPC collection for hematopoietic transplant.

This article is part of a review series on Stem cell niche. Other articles in the series can be found online at http://stemcellres.com/series/ stemcellniche

CXC4 chemokine receptor

granulocyte colony-stimulating factor

hypoxia inducible factor 1

hematopoietic progenitor cell

hematopoietic stem cell

hematopoietic stem and progenitor cell

mammalian target of rapamycin

receptor activator NF-B

stromal cell-derived factor 1

vascular cell adhesion molecule 1

late antigen 4.

The present work was supported by NIH grants HL069669 and HL096305 (to LMP). JH is supported by training grant HL007910.

Below are the links to the authors original submitted files for images.

The authors declare that they have no competing interests.

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Mobilization of hematopoietic stem cells from the bone ...

Comparison Between Bone Marrow or Peripheral Blood Stem Cells and Cord Blood Donated for Transplantation

Cord blood transplants, as all unrelated hematopoietic stem cell transplants, can be associated with serious complications, severe organ toxicity, and in some cases, death.

A transplant requires donation of a quart or more of bone marrow (mixed with blood).

After a formal search is started, it usually takes 2 or more months to transplant, if a donor is available.

When a match is found, it can take only a few days for confirmatory and special testing for shipment to the Transplant Center (less than 24 hours in an emergency).

Donor may be available to give a second transplant or to donate blood for T-cells if necessary.

Patient must begin conditioning before the bone marrow or peripheral bloods harvest. Coordination between donation and transplant is critical and complex.

Cord blood graft can be shipped to the transplant center before the patient enters the hospital and begins conditioning for transplantation. Coordination is simple. Cord blood units are shipped on demand.

No risk of transplanting a genetic disease.

There is a small probability that a rare, unrecognized genetic disease affecting the blood or immune system of the baby may be given with the cord blood transplant.

Generally requires a perfect match between donor and recipient for 8/8 HLA-A, -B, -C and -DRB1 antigens. Additional HLA factors (HLA-DQ and -DP) increasingly used to improve prognosis.

HLA-mismatched cord blood transplants are possible, making it easier to find a suitable match. Role of HLA-C, -DQ and -DP are not yet known.

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Comparison Between Bone Marrow or Peripheral Blood Stem ...

Introduction

The post-natal bone marrow has traditionally been seen as an organ composed of two main systems rooted in distinct lineagesthe hematopoietic tissue proper and the associated supporting stroma. The evidence pointing to a putative stem cell upstream of the diverse lineages and cell phenotypes comprising the bone marrow stromal system has made marrow the only known organ in which two separate and distinct stem cells and dependent tissue systems not only coexist, but functionally cooperate. Originally examined because of their critical role in the formation of the hematopoietic microenvironment (HME), marrow stromal cells later came to center stage with the recognition that they are the stem/progenitor cells of skeletal tissues. More recent data pointing to the unexpected differentiation potential of marrow stromal cells into neural tissue or muscle grant them membership in the diverse family of putative somatic stem cells. These cells exist in a number of post-natal tissues that display transgermal plasticity; that is, the ability to differentiate into cell types phenotypically unrelated to the cells in their tissue of origin.

The increasing recognition of the properties of marrow stromal cells has spawned a major switch in our perception of their nature, and ramifications of their potential therapeutic application have been envisioned and implemented. Yet, several aspects of marrow stromal cell biology remain in question and unsettled throughout this evolution both in general perspective and in detail, and have gained further appeal and interest along the way. These include the identity, nature, developmental origin and in vivo function of marrow stromal cells, and their amenability to ex vivo manipulation and in vivo use for therapy. Just as with other current members of the growing list of somatic stem cells, imagination is required to put a finger on the seemingly unlikely properties of marrow stromal cells, many of which directly confront established dogmas or premature inferences made from other more extensively studied stem cell systems.

Alexander Friedenstein, Maureen Owen, and their coworkers were the first to utilize in vitro culture and transplantation in laboratory animals, either in closed systems (diffusion chambers) or open systems (under the renal capsule, or subcutaneously) to characterize cells that compose the physical stroma of bone marrow [1-3]. Because there is very little extracellular matrix present in marrow, gentle mechanical disruption (usually by pipetting and passage through syringe needles of decreasing sizes) can readily dissociate stroma and hematopoietic cells into a single-cell suspension. When these cells are plated at low density, bone marrow stromal cells (BMSCs) rapidly adhere and can be easily separated from the nonadherent hematopoietic cells by repeated washing. With appropriate culture conditions, distinct colonies are formed, each of which is derived from a single precursor cell, the CFU-F.

The ratio of CFU-F in nucleated marrow cells, as determined by the colony-forming efficiency (CFE) assay [4], is highly dependent on the culture conditions, and there is a great deal of variability in the requirements from one animal species to another. In rodents, irradiated marrow feeder cells are absolutely required in addition to selected lots of serum in order to obtain the maximum number of assayable CFU-F (100% CFE), whereas CFE is feeder cell-independent in humans [5]. The mitogenic factors that are required to stimulate the proliferation of CFU-F are not completely known at this time, but do at least include platelet-derived growth factor (PDGF), epidermal growth factor (EGF), basic fibroblast growth factor, transforming growth factor-, and insulin-like growth factor-1 [6, 7]. Under optimal conditions, multi-colony-derived strains (where all colonies are combined by trypsinization) can undergo over 25 passages in vitro (more than 50 cell doublings), demonstrating a high capacity for self-replication. Therefore, billions of BMSCs can be generated from a limited amount of starting material, such as 1 ml of a bone marrow aspirate. Thus, the in vitro definition of BMSCs is that they are rapidly adherent and clonogenic, and capable of extended proliferation.

The heterogeneous nature of the BMSC population is immediately apparent upon examination of individual colonies. Typically this is exemplified by a broad range of colony sizes, representing varying growth rates, and different cell morphologies, ranging from fibroblast-like spindle-shaped cells to large flat cells. Furthermore, if such cultures are allowed to develop for up to 20 days, phenotypic heterogeneity is also noted. Some colonies are highly positive for alkaline phosphatase (ALP), while others are negative, and a third type is positive in the central region, and negative in the periphery [8]. Some colonies form nodules (the initiation of matrix mineralization) which can be identified by alizarin red or von Kossa staining for calcium. Yet others accumulate fat, identified by oil red O staining [9], and occasionally, some colonies form cartilage as identified by alcian blue staining [10].

Upon transplantation into a host animal, multi-colony-derived strains form an ectopic ossicle, complete with a reticular stroma supportive of myelopoiesis and adipocytes, and occasionally, cartilage [8, 11]. When single colony-derived BMSC strains (isolated using cloning cylinders) are transplanted, a proportion of them have the ability to completely regenerate a bone/marrow organ in which bone cells, myelosupportive stroma, and adipocytes are clonal and of donor origin, whereas hematopoiesis and the vasculature are of recipient origin [7] (Fig. 1). These results define the stem cell nature of the original CFU-F from which the clonal strain was derived. However, they also confirm that not all of the clonogenic cells (those cells able to proliferate to form a colony) are in fact multipotent stem cells. It must also be noted that it is the behavior of clonal strains upon transplantation, and not their in vitro phenotype, that provides the most reliable information on the actual differentiation potential of individual clones. Expression of osteogenic, chondrogenic, or adipogenic phenotypic markers in culture (detected either by mRNA expression or histochemical techniques), and even the production of mineralized matrix, does not reflect the degree of pluripotency of a selected clone in vivo [12]. Therefore, the identification of stem cells among stromal cells is only done a posteriori and only by using the appropriate assay. In this respect, chondrogenesis requires an additional comment. It is seldom observed in open transplantation assays, whereas it is commonly seen in closed systems such as diffusion chambers [11], or in micromass cultures of stromal cells in vitro [13], where locally low oxygen tensions, per se, permissive for chondrogenesis, are attained [14]. Thus, the conditions for transplantation or even in vitro assays are critical determinants of the range of differentiation characteristics that can be assessed.

FigureFigure 1.. Transplantation of ex vivo-expanded human BMSC into the subcutis of immunocompromised mice.A) Multi-colony and some single colony-derived strains attached to particles of hydroxyapatite/tricalcium phosphate ceramic (HA) form a complete bone/marrow organ composed of bone (B) encasing hematopoietic marrow (HP). B) The bone (B) and the stroma (S) are of human origin as determined by in situ hybridization using a human specific alu sequence as probe, while the hematopoietic cells are of recipient origin.

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The ability to isolate the subset of marrow stromal cells with the most extensive replication and differentiation potential would naturally be of utmost importance for both theoretical and applicative reasons. This requires definitive linkage of the multipotency displayed in transplantation assays with a phenotypic trait that could be assessed prior to, and independently of, any subsequent assays. Several laboratories have developed monoclonal antibodies using BMSCs as immunogen in order to identify one or more markers suitable for identification and sorting of stromal cell preparations [15-18]. To date, however, the isolation of a pure population of multipotent marrow stromal stem cells remains elusive. The nearest approximation has been the production of a monoclonal antibody, Stro-1, which is highly expressed by stromal cells that are clonogenic (Stro-1+bright), although a certain percentage of hematopoietic cells express low levels of the antigen (Stro-1+dull) [19]. In principle, the use of the same reagent in tissue sections would be valuable in establishing in vivo-in vitro correlation, and in pursuing the potential microanatomical niches, if not anatomical identity, of the cells that are clonogenic. The Stro-1 reagent has limited application in fixed and paraffin-embedded tissue. However, preliminary data using frozen sections suggest that the walls of the microvasculature in a variety of tissues are the main site of immunoreactivity (Fig. 2), a finding of potentially high significance (see below).

FigureFigure 2.. Immunolocalization of the Stro-1 epitope in the microvasculature of human thymus.A) CD34 localizes to endothelial cells (E) forming the lumen (L) of the blood vessel. B) Stro-1 localizes not only to endothelial cells, but also the perivascular cells of the blood vessel wall (BVW).

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Freshly isolated Stro-1+bright cells and multi-colony-derived BMSC strains, both of which contain but are not limited to multipotent stromal stem cells, have been extensively characterized for a long list of markers expressed by fibroblasts, myofibroblasts, endothelial cells, and hematopoietic cells in several different laboratories [20-24]. From these studies, it is apparent that the BMSC population at large shares many, but not all, properties of fibroblastic cells such as expression of matrix proteins, and interestingly, some markers of myofibroblastic cells, notably, the expression of -smooth muscle actin (-SMA) and some characteristics of endothelial cells such as endoglin and MUC-18. It has been claimed that the true mesenchymal stem cell can be isolated using rather standard procedures, and characterized using a long list of indeterminate markers [23]. However, in spite of this putative purification and extensive characterization, the resulting population was no more pure than multi-colony-derived strains isolated by simple, short-term adherence to plastic; the resulting clones displayed varying degrees of multipotentiality. Furthermore, the pattern of expressed markers in even clonal strains that are able to completely regenerate a bone/marrow organ in vivo is not identical, and changes as a function of time in culture. These results indicate that identifying the phenotypic fingerprint of a stromal stem cell may well be like shooting at a moving target, in that they seem to be constantly changing in response to their microenvironment, both in vitro and in vivo.

The primitive marrow stroma is established in development through a complex series of events that takes place following the differentiation of primitive osteogenic cells, the formation of the first bone, and the vascular invasion of bone rudiments [25]. This intimate relationship of the stromal cells with the marrow vascularity is also found in the adult marrow. In the post-natal skeleton, bone and bone marrow share a significant proportion of their respective vascular bed [26]. The medullary vascular network, much like the circulatory system of other organs, is lined by a continuous layer of endothelial cells and subendothelial pericytes [27]. In the arterial and capillary sections of this network, pericytes express both ALP (Fig. 3B, C, D, F, G) and -SMA (Fig. 3E), both of which are useful markers for their visualization in tissue sections. In the venous portion, cells residing on the abluminal side of the endothelium display a reticular morphology, with long processes emanating from the sinus wall into the adjacent hematopoietic cords where they establish close cell-cell contacts, that convey microenvironmental cues to maturing blood cells. These particular adventitial reticular cells express ALP (Fig. 3G) but not -SMA under normal steady-state conditions (Fig. 3H). In spite of this, but in view of their specific position along with the known diversity of pericytes in different sites, organs and tissues [28], reticular cells can be seen as bona fide specialized pericytes of venous sinusoids in the marrow. Hence, phenotypic properties of marrow pericytes vary along the different sections of the marrow microvascular network (arterial/capillary versus post-capillary venous sinusoids). In addition, adventitial reticular cells of venous sinusoids can accumulate lipid and convert to adipocytes, and they do so mainly under two circumstances: A) during growth of an individual skeletal segment when the expansion of the total marrow cavity makes available space in excess of what is required by hematopoietic cells, or B) independent of growth, when there is an abnormal or age-related numerical reduction of hematopoietic cells thereby making space redundant [29-31].

FigureFigure 3.. Anatomical and immunohistological relationship of marrow stromal cells to marrow pericytes.A) Marrow vascular structures as seen in a histological section of human adult bone marrow. hc = hematopoietic cells; ad = adipocytes; a = artery; VS = venous sinusoid; PCA = pre-capillary arteriole. Note the thin wall of the venous sinusoid. B) Semi-thin section from low-temperature processed glycol-methacrylate embedded human adult bone marrow reacted for ALP. Arrows point to three arterioles emerging from a parent artery (A). Note that while there is no ALP activity in the wall of the large size parent artery, a strong reaction is noted in the arteriolar walls. C, D) Details of the arterioles shown in A and B. Note that ALP activity is associated with pericytes (P). E) Section of human adult bone marrow immunolabeled for -SMA. Note the reactivity of an arteriolar wall, and the complete absence of reactivity in the hematopoietic cords (hc) interspersed between adipocytes (ad). F) Detail of the wall of a marrow venous sinusoid lined by thin processes of adventitial reticular cells (venous pericytes). Note the extension of cell processes apparently away from the wall of the venous sinusoid (vs) and into the adjacent hematopoietic cord ALP reaction. G, H) High power views of hematopoietic cords in sections reacted for ALP (G) and -SMA (H). Note the presence of ALP activity identifying reticular cells, and the absence of labeling for -SMA.

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The ability of reticular cells to convert to adipocytes makes them a unique and specialized pericyte. Production of a basement membrane by adipocytes endows the sinus with a more substantial basement membrane, likely reducing the overall permeability of the vessel. Furthermore, the dramatic increase in cell volume through the accumulation of lipid during adipose conversion collapses the lumen of the sinus. This may exclude an individual sinus from the circulation without causing its irreversible loss. In general, the loss of pericyte coating on a microvessel is associated with vessel regression by apoptosis, while a normal pericyte coating is thought to stabilize them and prevent vessel pruning [32]. Adipose conversion is thus a mechanism whereby the size and permeability of the overall sinusoidal system is reversibly regulated in the bone marrow. Not surprisingly, regions of bone marrow that are hematopoietically inactive are filled with fat.

Given the similar location of pericytes and stromal cells, the significance of -SMA expression, a marker of smooth muscle cells, in marrow stromal cells takes on new meaning, although its expression is variable, both in vitro and in vivo. -SMA expression is commonly observed in nonclonal, and some clonal cultures of marrow stromal cells [33], where it appears to be related to phases of active cell growth [34], and may reflect a myoid differentiation event, at least in vitro [35]. However, the phenotype of -SMA-expressing stromal cells in culture resembles that of pericytes and subintimal myoid cells rather than that of true smooth muscle cells [35]. In the steady-state normal bone marrow, -SMA expressing stromal cells other than those forming the pericyte/smooth muscle coats of arteries and capillaries are not seen. In contrast, -SMA+ stromal cells not associated with the vasculature are commonly observed in the fetal bone marrow [36, 37], that physically grows together with the bone encasing it. -SMA+ marrow stromal cells are likewise seen in conjunction with a host of hematological diseases [37], and in some bone diseases, such as hyperparathyroidism [37] and fibrous dysplasia (FD) of bone (Riminucci and Bianco, unpublished results). In some of these conditions, these cells have been interpreted as myofibroblasts [34, 37]. More interestingly, at least some of these conditions also feature an increased vascularity, possibly related to angiogenesis [38], and an increased number of CFU-F, quantitated as discussed above (Bianco, Kuznetsov, Robey, unpublished results). Taken together, these observations seem to indicate that -SMA expression in extravascular marrow stromal cells (other than arterial/ capillary pericytes) is related to growth or regeneration events in the marrow environment, which is in turn associated with angiogenesis.

Angiogenesis in all tissues involves the coordinated growth of endothelial cells and pericytes. Nascent endothelial tubes produce EGF and PDGF-B, which stimulate the growth and migration of pericytes away from the subintimal myoid cell layer of the vascular section. A precise ligand-receptor expression loop of PDGF-B produced by endothelial cells and expression of the cognate receptor on pericytes regulates the formation of a pericyte coating and its occurrence in physical continuity with the nascent vascular network [39]. Interestingly, PDGF-receptor beta and EGF receptor are two of the most abundant tyrosine kinase growth factor receptors in BMSCs, and PDGF-B and EGF have been found to stimulate proliferation of BMSCs [6, 40], indicating a physiological similarity between pericytes and BMSCs.

In bone, as in any other organ, angiogenesis is normally restricted to phases of developmentally programmed tissue growth, but may reappear in tissue repair and regeneration or proliferative/neoplastic diseases. During normal bone growth, endothelial cell growth, pericyte coverage, and bone formation by newly generated bone-forming cells occur in a precise spatial and temporal sequence, best visualized in metaphyseal growth plates. Growing endothelial tubes devoid of pericytes occupy the foremost 200 microns of the developing metaphysis [41]. Actively dividing abluminal pericytes and bone-forming osteoblasts are next in line. Progression of endochondral bone formation is dependent on efficient angiogenesis, and is blocked if angiogenesis is blocked, as illustrated by both experimental and pathological conditions. Experimentally, inhibition of VEGF signaling initiated by chondrocytes with blocking antibodies to the cognate receptor on growing blood vessels in the metaphysis results in a blockade not only of bone growth, but also of the related activities in the adjacent cartilage growth plates [42]. A remarkably similar event occurs naturally in rickets, and can be mimicked by microsurgical ablation of the metaphyseal vasculature [41].

Taking into account the similarities in their physical relationship to the vasculature, the cellular response to growth factors, and expression of similar markers lead one to suspect that marrow pericytes and marrow stromal cells are the same entity. Pericytes are perhaps one of the most elusive cell types in the body, and their significance as potential progenitor cells has been repeatedly surmised or postulated [28, 43-46]. Elegant as much as unconventional, experimental proof of their ability to generate cartilage and bone in vivo, for example, has been given in the past [47, 48]. Likewise, it has been shown that retinal pericytes form cartilage and bone (and express Stro-1) in vitro [49]. But, there has been little definitive understanding of the origin of this elusive cell type. Current evidence suggests that there is most likely more than one source of pericytes throughout development and growth. First, during development, pericytes may be recruited during angiogenesis or vasculogenesis from neighboring resident mesenchymal cells [50]. Secondly, as recently shown, pericytes may arise directly from endothelial cells or their progenitors [51, 52]. Third, they can be generated during angiogenesis, either pre- or post-natally, through replication, migration and differentiation of other pericytes downstream of the growing vascular bud [32, 39, 53, 54]. With regards to bone marrow, this implies that marrow pericytes might also be heterogeneous in their mode of development and origin. Some may be recruited during blood vessel formation from resident, preexisting osteogenic cells; others may originate from endothelial cells; still others may grow from preexisting pericytes during vascular growth. Interestingly, it would be predicted from this model that a hierarchy of marrow stromal/progenitor cells exists. Some would be osteogenic in nature, while others would not. If so, one would expect to find multipotent cells with markers of osteogenic commitment, and multipotent cells with endothelial/pericytic markers. With respect to the phenotypic characterization of clonal stromal cells, evidence supporting a dual origin is indeed available.

As described above, stromal cells can take on many forms such as cartilage, bone, myelosupportive stroma, or fat. This behavior of marrow stromal cells, both in vitro and in vivo, has perhaps offered the first glimpse of the property now widely referred to as plasticity. It was shown, for example, that clonal strains of marrow adipocytes could be directed to an osteogenic differentiation and form genuine bone in an in vivo assay [55, 56]. Earlier, the ability of marrow reticular cells to convert to adipocytes in vivo had been noted [29, 57]. A number of different studies have claimed that fully differentiated chondrocytes can dedifferentiate in culture and then shift to an osteogenic phenotype [58, 59], and that similar or correlated events can be detected in vivo [60]. All of these data highlight the non-irreversible nature of the differentiation of several cell types otherwise seen as end points of various pathways/lineages (i.e., reticular cells, osteoblasts, chondrocytes, and adipocytes). The primary implication of these findings has remained largely unnoticed until recently. Commitment and differentiation are not usually thought of as reversible, but rather as multistep, unidirectional and terminal processes. This concept is reflected in the basic layout of virtually every scheme in every textbook depicting the organization of a multilineage system dependent on a stem cell. Here, a hierarchy of progenitors of progressively restricted differentiation potential is recognized or postulated. Lineages are segregated, leaving no room for switching phenotype at a late stage of differentiation, no way of turning red blood cells into white blood cells, for example. In contrast, it seems that one can turn an adipocyte or a chondrocyte into an osteoblast, and nature itself seems to do this under specific circumstances. If so, then some kind of reversible commitment is maintained until very late in the history of a single cell of the stromal systema notable and yet unnoticed singularity of the system, with broad biological significance.

There is a real physiological need for plasticity of connective tissue cells, namely the need to adapt different tissues that reside next to one another during organ growth, for example [30, 61], and it is likely that nature has evolved mechanisms for maintaining plasticity which remain to be fully elucidated. One example may be the key transcription factor controlling osteogenic commitment, cbfa1 [62, 63], which is commonly if not constitutively expressed in stromal cells derived in culture from the post-natal marrow [12], and maintained during differentiation towards other cell types such as adipocytes. This is perhaps the most stringent proof that a cell committed to osteogenesis (as demonstrated by expression of the key gene of commitment) may still enter other pathways of differentiation that were thought to be alternative ones [61]. Whether one can isolate a multipotent cbfa1-negative (non-osteogenically committed) stromal cell is at present unclear. However, freshly isolated stromal cells sorted as Stro-1bright have been shown to be cbfa1-negative by reverse transcriptase-polymerase chain reaction (Gronthos and Simmons, unpublished results). Interestingly, these cells also exhibit several endothelial markers, although never a true endothelial phenotype [21, 22].

The fact that chondrocytes, osteoblasts, reticular cells, and adipocytes come from a single precursor cell carrying a marker of osteogenic commitment is consistent with the fact that all of these cell types are members of the same organ, even though of different tissues. A single skeletal segment contains all of these cell types either at different stages of its own organogenesis or simultaneously. Although heretical to some and novel to others, even the notion that each of these cell phenotypes can switch to another within the same family under specific circumstances is consistent with the development and maintenance of the organ from which they were derived. This kind of plasticity is thus orthodox, meaning that it remains within the context of the organ system.

Over the past 2 years, several studies have indicated or implied that progenitors can be found in a host of different post-natal tissues with the apparently unorthodox potential of differentiating into unrelated tissues. First, it was shown that the bone marrow contained systemically transplantable myogenic progenitors [64]. Second, it was shown that neural stem cells could reestablish hematopoiesis in irradiated mice [65]; third, that bone marrow cells could generate neural cells [66], and hepatocytes [67]; and fourth, that a neurogenic potential could be ascribed to marrow stromal cells [68, 69]. What is striking about these data is the developmentally distant nature of the source of these progenitors and their ultimate destination. Differentiation across germ layers violates a consolidated law of developmental biology. Although consolidated laws are not dogmas (which elicited the comment that germ layers are more important to embryologists than to embryos), it is still indisputable and remarkable that even in embryos, cells with transgermal potential only exist under strict temporal and spatial constraints, with the notable exception of neural crest cells, which in spite of their neuroectodermal nature generate a number of craniofacial mesodermal tissues including bone. Cells grown in culture from the inner cell mass self-renew and maintain totipotency in culture for extended periods of time. However, this is in a way an artifact, of which we know some whys and wherefores (feeder cell layers, leukemia inhibitory factor). Embryonic stem (ES) cells only remain multipotent and self-renewing in the embryo itself for a very short period of time, after which totipotent cells only exist in the germline.

Consequently, the first key question iswhere do the multipotent cells of post-natal organisms come from? All answers at this time are hypothetical at best. However, if marrow stromal cells are indeed members of a diffuse system of post-natal multipotent stem cells and they are at the same time vascular/pericytic in nature/origin, then a natural corollary would read that perhaps the microvasculature is a repository of multipotent cells in many, if not all, tissues [70]a hypothesis that is currently being tested.

A second question is that if multipotent cells are everywhere, or almost everywhere, then what are the mechanisms by which differentiated cells keep their multipotency from making every organ a teratoma? Phrased in another way, adult tissues must retain some kind of organizing ability previously thought of as specific to embryonic organizers. If indeed cells in the bone marrow are able to become muscle or liver or brain, then there must be mechanisms ensuring that there is no liver or brain or muscle in the marrow. Hence, signals for maintenance of a tissue's self must exist and be accomplished by differentiated cells. (That is, of course, if stem cells are not differentiated cells themselves).

A third question ishow much of the stemness (self-renewal and multipotency) observed in experimental systems is inherent to the cells that we manipulate, and how much is due to the manipulation? Are we discovering unknown and unexpected cells, or rather unknown and unexpected effects of manipulation of cells in culture? To what extent do cell culture conditions mimic the effects of an enucleated oocyte cytoplasm, which permits a somatic cell nucleus to generate an organism such as Dolly, the cloned sheep? For sure, a new definition of what a stem cell isa timely, and biotechnologically correct, oneshould incorporate the conditions under which phenomena are recorded, rather than guessing from ex vivo performance what the true in vivo properties are. This exercise also has important implications for understanding where and when stem cells come into action in physiology. Even for the mother of all stem cells, the ES cell, self-renewal and multipotency are limited to specific times and events in vivo, and are much less limited ex vivo. Are similar constraints operating for other stem cells? Marrow stromal stem cells for example, can be expanded extensively in culture, but the majority of them likely never divide in vivo once skeletal growth has ceased (except the few that participate in bone turnover, and perhaps in response to injury or disease). What physiological mechanism calls for resumption of a stem cell behavior in vivo in the skeleton and other systems?

All of these questions are important not only for philosophical or esoteric reasons, but also for applicative purposes. Knowing even a few of the answers will undoubtedly enable biotechnology to better harness the magical properties of stem cells for clinical applications.

In vivo transplantation under defined experimental conditions has been the gold standard for defining the differentiation potential of marrow stromal cells, and a cardinal element of their very discovery. Historically, studies on the transplantability of marrow stromal cells are inscribed into the general problem of bone marrow transplantation (BMT). The HME is created by transplantation of marrow stromal cell strains and allows for the ectopic development of a hematopoietic tissue at the site of transplantation. The donor origin of the microenvironment and the host origin of hematopoiesis make the ectopic ossicle a true reverse BMT.

Local transplantation of marrow stromal cells for therapeutic applications permits the efficient reconstruction of bone defects larger than those that would spontaneously heal (critical size). A number of preclinical studies in animal models have convincingly shown the feasibility of marrow stromal cell grafts for orthopedic purposes [71-77], even though extensive work lies ahead in order to optimize the procedures, even in their simplest applications. For example, the ideal ex vivo expansion conditions have yet to be determined, or the composition and structure of the ideal carrier, or the numbers of cells that are required for regeneration of a volume of bone.

In addition to utilizing ex vivo-expanded BMSCs for regeneration of bone and associated tissues, evidence of the unorthodox plasticity of marrow stromal cells has suggested their potential use for unorthodox transplantation; that is, for example, to regenerate neural cells or deliver required gene products at unorthodox sites such as the central nervous system (CNS) [78]. This could simplify an approach to cell therapy of the nervous system by eliminating the need for harvesting autologous human neural stem cells, an admittedly difficult procedure, although it is currently believed that heterologous cells may be used for the CNS, given the immune tolerance of the brain. Moreover, if indeed marrow stromal cells represent just a special case of post-natal multipotent stem cells, there is little doubt that they represent one of the most accessible sources of such cells for therapeutic use. The ease with which they are harvested (a simple marrow aspirate), and the simplicity of the procedures required for their culture and expansion in vitro may make them ideal candidates. For applicative purposes, understanding the actual differentiation spectrum of stromal stem cells requires further investigation. Besides neural cells, cardiomyocytes have been reported to represent another possible target of stromal cell manipulation and transplantation [79]. It also remains to be determined whether the myogenic progenitors found in the marrow [64] are indeed stromal (as some recent data would suggest, [80]) or non-stromal in nature [81], or both.

Given their residency in the marrow, and the prevailing view that marrow stromal cells fit into the hematopoietic paradigm, it was unavoidable that systemic transplantation of marrow stromal cells would be attempted [82] in order to cure more generalized skeletal diseases based on the successes of hematopoietic reconstitution by BMT. Yet major uncertainties remain in this area. Undoubtedly, the marrow stromal cell is the entity responsible for conveying genetic alterations into diseases of the skeleton. This is illustrated very well by the ability of these cells to recapitulate natural or targeted genetic abnormalities into abnormal bone formation in animal transplantation assays [83-85]. As such, they also represent a potential repository for therapy to alleviate bone disease. However, a significant rationale for the ability of stromal cells to colonize the skeleton once infused into the circulation is still missing.

The stroma is not transplanted along with hematopoiesis in standard BMT performed for hematological or oncological purposes [86-88]. Infusion of larger numbers of stromal cells than those present in cell preparations used for hematological BMT should be investigated further, as it might result, in principle, in limited engraftment. Stringent criteria must be adopted when assessing successful engraftment of systemically infused stromal cells [61]. The detection of reporter genes in tissue extracts or the isolation in culture of cells of donor origin does not prove cell engraftment; it proves cell survival. In this respect, it should be noted that even intra-arterial infusion of marrow stromal cells in a mouse limb may result in virtually no engraftment, even though abundant cells of donor origin are found impacted within the marrow microvascular network. Of note, these nonengrafted cells would routinely be described as engrafted by the use of any reporter gene or ex vivo culture procedure. Less than stringent definitions of stromal cells (for example, their identification by generic or nonspecific markers) must be avoided when attempting their detection in the recipient's marrow. Clear-cut evidence for the sustained integration in the target tissue of differentiated cells of donor origin must be provided. This is rarely the case in current studies claiming engraftment of marrow stromal cells to the skeleton. Some evidence for a limited engraftment of skeletal progenitors following systemic infusion has, however, been obtained in animal models [89, 90]. These data match similar evidence for the possible delivery of marrow-derived myogenic progenitors to muscle via the systemic circulation [64]. It should be kept in mind that both skeletal and muscle tissues are normally formed during development and growth by extravascular cells that exploit migratory processes not involving the circulation. Is there an independent circulatory route for delivery of progenitors to solid phase tissues, and if so, are there physiologically circulating mesodermal progenitors? From where would these cells originate, both in development and post-natal organisms, and how would they negotiate the vessel wall? Addressing these questions is mandatory and requires extensive preclinical work.

Even once these issues are addressed, kinetic considerations regarding skeletal growth and turnover represent another major hurdle that must be overcome in order to cure systemic skeletal diseases via systemic infusion of skeletal progenitors. Yet there is broad opportunity for the treatment of single clinical episodes within the context of skeletal disease. While curing osteogenesis imperfecta by replacing the entire population of mutated skeletal progenitors with normal ones may remain an unattainable goal, individual fractures or deformity in osteogenesis imperfecta or FD of bone could be successfully treated with ex vivo repaired stromal cells, for example. Towards this end, future work must focus on the feasibility of transducing or otherwise genetically correcting autologous mutated osteoprogenitors ex vivo, and studies are beginning to move in this direction.

Molecular engineering of cells, either transiently or permanently, has become a mainstay in cell and molecular biology, leading to many exciting insights into the role of a given protein in cell metabolism both in vitro and in vivo. Application of these techniques for correcting human deficiencies and disease is a challenge that is currently receiving much attention. BMSCs offer a unique opportunity to establish transplantation schemes to correct genetic diseases of the skeleton. They may be easily obtained from the future recipient, manipulated genetically and expanded in number before reintroduction. This eliminates not only the complications of xenografts, but also bypasses the limitations and risks connected with delivery of genetic repair material directly to the patient via pathogen-associated vectors. While a similar strategy may be applied to ES cells, the use of post-natal BMSCs is preferable considering that they can be used autologously, thereby avoiding possible immunological complications from a xenograft. Furthermore, there is far less concern of inappropriate differentiation as may occur with ES cell transplantation. Finally, ES cell transplantation is highly controversial, and it is likely that the ethical debate surrounding their usage will continue for quite some time.

Depending on the situation, there are several approaches that can be envisioned. If a short-lived effect is the goal, such as in speeding up bone regeneration, transient transduction would be the desired outcome, utilizing methods such as electroporation, chemical methods including calcium phosphate precipitation and lipofection, and plasmids and viral constructs such as adenovirus. Transducing BMSCs with adenoviral constructs containing BMP-2 has demonstrated at least partial efficacy of this approach in hastening bone regeneration in animal models [75, 91, 92]. Adenoviral techniques are attractive due to the lack of toxicity; however, the level at which BMSCs are transfected is variable, and problematic. It has been reported that normal, non-transformed BMSCs require 10 more infective agent than other cell types [93], which is often associated with cellular toxicity. Clearly, further optimization is needed for full implementation of this approach.

For treatment of recessive diseases in which a biological activity is either missing or diminished, long-lasting or permanent transduction is required, and has depended on the use of adeno-associated viruses, retroviruses, lentiviruses (a subclass of retrovirus), and more recently, adeno-retroviral chimeras [94]. These viruses are able to accommodate large constructs of DNA (up to 8 kb), and while retroviruses require active proliferation for efficient transfection, lentiviruses do not. Exogenous biological activity in BMSCs by transduction with retroviral constructs directing the synthesis of reporter molecules, interleukin 3, CD-2, Factor VIII, or the enzymes that synthesize L-DOPA has been reported [78, 95-102]. However, these studies also highlight some of the hurdles that must be overcome before this technology will become practical. The first hurdle is optimization of ex vivo transfection. It has been reported that lengthy ex vivo expansion (3-4 weeks) to increase cell numbers reduces transfectability of BMSCs, whereas short-term culture (10-12 days) does not [98]. Furthermore, high levels of transduction may require multiple rounds of transfection [95, 101]. The second hurdle relates to the durability of the desired gene expression. No reported study has extended beyond 4 months post-transplantation of transduced cells [99] (Gronthos, unpublished results), and in most instances, it has been reported that expression decreases with time [96], due to promoter inactivation [102] and/or loss of transduced cells (Mankani and Robey, unpublished results). While promising, these results point to the need for careful consideration of the ex vivo methods, choice of promoter to drive the desired biological activity, and assessment of the ability of the transduced BMSCs to retain their ability to self-maintain upon in vivo transplantation. It must also be pointed out that using retrovirally transduced BMSCs for this type of application, providing a missing or decreased biological activity, does not necessarily require that they truly engraft, as defined above. They may be able to perform this function by remaining resident without actually physically incorporating and functioning within a connective tissue. In this case, they can be envisioned as forming an in vivo biological mini-pump as a means of introducing a required factor, as opposed to standard means of oral or systemic administration.

Use of transduced BMSCs for the treatment of a dominant negative disease, in which there is actual expression of misfunctioning or inappropriate biological activity, is far more problematic, independent of whether we are able to deliver BMSCs systemically or orthotopically. In this case, an activity must be silenced such that it does not interfere with any normal activity that is present, or reintroduced by any other means. The most direct approach would be the application of homologous recombination, as applied to ES cells and generation of transgenic animals. The almost vanishing low rate of homologous recombination in current methodology, coupled with issues of the identification, separation, and expansion of such recombinants does not make this seem feasible in the near future. However, new techniques for increasing the rate of homologous recombinations are under development [103] which may make this approach more feasible. Another approach to gene therapy is based on the processes whereby mismatches in DNA heteroduplexes that arise sporadically during normal cell activity are automatically corrected. Genetic mutations could be targeted by introducing exogenous DNA with the desired sequence (either short DNA oligonucleotides or chimeric RNA/DNA oligonucleotides) which binds to homologous sequences in the genome forming a heteroduplex that is then rectified by a number of naturally occurring repair processes [104]. A third option exists using a specially constructed oligonucleotide that binds to the gene in question to form a triple helical structure, thereby disallowing gene transcription [105].

While it would be highly desirable to correct a genetic disease at the genomic level, mRNA represents another very significant target, and perhaps a more accessible one, to silence the activity of a dominant negative gene. Methods for inhibiting mRNA translation and/or increasing its degradation have been employed through the use of protein decoys to prevent association of a particular mRNA to the biosynthetic machinery and antisense sequences (either oligonucleotides or full-length sequences). Double-stranded RNA also induces rapid degradation of mRNA (termed RNA interference, RNAi) by a process that is not well understood [105]. However, eliminating mRNAs transcribed from a mutant allele with short or single-base mutations by these approaches would most likely not maintain mRNA from a normal allele. For this reason, hammerhead and hairpin ribozymes represent yet another alternative, based on their ability to bind to very specific sequences, and to cleave them and inactivate them from subsequent translation. Consequently, incorporating a mutant sequence, even one that transcribes a single base mutation, can direct a hammerhead or hairpin ribozyme to inactivate a very specific mRNA. This approach is currently being probed for its possible use in the treatment of osteogenesis imperfecta [106]. Taking this technology one step further, DNAzymes that mimic the enzymatic activity of ribozymes, which would be far more stable than ribozymes, are also being developed. Regardless of whether genomic or cytoplasmic sequences are the target of gene therapy, the efficacy of all of these new technologies will depend on: A) the efficiency at which the reagents are incorporated into BMSCs in the ex vivo environment; B) the selection of specific targets, and C) the maintenance of the ability of BMSCs to function appropriately in vitro.

In conclusion, the isolation of post-natal stem cells from a variety of tissues along with discovery of their unexpected capabilities has provided us with a new conceptual framework in which to both view them and use them. However, even with this new perspective, there is much to be done to better understand them: their origins, their relationships to one another, their ability to differentiate or re-differentiate, their physiological role during development, growth, and maturity, and in disease. These types of studies will most certainly require a great deal of interdisciplinary crosstalk between investigators in the areas of natal and post-natal development, and in different organ systems. Clearly, as these studies progress, open mindedness will be needed to better understand the nature of this exciting family of cells, as well as to better understand the full utilization of stem cells with or without genetic manipulation. Much to be learned. Much to be gained.

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Bone Marrow Stromal Stem Cells: Nature, Biology, and ...

Bone marrow is the tissue comprising the center of large bones.

It is the place where new blood cells are produced.

Bone marrow contains two types of stem cells: hemopoietic (which can produce blood cells) and stromal (which can produce fat, cartilage and bone).

There are two types of bone marrow: red marrow (also known as myeloid tissue) and yellow marrow.

Red blood cells, platelets and most white blood cells arise in red marrow; some white blood cells develop in yellow marrow.

The color of yellow marrow is due to the much higher number of fat cells.

Both types of bone marrow contain numerous blood vessels and capillaries.

At birth, all bone marrow is red.

With age, more and more of it is converted to the yellow type.

Adults have on average about 2.6kg (5.7lbs) of bone marrow, with about half of it being red.

Red marrow is found mainly in the flat bones such as hip bone, breast bone, skull, ribs, vertebrae and shoulder blades, and in the cancellous ("spongy") material at the proximal ends of the long bones femur and humerus.

Pink Marrow is found in the hollow interior of the middle portion of long bones.

There are several serious diseases involving bone marrow.

In cases of severe blood loss, the body can convert yellow marrow back to red marrow in order to increase blood cell production.

The normal bone marrow architecture can be displaced by malignancies or infections such as tuberculosis, leading to a decrease in the production of blood cells and blood platelets.

In addition, cancers of the hematologic progenitor cells in the bone marrow can arise; these are the leukemias.

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Bone marrow stem-cells - ScienceDaily

In Diamond Blackfan anemia (DBA), the bone marrow (the center of the bone where blood cells are made) does not make enough red blood cells. Red blood cells carry oxygen to all of the organs in the body. When the number of red blood cells is low, the organs in the body may not get the oxygen they need.

A stem cell transplant can help restore the marrows ability to make red blood cells, and it is currently the only known cure for DBA.

However, physical problems associated with DBA but not related to the bone marrow, such as a cleft palate or a heart defect, will not change. In addition, the persons genes will still have DBA, so there is still a 50 percent chance of passing the disorder to any future children, if fertility is retained.

Stem cell transplant is an expensive and potentially dangerous procedure that can lead to death or severe chronic illness in some patients. For this reason, it typically is not a first line treatment. Other treatments, such as steroid medicine (corticosteroid) therapy and blood transfusion therapy, tend to be used first, if possible. Before deciding to have a transplant, people with DBA should discuss the pros and cons of this procedure with their medical team.

All of the blood cells in the body start out as immature cells called blood-forming stem cells. Stem cells are able to grow into other blood cells that mature and function as needed in the body. Stem cells create the three main types of blood cells: red blood cells that carry oxygen throughout the body, white blood cells that fight infection, and platelets that help the blood to clot and prevent abnormal bleeding.

Stem cells are located in three placesbone marrow (the spongy center of the bone where blood cells are made), peripheral blood (found in blood vessels throughout the body), and cord blood (found in the umbilical cord and collected after a babys birth). Stem cells for transplantation are obtained from any of these three places.

A stem cell transplant (also commonly referred to as a bone marrow transplant), takes healthy stem cells from a donor and gives them to the patient through a central line in a vein in the chest. The bag of stem cells usually looks similar to a bag of blood used for blood transfusion. This is because it contains red blood cells. The goal of a stem cell transplant is to replace unhealthy stem cells with new healthy ones. If all goes well, these healthy stem cells find their way to the bone marrow and begin to function and produce blood cells normally (called an engraft). It often takes several weeks for this to happen.

For a person to be a donor, the donated stem cells must closely match the patients Human Leukocyte Antigen (HLA) type. HLA markers are special proteins found on most cells in the body. The immune system uses these proteins or markers to recognize which cells belong in the body and which do not. These markers are inherited from both parents. Special tests called HLA typing or HLA tissue typing determines whether the patient and the donor cells match.

Close family members such as brothers and sisters (but rarely parents) are often used as donors because they are most likely to match the patients tissue type. Each sibling who has the same parents has a 25 percent chance of matching the patients tissue type. However, if a sibling also has one of the DBA genes, it will be passed to the recipient during the transplant. It is important to screen potential donors for DBA genes because there is a risk of transfer from a sibling who has the gene for DBA, but who has no symptoms.

If there is not a brother or sister or other family member who is a match for the patient, the transplant center can check the National Marrow Donor Program (NMDP) registry for an unrelated matching donor. In some instances, unrelated donors may be adequately matched and able to donate. However, the rate of successful transplant from matched unrelated donors (MUDs) is lower. The best scenario is an identically matched, sibling who does not have DBA. The National Marrow Donor Program (NMDP) is a database containing the tissue types of more than six million potential volunteer donors. Visit the program online to learn more: http://www.marrow.org/index.html.

For DBA patients, a stem cell transplant is intended to restore the marrows ability to make red blood cells. Once the body starts producing red blood cells, the patient may experience a decrease in signs and symptoms of anemia, such as tiredness and paleness. Often times, stem cell transplant may result in a cure of DBA and, when successful, may often extend a persons life and improve the quality of life they are able to enjoy. The person will no longer require long-term steroid medicine or blood transfusions. The persons blood type will actually change to that of the donor.

A stem cell transplant is a complex procedure with risks. Although some people with DBA experience few problems with transplant, others experience many problems and must endure frequent tests and hospitalizations. Before a stem cell transplant, the patient receives chemotherapy and occasionally radiation therapy to destroy their unhealthy stem cells. This is called a preparative regimen. Some side effects, such as nausea, vomiting, fatigue, loss of appetite, mouth sores, hair loss, and skin reactions may be due to the preparative regimen.

Several complications, some potentially fatal, can occur as a result of a stem cell transplant:

After the transplant, before the new marrow has started to grow, the number of white blood cells is low and the immune system (how the body fights infection and stays healthy) is very weak. During this time, the body is susceptible to infections, sometimes from the bacteria that live in the patients own body. Therefore, infections that normally would not be harmful can be very serious, and patients can die of them. Bacterial, viral, and fungal infections are often seen following transplant.

Graft-versus-host disease (GVHD) occurs when the new stem cells (from the donor) do not recognize the patients cells and attacks them, leading to skin rashes, diarrhea, or liver abnormalities. GVHD can be acute or chronic and range in severity from mild to moderate to severe. Medicines are given to prevent GVHD. Mild and moderate GVHD can be treated successfully with drugs and does not increase the risk of the patient dying. The most severe degree of GVHD is less frequent, but very serious, and patients can die of this complication. A close match between the donor and recipient will reduce the risk for GVHD, thereby allowing a greater chance for the donor stem cells to produce normal blood cells without complications.

Some of the more common long-term risks of stem cell transplant include infertility (the inability to produce children) and cataracts (clouding of the lens of the eye, which can be fixed with surgery). Less common effects include long-term damage to organs such as the liver, kidneys, lungs, or heart, and the occurrence of cancers.

After the transplant regular check-ups are needed to identify and take care of any problems that may arise after a patient has a stem cell transplant. Initially, follow-up care involves clinic visits once or twice a week with platelet or blood transfusions, as needed. Long-term follow-up is necessary to maintain a healthy lifestyle, ensure that the DBA continues to be in remission, and ensure that any late effects of the transplant or DBA are caught early. During long-term follow up, growth and development, immunizations, fertility, and mental and physical health are monitored.

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Stem | Treatments | DBA | NCBDDD | CDC

Summary

Mesenchymal stem cells (MSCs) from bone marrow are main cell source for tissue repair and engineering, and vehicles of cell-based gene therapy. Unlike other species, mouse bone marrow derived MSCs (BM-MSCs) are difficult to harvest and grow due to the low MSCs yield. We report here a standardised, reliable, and easy-to-perform protocol for isolation and culture of mouse BM-MSCs. There are five main features of this protocol. (1) After flushing bone marrow out of the marrow cavity, we cultured the cells with fat mass without filtering and washing them. Our method is simply keeping the MSCs in their initial niche with minimal disturbance. (2) Our culture medium is not supplemented with any additional growth factor. (3) Our method does not need to separate cells using flow cytometry or immunomagnetic sorting techniques. (4) Our method has been carefully tested in several mouse strains and the results are reproducible. (5) We have optimised this protocol, and list detailed potential problems and trouble-shooting tricks. Using our protocol, the isolated mouse BM-MSCs were strongly positive for CD44 and CD90, negative CD45 and CD31, and exhibited tri-lineage differentiation potentials. Compared with the commonly used protocol, our protocol had higher success rate of establishing the mouse BM-MSCs in culture. Our protocol may be a simple, reliable, and alternative method for culturing MSCs from mouse bone marrow tissues.

Mesenchymal stem cells (MSCs) are multipotent stem cells that have the potential to self-renew and differentiate into a variety of specialised cell types such as osteoblasts, chondrocytes, adipocytes, and neurons [1]and[2]. MSCs are easily accessible, expandable, immunosuppressive and they do not elicit immediate immune responses [3]and[4]. Therefore, MSCs are an attractive cell source for tissue engineering and vehicles of cell therapy.

MSCs can be isolated from various sources such as adipose tissue, tendon, peripheral blood, and cord blood [5], [6]and[7]. Bone marrow (BM) is the most common source of MSCs. MSCs have been successfully isolated and characterised from many species including mouse, rat, rabbit, dog, sheep, pig, and human [8], [9], [10], [11]and[12]. Mice are one of the most commonly used experimental animals in biology and medicine primarily because they are mammals, small, inexpensive, easily maintained, can reproduce quickly, and share a high degree of homology with humans [13]. However, the isolation and purification of MSCs from mouse bone marrow is more difficult than other species due to their heterogeneity and low percentage in the bone marrow [1], [14]and[15].

Two main stem cell populations and their progenies, haematopoietic stem cells and BM-MSCs, are the main residents of bone marrow [1]and[15]. BM-MSCs are usually isolated and purified through their physical adherence to the plastic cell culture plate [16]. Several techniques have been used to purify or enrich MSCs including antibody-based cell sorting [17], low and high-density culture techniques [18]and[19], positive and negative selection method [20], frequent medium changes [21], and enzymatic digestion approach [22]. However, they all had some short falls: the standard MSCs culture method based on plastic adherence has been confirmed to have lower successful rate [23]; whereas the cell sorting approach reduced the osteogenic potentials of MSCs [17]. Negative selection method leads to granulocytemonocyte lineage cells reappearing after 1 week of culture [24]. Cells obtained using a positive selection method show higher proliferation ability compared with the negative selection method, but the method was only repeated in the C57B1/6 mice and failed to repeat in other strains of mice [25]. Frequent medium change method is inconvenient because it is required to change the culture medium every 8 hours during the first 72 hours of the initial culture [21]. Therefore, an easy and effective protocol for isolation of mouse BM-MSCs is needed.

Reagents used included: 0.25% trypsinEDTA (1) with phenol red; penicillinstreptomycin neomycin (PSN; Life Technologies, Carlsbad, CA, USA) antibiotic mixture; foetal bovine serum, qualified, heat-inactivated (Life Technologies); minimal essential medium (MEM) , nucleosides, powder (Life Technologies); and NaHCO3 (SigmaAldrich, St Louis, MO, USA).

Stock of -MEM was made up with 1 bag of -MEM powder (1L) and 2.2g NaHCO3 in 1000mL of Milli-Q water, adjusted to pH 7.2, filtered to sterilise, and stored for 12 weeks at 4C. Complete -MEM medium was -MEM medium stock supplemented with 15% foetal bovine serum and 1% PSN, stored at 4C. Phosphate-buffered saline (PBS) included: NaCl 8.0g, KCl 0.2g, KH2PO4 0.24g, and Na2HPO4 1.44g in 1L Milli-Q water (pH 7.4, sterilised and stored at 4C).

In this study, two mouse strains (ICR and C57) with different ages (4 weeks and 8 weeks, males and females) were tested using our protocol. All mice were purchased from and housed in a designated and government approved animal facility at The Chinese University Hong Kong, Hong Kong SAR, China, in according to The Chinese University Hong Kong's animal experimental regulations. All efforts were made to minimise animal suffering.

Mice aged 4 weeks or 8 weeks are terminated by cervical dislocation and placed in a 100-mm cell culture dish (Becton Dickinson, Franklin Lakes, NJ, USA), where the whole body is soaked in 70% (v/v) ethanol for 2 minutes, and then the mouse is transferred to a new dish (Fig.1A). Four claws are dissected at the ankle and carpal joints, and incisions made around the connection between hindlimbs and trunk, forelimbs, and trunk. The whole skin is then removed from the hind limbs and forelimbs by pulling toward the cutting site of the claw. Muscles, ligaments, and tendons are carefully disassociated from tibias, femurs, and humeri using microdissecting scissors and surgical scalpel. Tibias, femurs, and humeri are dissected by cutting at the joints, and the bones are transferred onto sterile gauze. Bones are carefully scrubbed to remove the residual soft tissues (Fig.1B), and transferred to a 100-mm sterile culture dish with 10mL complete -MEM medium on ice (Fig.1C). All samples are processed within 30 minutes following animal death to ensure high cell viability. The soft tissues are completely dissociated from the bones to avoid contamination.

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An improved protocol for isolation and culture of ...

Introduction

A bone marrow transplant, alsoknown as a haemopoietic stem cell transplant, replaces damaged bone marrow with healthy bone marrow stem cells.

Bone marrow is aspongytissue found in the hollow centres of some bones. It contains specialist stem cells, which produce the body's blood cells.

Stem cells in bone marrow produce three important types of blood cells:

Bone marrow transplants are often needed to treat conditions thatdamage bone marrow. If bone marrow is damaged, it is no longer able to produce normal blood cells. The new stem cells take over blood cellproduction.

Conditions that bone marrow transplants are used to treat include:

Read more about why a bone marrow transplantis needed.

A bone marrow transplant involves taking healthy stem cells from the bone marrow of one person and transferring them to the bone marrow of another person.

In some cases, it may be possible to take the bone marrow from your own body. This is known as an autologous transplantation. Before it is returned, the bone marrow is cleared of any damaged or diseased cells.

A bone marrowtransplant has five stages. These are:

Having a bone marrow transplant can be an intensive and challenging experience. Many people take up to a year to fully recover from the procedure.

Read more about what happens during a bone marrow transplant.

Bone marrow transplants are usually only recommended if:

Read more about who can have a bone marrow transplant.

Bone marrow transplants arecomplicated procedures with significant risks.

In some cases, the transplanted cells (graft cells) recognise the recipient's cells as "foreign"and try to attack them. This is known as graft versus host disease (GvHD).

The risk of infectionis alsoincreased because your immune system is weakened when you're conditioned (prepared) for the transplant.

Read more about the risks of having a bone marrow transplant.

It's nowpossible to harvest stem cells from sources other than bone marrow.

Peripheral blood stem cell donation involves injectinga medicine into the donor's blood thatcauses the stem cells to moveout of the bone marrow and into the bloodstream where theycan be harvested (collected).

The advantage of this type of stem cell donation is that the donor doesn't needa general anaesthetic.

Stem cells can also be collectedfrom the placenta and umbilical cord of a newborn baby and stored in a laboratory until they're needed.

Cord blood stem cells are very usefulbecause they don't need to be as closely matched as bone marrow or peripheral blood stem cells for a successful outcome.

Find out more about theNHS Cord Blood Bank(external link).

Page last reviewed: 18/02/2014

Next review due: 18/02/2016

Originally posted here:
Bone marrow transplant - NHS Choices

Hematopoietic stem cell transplantation (HSCT) is the transplantation of multipotent hematopoietic stem cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood. It may be autologous (the patient's own stem cells are used) or allogeneic (the stem cells come from a donor). It is a medical procedure in the field of hematology, most often performed for patients with certain cancers of the blood or bone marrow, such as multiple myeloma or leukemia. In these cases, the recipient's immune system is usually destroyed with radiation or chemotherapy before the transplantation. Infection and graft-versus-host disease are major complications of allogeneic HSCT.

Hematopoietic stem cell transplantation remains a dangerous procedure with many possible complications; it is reserved for patients with life-threatening diseases. As survival following the procedure has increased, its use has expanded beyond cancer, such as autoimmune diseases.[1][2]

Indications for stem cell transplantation are as follows:

Many recipients of HSCTs are multiple myeloma[3] or leukemia patients[4] who would not benefit from prolonged treatment with, or are already resistant to, chemotherapy. Candidates for HSCTs include pediatric cases where the patient has an inborn defect such as severe combined immunodeficiency or congenital neutropenia with defective stem cells, and also children or adults with aplastic anemia[5] who have lost their stem cells after birth. Other conditions[6] treated with stem cell transplants include sickle-cell disease, myelodysplastic syndrome, neuroblastoma, lymphoma, Ewing's sarcoma, desmoplastic small round cell tumor, chronic granulomatous disease and Hodgkin's disease. More recently non-myeloablative, "mini transplant(microtransplantation)," procedures have been developed that require smaller doses of preparative chemo and radiation. This has allowed HSCT to be conducted in the elderly and other patients who would otherwise be considered too weak to withstand a conventional treatment regimen.

A total of 50,417 first hematopoietic stem cell transplants were reported as taking place worldwide in 2006, according to a global survey of 1327 centers in 71 countries conducted by the Worldwide Network for Blood and Marrow Transplantation. Of these, 28,901 (57 percent) were autologous and 21,516 (43 percent) were allogeneic (11,928 from family donors and 9,588 from unrelated donors). The main indications for transplant were lymphoproliferative disorders (54.5 percent) and leukemias (33.8 percent), and the majority took place in either Europe (48 percent) or the Americas (36 percent).[7] In 2009, according to the World Marrow Donor Association, stem cell products provided for unrelated transplantation worldwide had increased to 15,399 (3,445 bone marrow donations, 8,162 peripheral blood stem cell donations, and 3,792 cord blood units).[8]

Autologous HSCT requires the extraction (apheresis) of haematopoietic stem cells (HSC) from the patient and storage of the harvested cells in a freezer. The patient is then treated with high-dose chemotherapy with or without radiotherapy with the intention of eradicating the patient's malignant cell population at the cost of partial or complete bone marrow ablation (destruction of patient's bone marrow function to grow new blood cells). The patient's own stored stem cells are then transfused into his/her bloodstream, where they replace destroyed tissue and resume the patient's normal blood cell production. Autologous transplants have the advantage of lower risk of infection during the immune-compromised portion of the treatment since the recovery of immune function is rapid. Also, the incidence of patients experiencing rejection (graft-versus-host disease) is very rare due to the donor and recipient being the same individual. These advantages have established autologous HSCT as one of the standard second-line treatments for such diseases as lymphoma.[9]

However, for others cancers such as acute myeloid leukemia, the reduced mortality of the autogenous relative to allogeneic HSCT may be outweighed by an increased likelihood of cancer relapse and related mortality, and therefore the allogeneic treatment may be preferred for those conditions.[10] Researchers have conducted small studies using non-myeloablative hematopoietic stem cell transplantation as a possible treatment for type I (insulin dependent) diabetes in children and adults. Results have been promising; however, as of 2009[update] it was premature to speculate whether these experiments will lead to effective treatments for diabetes.[11]

Allogeneics HSCT involves two people: the (healthy) donor and the (patient) recipient. Allogeneic HSC donors must have a tissue (HLA) type that matches the recipient. Matching is performed on the basis of variability at three or more loci of the HLA gene, and a perfect match at these loci is preferred. Even if there is a good match at these critical alleles, the recipient will require immunosuppressive medications to mitigate graft-versus-host disease. Allogeneic transplant donors may be related (usually a closely HLA matched sibling), syngeneic (a monozygotic or 'identical' twin of the patient - necessarily extremely rare since few patients have an identical twin, but offering a source of perfectly HLA matched stem cells) or unrelated (donor who is not related and found to have very close degree of HLA matching). Unrelated donors may be found through a registry of bone marrow donors such as the National Marrow Donor Program. People who would like to be tested for a specific family member or friend without joining any of the bone marrow registry data banks may contact a private HLA testing laboratory and be tested with a mouth swab to see if they are a potential match.[12] A "savior sibling" may be intentionally selected by preimplantation genetic diagnosis in order to match a child both regarding HLA type and being free of any obvious inheritable disorder. Allogeneic transplants are also performed using umbilical cord blood as the source of stem cells. In general, by transfusing healthy stem cells to the recipient's bloodstream to reform a healthy immune system, allogeneic HSCTs appear to improve chances for cure or long-term remission once the immediate transplant-related complications are resolved.[13][14][15]

A compatible donor is found by doing additional HLA-testing from the blood of potential donors. The HLA genes fall in two categories (Type I and Type II). In general, mismatches of the Type-I genes (i.e. HLA-A, HLA-B, or HLA-C) increase the risk of graft rejection. A mismatch of an HLA Type II gene (i.e. HLA-DR, or HLA-DQB1) increases the risk of graft-versus-host disease. In addition a genetic mismatch as small as a single DNA base pair is significant so perfect matches require knowledge of the exact DNA sequence of these genes for both donor and recipient. Leading transplant centers currently perform testing for all five of these HLA genes before declaring that a donor and recipient are HLA-identical.

Race and ethnicity are known to play a major role in donor recruitment drives, as members of the same ethnic group are more likely to have matching genes, including the genes for HLA.[16]

As of 2013[update], there were at least two commercialized allogeneic cell therapies, Prochymal and Cartistem.[17]

To limit the risks of transplanted stem cell rejection or of severe graft-versus-host disease in allogeneic HSCT, the donor should preferably have the same human leukocyte antigens (HLA) as the recipient. About 25 to 30 percent of allogeneic HSCT recipients have an HLA-identical sibling. Even so-called "perfect matches" may have mismatched minor alleles that contribute to graft-versus-host disease.

In the case of a bone marrow transplant, the HSC are removed from a large bone of the donor, typically the pelvis, through a large needle that reaches the center of the bone. The technique is referred to as a bone marrow harvest and is performed under general anesthesia.

Peripheral blood stem cells[18] are now the most common source of stem cells for allogeneic HSCT. They are collected from the blood through a process known as apheresis. The donor's blood is withdrawn through a sterile needle in one arm and passed through a machine that removes white blood cells. The red blood cells are returned to the donor. The peripheral stem cell yield is boosted with daily subcutaneous injections of Granulocyte-colony stimulating factor, serving to mobilize stem cells from the donor's bone marrow into the peripheral circulation.

It is also possible to extract stem cells from amniotic fluid for both autologous or heterologous use at the time of childbirth.

Umbilical cord blood is obtained when a mother donates her infant's umbilical cord and placenta after birth. Cord blood has a higher concentration of HSC than is normally found in adult blood. However, the small quantity of blood obtained from an Umbilical Cord (typically about 50 mL) makes it more suitable for transplantation into small children than into adults. Newer techniques using ex-vivo expansion of cord blood units or the use of two cord blood units from different donors allow cord blood transplants to be used in adults.

Cord blood can be harvested from the Umbilical Cord of a child being born after preimplantation genetic diagnosis (PGD) for human leucocyte antigen (HLA) matching (see PGD for HLA matching) in order to donate to an ill sibling requiring HSCT.

Unlike other organs, bone marrow cells can be frozen (cryopreserved) for prolonged periods without damaging too many cells. This is a necessity with autologous HSC because the cells must be harvested from the recipient months in advance of the transplant treatment. In the case of allogeneic transplants, fresh HSC are preferred in order to avoid cell loss that might occur during the freezing and thawing process. Allogeneic cord blood is stored frozen at a cord blood bank because it is only obtainable at the time of childbirth. To cryopreserve HSC, a preservative, DMSO, must be added, and the cells must be cooled very slowly in a controlled-rate freezer to prevent osmotic cellular injury during ice crystal formation. HSC may be stored for years in a cryofreezer, which typically uses liquid nitrogen.

The chemotherapy or irradiation given immediately prior to a transplant is called the conditioning regimen, the purpose of which is to help eradicate the patient's disease prior to the infusion of HSC and to suppress immune reactions. The bone marrow can be ablated (destroyed) with dose-levels that cause minimal injury to other tissues. In allogeneic transplants a combination of cyclophosphamide with total body irradiation is conventionally employed. This treatment also has an immunosuppressive effect that prevents rejection of the HSC by the recipient's immune system. The post-transplant prognosis often includes acute and chronic graft-versus-host disease that may be life-threatening. However, in certain leukemias this can coincide with protection against cancer relapse owing to the graft versus tumor effect.[19]Autologous transplants may also use similar conditioning regimens, but many other chemotherapy combinations can be used depending on the type of disease.

A newer treatment approach, non-myeloablative allogeneic transplantation, also termed reduced-intensity conditioning (RIC), uses doses of chemotherapy and radiation too low to eradicate all the bone marrow cells of the recipient.[20]:320321 Instead, non-myeloablative transplants run lower risks of serious infections and transplant-related mortality while relying upon the graft versus tumor effect to resist the inherent increased risk of cancer relapse.[21][22] Also significantly, while requiring high doses of immunosuppressive agents in the early stages of treatment, these doses are less than for conventional transplants.[23] This leads to a state of mixed chimerism early after transplant where both recipient and donor HSC coexist in the bone marrow space.

Decreasing doses of immunosuppressive therapy then allows donor T-cells to eradicate the remaining recipient HSC and to induce the graft versus tumor effect. This effect is often accompanied by mild graft-versus-host disease, the appearance of which is often a surrogate marker for the emergence of the desirable graft versus tumor effect, and also serves as a signal to establish an appropriate dosage level for sustained treatment with low levels of immunosuppressive agents.

Because of their gentler conditioning regimens, these transplants are associated with a lower risk of transplant-related mortality and therefore allow patients who are considered too high-risk for conventional allogeneic HSCT to undergo potentially curative therapy for their disease. The optimal conditioning strategy for each disease and recipient has not been fully established, but RIC can be used in elderly patients unfit for myeloablative regimens, for whom a higher risk of cancer relapse may be acceptable.[20][22]

After several weeks of growth in the bone marrow, expansion of HSC and their progeny is sufficient to normalize the blood cell counts and re-initiate the immune system. The offspring of donor-derived hematopoietic stem cells have been documented to populate many different organs of the recipient, including the heart, liver, and muscle, and these cells had been suggested to have the abilities of regenerating injured tissue in these organs. However, recent research has shown that such lineage infidelity does not occur as a normal phenomenon[citation needed].

HSCT is associated with a high treatment-related mortality in the recipient (1 percent or higher)[citation needed], which limits its use to conditions that are themselves life-threatening. Major complications are veno-occlusive disease, mucositis, infections (sepsis), graft-versus-host disease and the development of new malignancies.

Bone marrow transplantation usually requires that the recipient's own bone marrow be destroyed ("myeloablation"). Prior to "engraftment" patients may go for several weeks without appreciable numbers of white blood cells to help fight infection. This puts a patient at high risk of infections, sepsis and septic shock, despite prophylactic antibiotics. However, antiviral medications, such as acyclovir and valacyclovir, are quite effective in prevention of HSCT-related outbreak of herpetic infection in seropositive patients.[24] The immunosuppressive agents employed in allogeneic transplants for the prevention or treatment of graft-versus-host disease further increase the risk of opportunistic infection. Immunosuppressive drugs are given for a minimum of 6-months after a transplantation, or much longer if required for the treatment of graft-versus-host disease. Transplant patients lose their acquired immunity, for example immunity to childhood diseases such as measles or polio. For this reason transplant patients must be re-vaccinated with childhood vaccines once they are off immunosuppressive medications.

Severe liver injury can result from hepatic veno-occlusive disease (VOD). Elevated levels of bilirubin, hepatomegaly and fluid retention are clinical hallmarks of this condition. There is now a greater appreciation of the generalized cellular injury and obstruction in hepatic vein sinuses, and hepatic VOD has lately been referred to as sinusoidal obstruction syndrome (SOS). Severe cases of SOS are associated with a high mortality rate. Anticoagulants or defibrotide may be effective in reducing the severity of VOD but may also increase bleeding complications. Ursodiol has been shown to help prevent VOD, presumably by facilitating the flow of bile.

The injury of the mucosal lining of the mouth and throat is a common regimen-related toxicity following ablative HSCT regimens. It is usually not life-threatening but is very painful, and prevents eating and drinking. Mucositis is treated with pain medications plus intravenous infusions to prevent dehydration and malnutrition.

Graft-versus-host disease (GVHD) is an inflammatory disease that is unique to allogeneic transplantation. It is an attack of the "new" bone marrow's immune cells against the recipient's tissues. This can occur even if the donor and recipient are HLA-identical because the immune system can still recognize other differences between their tissues. It is aptly named graft-versus-host disease because bone marrow transplantation is the only transplant procedure in which the transplanted cells must accept the body rather than the body accepting the new cells. Acute graft-versus-host disease typically occurs in the first 3 months after transplantation and may involve the skin, intestine, or the liver. High-dose corticosteroids such as prednisone are a standard treatment; however this immuno-suppressive treatment often leads to deadly infections. Chronic graft-versus-host disease may also develop after allogeneic transplant. It is the major source of late treatment-related complications, although it less often results in death. In addition to inflammation, chronic graft-versus-host disease may lead to the development of fibrosis, or scar tissue, similar to scleroderma; it may cause functional disability and require prolonged immunosuppressive therapy. Graft-versus-host disease is usually mediated by T cells, which react to foreign peptides presented on the MHC of the host[citation needed].

Graft versus tumor effect (GVT) or "graft versus leukemia" effect is the beneficial aspect of the Graft-versus-Host phenomenon. For example, HSCT patients with either acute, or in particular chronic, graft-versus-host disease after an allogeneic transplant tend to have a lower risk of cancer relapse.[25][26] This is due to a therapeutic immune reaction of the grafted donor T lymphocytes against the diseased bone marrow of the recipient. This lower rate of relapse accounts for the increased success rate of allogeneic transplants, compared to transplants from identical twins, and indicates that allogeneic HSCT is a form of immunotherapy. GVT is the major benefit of transplants that do not employ the highest immuno-suppressive regimens.

Graft versus tumor is mainly beneficial in diseases with slow progress, e.g. chronic leukemia, low-grade lymphoma, and some cases multiple myeloma. However, it is less effective in rapidly growing acute leukemias.[27]

If cancer relapses after HSCT, another transplant can be performed, infusing the patient with a greater quantity of donor white blood cells (Donor lymphocyte infusion).[27]

Patients after HSCT are at a higher risk for oral carcinoma. Post-HSCT oral cancer may have more aggressive behavior with poorer prognosis, when compared to oral cancer in non-HSCT patients.[28]

Prognosis in HSCT varies widely dependent upon disease type, stage, stem cell source, HLA-matched status (for allogeneic HCST) and conditioning regimen. A transplant offers a chance for cure or long-term remission if the inherent complications of graft versus host disease, immuno-suppressive treatments and the spectrum of opportunistic infections can be survived.[13][14] In recent years, survival rates have been gradually improving across almost all populations and sub-populations receiving transplants.[29]

Mortality for allogeneic stem cell transplantation can be estimated using the prediction model created by Sorror et al.,[30] using the Hematopoietic Cell Transplantation-Specific Comorbidity Index (HCT-CI). The HCT-CI was derived and validated by investigators at the Fred Hutchinson Cancer Research Center (Seattle, WA). The HCT-CI modifies and adds to a well-validated comorbidity index, the Charlson Comorbidity Index (CCI) (Charlson et al.[31]) The CCI was previously applied to patients undergoing allogeneic HCT but appears to provide less survival prediction and discrimination than the HCT-CI scoring system.

The risks of a complication depend on patient characteristics, health care providers and the apheresis procedure, and the colony-stimulating factor used (G-CSF). G-CSF drugs include filgrastim (Neupogen, Neulasta), and lenograstim (Graslopin).

Filgrastim is typically dosed in the 10 microgram/kg level for 45 days during the harvesting of stem cells. The documented adverse effects of filgrastim include splenic rupture (indicated by left upper abdominal or shoulder pain, risk 1 in 40000), Adult respiratory distress syndrome (ARDS), alveolar hemorrage, and allergic reactions (usually expressed in first 30 minutes, risk 1 in 300).[32][33][34] In addition, platelet and hemoglobin levels dip post-procedure, not returning to normal until one month.[34]

The question of whether geriatrics (patients over 65) react the same as patients under 65 has not been sufficiently examined. Coagulation issues and inflammation of atherosclerotic plaques are known to occur as a result of G-CSF injection.[33] G-CSF has also been described to induce genetic changes in mononuclear cells of normal donors.[33] There is evidence that myelodysplasia (MDS) or acute myeloid leukaemia (AML) can be induced by GCSF in susceptible individuals.[35]

Blood was drawn peripherally in a majority of patients, but a central line to jugular/subclavian/femoral veins may be used in 16 percent of women and 4 percent of men. Adverse reactions during apheresis were experienced in 20 percent of women and 8 percent of men, these adverse events primarily consisted of numbness/tingling, multiple line attempts, and nausea.[34]

A study involving 2408 donors (1860 years) indicated that bone pain (primarily back and hips) as a result of filgrastim treatment is observed in 80 percent of donors by day 4 post-injection.[34] This pain responded to acetaminophen or ibuprofen in 65 percent of donors and was characterized as mild to moderate in 80 percent of donors and severe in 10 percent.[34] Bone pain receded post-donation to 26 percent of patients 2 days post-donation, 6 percent of patients one week post-donation, and <2 percent 1 year post-donation. Donation is not recommended for those with a history of back pain.[34] Other symptoms observed in more than 40 percent of donors include myalgia, headache, fatigue, and insomnia.[34] These symptoms all returned to baseline 1 month post-donation, except for some cases of persistent fatigue in 3 percent of donors.[34]

In one metastudy that incorporated data from 377 donors, 44 percent of patients reported having adverse side effects after peripheral blood HSCT.[35] Side effects included pain prior to the collection procedure as a result of GCSF injections, post-procedural generalized skeletal pain, fatigue and reduced energy.[35]

A study that surveyed 2408 donors found that serious adverse events (requiring prolonged hospitalization) occurred in 15 donors (at a rate of 0.6 percent), although none of these events were fatal.[34] Donors were not observed to have higher than normal rates of cancer with up to 48 years of follow up.[34] One study based on a survey of medical teams covered approximately 24,000 peripheral blood HSCT cases between 1993 and 2005, and found a serious cardiovascular adverse reaction rate of about 1 in 1500.[33] This study reported a cardiovascular-related fatality risk within the first 30 days HSCT of about 2 in 10000. For this same group, severe cardiovascular events were observed with a rate of about 1 in 1500. The most common severe adverse reactions were pulmonary edema/deep vein thrombosis, splenic rupture, and myocardial infarction. Haematological malignancy induction was comparable to that observed in the general population, with only 15 reported cases within 4 years.[33]

Georges Math, a French oncologist, performed the first European bone marrow transplant in November 1958 on five Yugoslavian nuclear workers whose own marrow had been damaged by irradiation caused by a criticality accident at the Vina Nuclear Institute, but all of these transplants were rejected.[36][37][38][39][40] Math later pioneered the use of bone marrow transplants in the treatment of leukemia.[40]

Stem cell transplantation was pioneered using bone-marrow-derived stem cells by a team at the Fred Hutchinson Cancer Research Center from the 1950s through the 1970s led by E. Donnall Thomas, whose work was later recognized with a Nobel Prize in Physiology or Medicine. Thomas' work showed that bone marrow cells infused intravenously could repopulate the bone marrow and produce new blood cells. His work also reduced the likelihood of developing a life-threatening complication called graft-versus-host disease.[41]

The first physician to perform a successful human bone marrow transplant on a disease other than cancer was Robert A. Good at the University of Minnesota in 1968.[42] In 1975, John Kersey, M.D., also of the University of Minnesota, performed the first successful bone marrow transplant to cure lymphoma. His patient, a 16-year-old-boy, is today the longest-living lymphoma transplant survivor.[43]

At the end of 2012, 20.2 million people had registered their willingness to be a bone marrow donor with one of the 67 registries from 49 countries participating in Bone Marrow Donors Worldwide. 17.9 million of these registered donors had been ABDR typed, allowing easy matching. A further 561,000 cord blood units had been received by one of 46 cord blood banks from 30 countries participating. The highest total number of bone marrow donors registered were those from the USA (8.0 million), and the highest number per capita were those from Cyprus (15.4 percent of the population).[44]

Within the United States, racial minority groups are the least likely to be registered and therefore the least likely to find a potentially life-saving match. In 1990, only six African-Americans were able to find a bone marrow match, and all six had common European genetic signatures.[45]

Africans are more genetically diverse than people of European descent, which means that more registrations are needed to find a match. Bone marrow and cord blood banks exist in South Africa, and a new program is beginning in Nigeria.[45] Many people belonging to different races are requested to donate as there is a shortage of donors in African, Mixed race, Latino, Aboriginal, and many other communities.

In 2007, a team of doctors in Berlin, Germany, including Gero Htter, performed a stem cell transplant for leukemia patient Timothy Ray Brown, who was also HIV-positive.[46] From 60 matching donors, they selected a [CCR5]-32 homozygous individual with two genetic copies of a rare variant of a cell surface receptor. This genetic trait confers resistance to HIV infection by blocking attachment of HIV to the cell. Roughly one in 1000 people of European ancestry have this inherited mutation, but it is rarer in other populations.[47][48] The transplant was repeated a year later after a leukemia relapse. Over three years after the initial transplant, and despite discontinuing antiretroviral therapy, researchers cannot detect HIV in the transplant recipient's blood or in various biopsies of his tissues.[49] Levels of HIV-specific antibodies have also declined, leading to speculation that the patient may have been functionally cured of HIV. However, scientists emphasise that this is an unusual case.[50] Potentially fatal transplant complications (the "Berlin patient" suffered from graft-versus-host disease and leukoencephalopathy) mean that the procedure could not be performed in others with HIV, even if sufficient numbers of suitable donors were found.[51][52]

In 2012, Daniel Kuritzkes reported results of two stem cell transplants in patients with HIV. They did not, however, use donors with the 32 deletion. After their transplant procedures, both were put on antiretroviral therapies, during which neither showed traces of HIV in their blood plasma and purified CD4 T cells using a sensitive culture method (less than 3 copies/mL). However, the virus was once again detected in both patients some time after the discontinuation of therapy.[53]

Since McAllister's 1997 report on a patient with multiple sclerosis (MS) who received a bone marrow transplant for CML,[54] there have been over 600 reports of HSCTs performed primarily for MS.[55] These have been shown to "reduce or eliminate ongoing clinical relapses, halt further progression, and reduce the burden of disability in some patients" that have aggressive highly active MS, "in the absence of chronic treatment with disease-modifying agents".[55]

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Hematopoietic stem cell transplantation - Wikipedia, the ...

Corresponding Author: Joshua M. Hare, MD, The Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, Biomedical Research Bldg/Room 908, PO Box 016960 (R-125), Miami, FL 33101 (jhare@med.miami.edu).

Published Online: November 6, 2012. doi:10.1001/jama.2012.25321

Author Contributions:Dr Hare had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Hare, Gerstenblith, DiFede Velazquez, George, Mendizabal, McNiece, Heldman.

Acquisition of data: Hare, Fishman, Gerstenblith, DiFede Velazquez, Zambrano, Suncion, Tracy, Johnston, Brinker, Breton, Davis-Sproul, Byrnes, George, Lardo, Mendizabal, Lowery, Wong Po Foo, Ruiz, Amador, Da Silva, McNiece, Heldman.

Analysis and interpretation of data: Hare, Fishman, Zambrano, Suncion, Tracy, Ghersin, Lardo, Schulman, Mendizabal, Altman, Ruiz, Amador, Da Silva, McNiece, Heldman.

Drafting of the manuscript: Hare, Fishman, Ghersin, Mendizabal, Ruiz, Amador, Heldman.

Critical revision of the manuscript for important intellectual content: Hare, Fishman, Gerstenblith, DiFede Velazquez, Suncion, Tracy, Johnston, Brinker, Breton, Davis-Sproul, Schulman, Byrnes, Geroge, Lardo, Mendizabal, Lowery, Rouy, Altman, Wong Po Foo, Ruiz, Da Silva, McNiece, Heldman.

Statistical analysis: Hare, Mendizabal, McNiece, Heldman.

Obtained funding: Hare, Lardo.

Administrative, technical, or material support: Hare, DiFede Velazquez, Zambrano, Suncion, Ghersin, Johnston, Breton, Davis-Sproul, Schulman, Byrnes, Lowery, Rouy, Altman, Wong Po Foo, Da Silva, McNiece, Heldman.

Study supervision: Hare, Fishman, Gerstenblith, Tracy, George, Schulman, Altman, Da Silva, McNiece, Heldman.

Conflict of Interest Disclosures: All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Dr Hare reported having a patent for cardiac cell-based therapy, receiving research support from and being a board member of Biocardia, having equity interest in Vestion Inc, and being a consultant for Kardia. Dr George reported serving on the board of GE Healthcare, consulting for ICON Medical Imaging, and receiving trademark royalties for fluoroperfusion imaging. Mr Mendizabal is an employee of EMMES Corporation. Drs Rouy, Altman, and Wong Po Foo are employees of Biocardia Inc. Dr McNiece reported being a consultant and board member of Proteonomix Inc. Dr Heldman reported having a patent for cardiac cell-based therapy, receiving research support from and being a board member of Biocardia, and having equity interest in Vestion Inc. No other authors reported any financial disclosures.

Funding/Support: This study was funded by the US National Heart, Lung, and Blood Institute (NHLBI) as part of the Specialized Centers for Cell-Based Therapy U54 grant (U54HL081028-01). Dr Hare is also supported by National Institutes of Health (NIH) grants RO1 HL094849, P20 HL101443, RO1 HL084275, RO1 HL107110, RO1 HL110737, and UM1HL113460. The NHLBI provided oversight of the clinical trial through the independent Gene and Cell Therapy Data and Safety Monitoring Board (DSMB). Biocardia Inc provided the Helical Infusion Catheters for the conduct of POSEIDON.

Role of the Sponsors: The NHLBI, NIH, and Biocardia Inc had no role in the design and conduct of the study; in the collection, management, analysis, and interpretation of the data; or in the preparation, review, or approval of the manuscript.

Additional Contributions: We thank the NHLBI Gene and Cell Therapy DSMB, the patients who participated in this trial, the bone marrow donors, the staff of the cardiac catheterization laboratories at the University of Miami Hospital and The Johns Hopkins Hospital. Erica Anderson, MA (EMMES Corporation), provided data management and Hongwei Tang, MD (TeraRecon Inc), provided consultation regarding CT imaging analysis. Ms Anderson received compensation for her contribution via the Specialized Centers for Cell-Based Therapy grant. Dr Tang did not receive any compensation for his contribution.

This article was corrected for errors on July 19, 2013.

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JAMA | Comparison of Allogeneic vs Autologous Bone Marrow ...

Abstract Introduction

Bone marrow mesenchymal stem cells (BMMSCs) are a heterogeneous population of postnatal precursor cells with the capacity of adhering to culture dishes generating colony-forming unit-fibroblasts (CFU-F). Here we identify a new subset of BMMSCs that fail to adhere to plastic culture dishes and remain in culture suspension (S-BMMSCs).

To catch S-BMMSCs, we used BMMSCs-produced extracellular cell matrix (ECM)-coated dishes. Isolated S-BMMSCs were analyzed by in vitro stem cell analysis approaches, including flow cytometry, inductive multiple differentiation, western blot and in vivo implantation to assess the bone regeneration ability of S-BMMSCs. Furthermore, we performed systemic S-BMMSCs transplantation to treat systemic lupus erythematosus (SLE)-like MRL/lpr mice.

S-BMMSCs are capable of adhering to ECM-coated dishes and showing mesenchymal stem cell characteristics with distinction from hematopoietic cells as evidenced by co-expression of CD73 or Oct-4 with CD34, forming a single colony cluster on ECM, and failure to differentiate into hematopoietic cell lineage. Moreover, we found that culture-expanded S-BMMSCs exhibited significantly increased immunomodulatory capacities in vitro and an efficacious treatment for SLE-like MRL/lpr mice by rebalancing regulatory T cells (Tregs) and T helper 17 cells (Th17) through high NO production.

These data suggest that it is feasible to improve immunotherapy by identifying a new subset BMMSCs.

Bone marrow mesenchymal stem cells (BMMSCs) are hierarchical postnatal stem/progenitor cells capable of self-renewing and differentiating into osteoblasts, chondrocytes, adipocytes, and neural cells [1,2]. BMMSCs express a unique surface molecule profile, including expression of STRO-1, CD29, CD73, CD90, CD105, CD146, Octamer-4 (Oct4), and stage-specific embryonic antigen-4 (SSEA4) [3,4]. It is generally believed that BMMSCs are negative for hematopoietic cell markers such as CD14 and CD34 [5-13]. BMMSCs have been widely used for tissue engineering [14-16]. Recently, a growing body of evidence has indicated that BMMSCs produce a variety of cytokines and display profound immunomodulatory properties [17-19], perhaps by inhibiting the proliferation and function of several major immune cells, such as natural killer cells, dendritic cells, and T and B lymphocytes [17-20]. These unique properties make BMMSCs of great interest for clinical applications in the treatment of different immune disorders [17,21-24].

BMMSCs are thought to be derived from the bone marrow stromal compartment, initially appearing as adherent, single colony clusters (colony-forming unit-fibroblasts [CFU-F]), and subsequently proliferating on culture dishes [25]. To date, the CFU-F assay has been considered one of the gold standards for determining the incidence of clonogenic BMMSC [26,27]. Since BMMSC are a heterogeneous population of stem cells, it is critical to identify whether BMMSC contain unique cell subsets with distinctive functions, analogous to the hematopoietic stem/progenitor cell system. In this study, we identified a subset of mouse BMMSCs in culture suspension and determined their immunomodulatory characteristics.

Female C3H/HeJ, C57BL/6J, and C3MRL-Faslpr/J mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Female immunocompromised mice (Beige nude/nude XIDIII) were purchased from Harlan (Indianapolis, IN, USA). All animal experiments were performed under the institutionally approved protocols for the use of animal research (USC #10874 and 10941).

Anti Oct4, SSEA4, Runx2, OCN, active catenin and catenin were purchased from Millipore (Billerica, MA, USA). Anti alkaline phosphatase (ALP) antibody was purchased from Abcam (Cambridge, MA, USA). Anti Sca-1-PE, CD34-PE, CD34-FITC, CD45-PE, CD73-PE, CD4-PerCP, CD8-FITC, CD25-APC, CD3 and CD28 antibodies were purchased from BD Bioscience (San Jose, CA, USA). Anti Foxp3-PE, IL17-PE, and IFN-APC antibodies were purchased from eBioscience (San Diego, CA, USA). Unconjugated anti CD34, CD73, and CD105, NOS2 were purchased from Santa Cruz Biosciences (Santa Cruz, CA, USA). Anti actin antibody was purchased from Sigma (St. Louis, MO, USA).

The single suspension of bone marrow derived all nucleated cells (ANCs) from femurs and tibias were seeded at a density of 15 106 into 100 mm culture dishes (Corning, NY, USA) at 37C and 5% CO2. Non-adherent cells were removed after two days and attached cells were maintained for 16 days in alpha minimum essential medium (-MEM, Invitrogen, Grand Island, NY, USA) supplemented with 20% fetal bovine serum (FBS, Equitech-bio, Kerrville, TX, USA), 2 mM L-glutamine, 55 M 2-mercaptoethanol, 100 U/ml penicillin, and 100 g/ml streptomycin (Invitrogen). Colony-forming attached cells were passed once for further experimental use.

ECM coated dishes were prepared as described previously [28]. Briefly, 100% confluence of BMMSCs was cultured in medium with 100 nM L-ascorbic acid phosphate (Wako Pure Chemical, Richmond, VA, USA). After two weeks, cultures were washed with PBS and incubated with 0.005% Triton X-100 (Sigma) for 15 minutes at room temperature to remove cells. The ECM was treated with DNase I (100 units/ml; Sigma) for 1 hour at 37C. The ECM was washed with PBS three times and stored in 2 ml of PBS containing 100 U/ml penicillin, 100 g/ml streptomycin and 0.25 g/ml fungizone (Invitrogen) at 4C.

Bone marrow-derived ANCs (15 106) were seeded into 100 mm culture dishes and cultured for two days. The culture supernatant with floating cells was collected and centrifuged to obtain putative non-attached BMMSCs. The cells were re-seeded at indicated numbers on ECM-coated dishes. After 2 days, the floating cells in the cultures were removed with PBS and the attached cells on ECM were maintained for an additional 14 days. Colony-forming attached cells were passed once and sub-cultured on regular plastic culture dishes for further experiments. For some stem cell characterization analyses, we collected SSEA4 positive S-BMMSCs using the MACS magnetic separation system (Milteny Biotech, Auburn, CA, USA) and expanded in the cultures.

One million cells of ANCs from bone marrow were seeded on a T-25 cell culture flask (Nunc, Rochester, NY, USA). After 16 days, the cultures were washed with PBS and stained with 1% toluidine blue solution in 2% paraformaldehyde (PFA). A cell cluster that had more than 50 cells was counted as a colony under microscopy. The colony number was counted in five independent samples per each experimental group.

The proliferation of BMMSCs and S-BMMSCs was performed using the bromodeoxyuridine (BrdU) incorporation assay. Each cell population (1 104 cells/well) was seeded on two-well chamber slides (Nunc) and cultured for two to three days. The cultures were incubated with BrdU solution (1:100) (Invitrogen) for 20 hours, and stained with a BrdU staining kit (Invitrogen). BrdU-positive and total cell numbers were counted in ten images per subject. The BrdU assay was repeated in five independent samples for each experimental group.

A total of 0.5 106 cells of BMMSCs and S-BMMSCs was seeded on 60 mm culture dishes at the first passage. Upon reaching confluence, the cells were passaged at the same cell density. The population doubling was calculated at every passage according to the equation: log2 (number of harvested cells/number of seeded cells). The finite population doublings were determined by cumulative addition of total numbers generated from each passage until the cells ceased dividing.

BMMSCs or S-BMMSCs (0.2 106 cells) were incubated with 1 g of R-Phycoerythrin (PE). (PE)-conjugated antibodies or isotype-matched control immunoglobulin Gs (IgGs) (Southern Biotech, Birmingham, AL, USA) at 4C for 45 minutes. Samples were analyzed by a fluorescence-activated cell sorting (FACS)Calibur flow cytometer (BD Bioscience). For dual color analysis, the cells were treated with PE-conjugated and fluorescein isothiocyanate (FITC)-conjugated antibodies or isotype-matched control IgGs (1 g each). The cells were analyzed on FACSCalibur (BD Bioscience).

The cells subcultured on eight-well chamber slides (Nunc) (2 103/well) were fixed with 4% PFA. The samples were incubated with the specific or isotype-matched mouse antibodies (1:200) overnight at 4C, and treated with Rhodamine-conjugated secondary antibodies (1:400, Jackson ImmunoResearch, West Grove, PA, USA; Southern Biotechnology, Birmingham, AL, USA). Finally, chamber slides were mounted using Vectashield mounting medium containing 4', 6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA).

A total of 4.0 106 cells was mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) ceramic powders (40 mg, Zimmer Inc., Warsaw, IN, USA) and subcutaneously transplanted into eight-week-old immunocompromised mice. After eight weeks, the transplants were harvested, fixed in 4% PFA and then decalcified with 5% ethylenediaminetetraacetic acid (EDTA; pH 7.4), followed by paraffin embedding. The paraffin sections were stained with H & E and analyzed by an NIH Image-J. The newly-formed mineralized tissue area from five fields was calculated and shown as a percentage to total tissue area.

BMMSCs and S-BMMSCs were cultured under osteogenic culture conditions containing 2 mM -glycerophosphate (Sigma), 100 M L-ascorbic acid 2-phosphate and 10 nM dexamethasone (Sigma). After induction, the cultures were stained with alizarin red or alkaline phosphatase.

For adipogenic induction, 500 nM isobutylmethylxanthine, 60 M indomethacin, 500 nM hydrocortisone, 10 g/ml insulin (Sigma), 100 nM L-ascorbic acid phosphate were added to the culture medium. After 10 days, the cultured cells were stained with Oil Red-O and positive cells were quantified by using an NIH Image-J. Total RNA was also isolated from cultures after 10 days induction for further experiments.

For chondrogenic induction, 1 106 cell pellets were cultured under chondrogenic medium containing 15% FBS, 1% ITS (BD), 100 nM dexamethasone, 2 mM pyruvate (SIGMA), and 10 ng/ml transforming growth factor beta 1 (TGF1) in (D)MEM (Invitrogen) for threeweeks. Cell pellets were harvested at three weeks post induction, fixed overnight with 4% PFA and then, sections were prepared for staining.

Extraction of total RNA and RT-PCR were performed according to standard procedures. Primer information is described in Additional materials and methods [see Additional file 1].

Additional file 1. Figures S1 to S8 and Additional materials and methods. Figure S1. ECM coated dish could capture a greater number of CFU-F. CFU-f number in ECM coated dish compared to regular dish. Figure S2. CD45-CD34-BMMSCs showed similar property with S-BMMSCs. (A) CFU-f number. (B) Flow cytometric analysis. Figure S3. S-BMMSCs extended survival rate of lethal dose of irradiated mice. The life span of irradiated mice. Figure S4. Osteoclast activity in S-BMMSC-treated MRL/lpr mice. (A) Osteoclast number. (B) sRANKL level. (C) CTX level. Figure S5. L-NMMA pre-treated BMMSC transplantation failed to ameliorate disease phenotype of MRL/lpr mice. (A) Anti dsDNA (IgG) level. (B) Anti dsDNA (IgM) level. (C) Urine protein level. (D) Tregs level. (E) Th17 level. (F) Ratio between Tregs/Th17. Figure S6. Inhibition of NO production in BMMSCs. (A) NO level with inhibitors. (B) iNOS level by western blot. Figure S7. Endogenous S-BMMSCs in mice bone marrow. (A) Cell sorting result. (B) CFU-f number. (C) Osteogenic differentiation in vitro. (D) NO level. Figure S8. Human bone marrow contains S-BMMSCs (hS-BMMSCs). (A) NO level. (B) Kynurenine production. (C) Kynurenine production in co-culture system. (D) T cell apoptosis induction by hS-BMMSCs. Additional materials and methods describe about TRAP staining, Histomotry, Rescue lethal dose irradiated mice, and Isolation of CD34+CD73+ double positive cells.

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A total of 20 g of protein was used and SDS-PAGE and western blotting were performed according to standard procedures. Detailed procedures are described in Additional materials and methods [see Additional file 1]. -actin on the same membrane served as the loading control.

BMMSCs and S-BMMSCs were cultured onto 35 mm low attach culture dishes (2 104/dish, STEMCELL Technologies, Vancouver, BC, V5Z 1B3, Canada) under hematopoietic differentiation medium (STEMCELL Technologies) with or without erythropoietin (EPO; 3 U/mL) for seven days. Whole bone marrow cells and linage negative bone marrow cells (Linage-cells) were used as positive controls. The results are representative of five independent experiments.

S-BMMSCs and BMMSCs were treated with 1 mM L-NG-monomethyl-arginine (L-NMMA) (Cayman Chemical, Ann Arbor, MI, USA) or 0.2 mM 1400 W (Cayman Chemical) to inhibit total nitric oxide synthase (NOS) or inducible nitric oxide synthase (iNOS), respectively.

BMMSCs (0.2 106/well) were cultured on 24-well plates with or without cytokines (IFN, 25 ng/ml; IL-1, 5 ng/ml, R&D Systems, Minneapolis, MN, USA) and chemicals (L-NMMA, 1 mM; 1400 W, 0.2 mM) at the indicated concentration and days. The supernatant from each culture was collected and nitric oxide concentration measured using a Total Nitric Oxide and Nitrate/Nitrite Parameter Assay kit (R&D Systems) according to the manufacturer's instruction.

The transwell system (Corning) was used for co-culture experiments. A total of 0.2 106 of S-BMMSCs or BMMSCs was seeded on each lower chamber. Activated spleen cells (1 106/chamber), which were pre-stimulated with plate-bound anti CD3 antibody (3 g/ml) and soluble anti CD28 antibody (2 g/ml) for two days, were loaded in the upper chambers. Both chambers were filled with a complete medium containing (D)MEM (Lonza, CH-4002 Basel, Switzerland) with 10% heat-inactivated FBS, 50 M 2-mercaptoethanol, 10 mM HEPES, 1 mM sodium pyruvate (Sigma), 1% non-essential amino acid (Cambrex, East Rutherford, NJ, USA), 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin. To measure the spleen cells viability, cell counting kit-8 (Dojindo Molecular Technologies, Rockville, MD, USA) was used. For apoptosis of spleen cells analyses, Annexin V-PE apoptosis detection kits I (BD Bioscience) were used and analyzed on FACSCalibur (BD Bioscience).

CD4+CD25- T-lymphocytes (1 106/well), collected using a CD4+CD25+ Treg isolation kit (Miltenyi Biotec), were pre-stimulated with plate-bound anti CD3 antibody (3 g/ml) and soluble anti CD28 antibody (2 g/ml) for two days. These activated T-lymphocytes were loaded on 0.2 106 BMMSCs or S-BMMSCs cultures with recombinant human TFG1 (2 ng/ml) (R&D Systems) and recombinant mouse IL2 (2 ng/ml) (R&D Systems). For Th17 induction, recombinant human TFG1 (2 ng/ml) and recombinant mouse IL6 (50 ng/ml) (Biolegend, San Diego, CA, USA) were added. After three days, cells in suspension were collected and stained with anti CD4-PerCP, anti CD8a-FITC, anti CD25-APC antibodies (each 1 g) for 45 minutes on ice under dark conditions. The cells were then stained with anti Foxp3-PE antibody (1 g) using a Foxp3 staining buffer kit (eBioscience) for cell fixation and permeabilization. For Th17, cells in suspension were stained with anti CD4-FITC (1g, Biolegend) for 45 minutes on ice under dark conditions followed by intercellular staining with anti-IL 17 antibody (1g, Biolegend) using a Foxp3 staining buffer kit. The cells were analyzed on FACSCalibur.

Under general anesthesia, C3H/HeJ-derived BMMSCs, S-BMMSCs, L-NMMA pre-treated BMMSCs (1 mM for five days), or CD34+/CD73+ double sorted cells (0.1 106 cells/10 g body weight) were infused into MRL/lpr mice via the tail vein at 10 weeks of age (n = 6 each group). In the control group, MRL/lpr mice received PBS (n = 5). All mice were sacrificed at two weeks post transplantation for further analysis. The protein concentration in urine was measured using a Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA).

Peripheral blood serum samples were collected from mice. Autoantibodies, sRANKL and CTX were analyzed by ELISA using commercially available kits (anti-dsDNA antibodies and ANA; alpha diagnostics, albumin and sRANKL; R&D Systems, CTX; Nordic Bioscience Diagnostics, Herlev, Rigion Hovedstaden, Denmark) according to their manufactures' instructions. The results were averaged in each group. The intra-group differences were calculated between the mean values.

To detect Tregs, peripheral blood mononuclear cells (PBMNCs) (1 106) were treated with PerCP-conjugated anti-CD4, FITC-conjugated anti-CD8a, APC-conjugated anti-CD25 antibodies, and stained with R-PE-conjugated anti-Foxp3 antibody using a Foxp3 staining buffer kit (eBioscience). To measure Th17 cells, PBMNCs (1 106) were incubated with PerCP-conjugated anti-CD4, FITC-conjugated anti-CD8a, followed by treatment with R-PE-conjugated anti-IL-17 and APC-conjugated anti-IFN antibodies using a Foxp3 staining buffer kit. The cells were then analyzed on FACSCalibur .

Student's t-test was used to analyze statistical difference. P values less than 0.05 were considered significant.

To determine whether a subset of BMMSCs remain in culture suspension, ANCs (15 106 cells) from bone marrow were plated onto regular plastic culture dishes for two days and all non-attached cells were subsequently transplanted into immunocompromised mice subcutaneously using HA/TCP as a carrier. At eight weeks post-transplantation, newly formed bone was identified in the transplants by H & E staining (Figure 1A), suggesting that the BMMSC culture suspension may contain cells with a capacity to differentiate into bone forming cells. In vitro studies indicated that ECM produced by culture-expanded BMMSCs (BMMSC-ECM) could capture higher numbers of CFU-Fs when compared to plastic cultures [see Additional file 1, Figure S1] [28]. Thus, we collected culture supernatant with floating cells at two days post CFU-F culture and re-loaded it onto BMMSC-ECM-coated dishes (Figure 1B). A subset of BMMSCs in the suspension (S-BMMSCs) was able to adhere to the BMMSC-ECM and form CFU-F (Figure 1B), at a lower incidence compared to the number of CFU-F generated from regular BMMSCs (Figure 1C). In order to characterize the stem cell properties of S-BMMSCs, we collected SSEA4-positive S-BMMSCs and assessed their proliferation rate by BrdU incorporation. We found that S-BMMSCs had a significantly elevated BrdU uptake rate compared to regular BMMSCs (Figure 1D). In addition, we used a continuous cell culture assay to indicate that SSEA4-positive S-BMMSCs acquired a significantly increased number of population doublings (Figure 1E). These data imply that S-BMMSCs are distinct from regular BMMSCs in terms of attachment, proliferation, and self-renewal.

Figure 1. Identification of suspension BMMSCs (S-BMMSCs). (A) Hypothetical model indicates that bone marrow all nucleated cells (ANCs) were seeded at 15 106 into 100 mm culture dishes and incubated for two days at 37C with 5% CO2, and subsequently non-attached cells from culture suspension were transplanted into immunocompromised mice subcutaneously using hydroxyapatite tricalcium phosphate (HA) as a carrier for eight weeks. Newly formed bone (B) by osteoblasts (arrow heads) and associated connective tissue (C) were detected in this non-attached cell transplants by H & E staining. Bar = 100 m. (B) Hypothetical model of isolating S-BMMSCs. BMMSCs usually attach on culture dishes within two days; however, a small portion of BMMSCs in ANCs failed to attach to the dishes and remained in the suspension. The suspensions containing putative non-attached BMMSCs were collected and transferred to the extracellular matrix (ECM) coated dish with generating single colony clusters (CFU-F). These ECM-attached BMMSCs (S-BMMSCs) were sub-cultured on regular plastic culture dishes for additional experiments. (C) The number of plastic attached CFU-F from ANCs (1.5 106 cells) is more than seven-fold higher than that derived from BMMSC-ECM adherent S-BMMSCs. (D) Proliferation rates of S-BMMSCs and BMMSCs were assessed by BrdU incorporation for 24 hours. The percentage of positive cells is significantly increased in S-BMMSCs when compared to BMMSCs. (E) S-BMMSCs exhibit a significant increase in population doublings when compared to BMMSCs. The results are representative of five independent experiments. Scale bars = 50 m. ***P <0.001. The graph bar represents mean SD. BMMSCs, bone marrow mesenchymal stem cells; BrdU, bromodeoxyuridine; S-BMMSCs, BMMSCs in suspension; SD, standard deviation.

To examine the multipotent differentiation potential, we showed that S-BMMSCs are analogous to BMMSCs in their expression of alkaline phosphatase (ALP), mineralized nodule accumulation under the osteogenic inductive cultures, and bone regeneration when transplanted into immunocompromised mice using HA/TCP as a carrier (Figures 2A and 2B). Furthermore, we showed that S-BMMSCs were similar to regular BMMSCs in forming Oil red-O positive fat cells under adipogenic inductive conditions, which was associated with expression of the adipogenic genes, peroxisome proliferator-activated receptor gamma 2 (ppar2) and lipoprotein lipase (lpl) (Figures 2C and 2D). Parallel studies showed a similar capacity between S-BMMSCs and regular BMMSCs to differentiate into chondrocytes under chondrogenic inductive conditions, associated with the expression of proteoglycan, trichrome positive collagen, and type II collagen (Figure 2E). Collectively, these data confirm that S-BMMSCs are a subset of BMMSCs.

Figure 2. Multipotent differentiation of S-BMMSCs. (A) Alizarin Red S and alkaline phosphatase (ALP) staining showed that S-BMMSCs were similar to regular BMMSCs in osteogenic differentiation in vitro. (B) S-BMMSCs or regular BMMSCs (4 106 cells/transplant) were transplanted into immunocompromised mice using HA/TCP (HA) as a carrier for eight weeks. Bone formation was detected in S-BMMSC and BMMSC transplants, evidenced by H & E staining. HA, hydroxyapatite tricalcium phosphate; B, bone; M, bone marrow; CT, connective tissue. Bar = 50 m. (C-D) S-BMMSCs are capable of forming Oil Red O positive cells (C) and expression of ppar2 and lpl mRNA as seen in regular BMMSCs (D). Glyceraldehyde 3-phosphate dehydrogenase (gapdh) was used as an internal control. The results are representative of five independent experiments. Scale bars = 100 m. (E) Chondrogenic differentiation was assessed by Alcian blue staining for acidic sulfated mucosubstances, Pollak's Trichrome staining for collagen, and immunohistochemical staining for collagen type II. S-BMMSCs were able to differentiate into chondrocytes as observed in regular BMMSCs. Bar = 50 m. The results are representative of three independent experiments. The graph bar represents mean SD. BMMSCs, bone marrow mesenchymal stem cells; S-BMMSCs, BMMSCs in suspension; SD, standard deviation.

By flow cytometric analysis, S-BMMSCs expressed mesenchymal stem cell markers at the same level as regular BMMSCs (Figure 3A). Interestingly, 23.4% of S-BMMSCs expressed CD34, a hematopoietic stem cell (HSC) and endothelial cell marker, whereas 0.2% of BMMSCs expressed CD34 (Figure 3A). BMMSCs (21.4%) and S-BMMSCs (31.2%) expressed CD45, another hematopoietic marker, at passage 2 (Figure 3A). Both BMMSCs and S-BMMSCs were negative to CD11b antibody staining (data not shown), excluding the possibility that S-BMMSCs are derived from monocyte/macrophage lineage cells. Importantly, CD34+ S-BMMSCs co-expressed BMMSC-associated markers CD73 or Octamer-4 (Oct4), as evidenced by flow cytometric analysis (Figure 3B). Western blot analysis confirmed that S-BMMSCs expressed CD34, CD73, and CD105 (Figure 3C), and regular BMMSCs expressed CD73 and CD105 but lacked CD34 expression (Figure 3C). Whole bone marrow cells (BMC) were used as positive control. S-BMMSCs also showed a continued expression of CD34 from passage one to five; however, the expression levels appear reduced after passage three (Figure 3D). In order to further verify CD34 expression in S-BMMSCs, immunocytostaining analyses were performed to show co-expression of CD34 with mesenchymal markers CD73 (Figure 3E) in contrast to regular BMMSCs that were negative for anti-CD34 antibody staining (Figure 3E).

Figure 3. S-BMMSCs express CD34. (A) Flow cytometric analysis showed that regular BMMSCs fail to express CD34, but are positive for CD45 antibody staining (21.4%). However, S-BMMSCs express both CD34 (23.4%) and CD45 (31.2%). (B) Flow cytometric analysis also showed that CD34+ S-BMMSCs were positive for anti CD73 (13.8%) and Oct4 (13.4%) antibody staining. IgG isotype staining groups were used as negative controls. (C, D) Western blot analysis indicated that S-BMMSCs express CD34 and mesenchymal surface molecules CD73 and CD105. In contrast, regular BMMSCs only express CD73 and CD105 (C). S-BMMSCs express CD34 at passage one to five (D). -actin was used as a sample loading control. BMC, whole bone marrow ANC. (E) Immunocytostaining confirmed that S-BMMSCs are double positive for CD34/CD73 (triangle). Regular BMMSCs are negative for CD34 antibody staining and only positive for anti CD73 antibody staining. Bar = 100 m. (F) Both BMMSCs and S-BMMSCs failed to differentiate into hematopoietic lineage under hematopoietic inductive conditions with EPO (upper panel) or without EPO (lower panel). Whole bone marrow cells and lineage negative cells were used as positive (yellow arrowheads) control. Bar = 100 m. ANC, all nucleated cells; BMMSCs, bone marrow mesenchymal stem cells; EPO, erythropoietin; S-BMMSCs, BMMSCs in suspension.

It is generally believed that CD34 expression is associated with HSCs and endothelial populations. HSCs can differentiate into all the blood cell lineages and rescue lethally irradiated subjects. Thus, we cultured S-BMMSCs and regular BMMSCs in hematopoietic differentiation medium and determined that these mesenchymal cells failed to differentiate into a hematopoietic cell lineage compare to bone marrow cells that formed myeloid and erythroid colony forming clusters (Figure 3F). In addition, CD45-CD34-BMMSCs showed an ability similar to that of S-BMMSCs in colony forming and expressing surface marker as MSC [see Additional file 1, Figure S2]. Furthermore, we infused S-BMMSCs systemically to rescue lethally irradiated mice and found that S-BMMSCs, but not regular BMMSCs, could extend the lifespan of lethally irradiated mice [see Additional file 1, Figure S3]. However, S-BMMSCs failed to rescue lethally irradiated mice, as shown in the whole bone marrow cell group [see Additional file 1, Figure S3]. These data provid further evidence that CD34 expression in S-BMMSCs is not due to HSC contamination.

Since the immunomodulation property of MSCs is one of the essential factors for MSC characterization, allogenic S-BMMSC transplantation into MRL/lpr mice was performed (Figure 4A). Two weeks after transplantation, both S-BMMSCs and BMMSCs were capable of ameliorating SLE-induced glomerular basal membrane disorder (yellow arrow, Figure 4B) and reducing the urine protein level (Figure 4C). It appeared that S-BMMSCs were superior compared to BMMSCs in terms of reducing the overall urine protein levels (Figure 4C). As expected, MRL/lpr mice showed remarkably increased levels of autoantibodies, including anti-double strand DNA (dsDNA) IgG and IgM antibodies (Figures 4D and 4E) and anti-nuclear antibody (ANA; Figure 4F) in the peripheral blood serum. Although S-BMMSC and BMMSC infusion showed significantly decreased serum levels of anti-dsDNA IgG, IgM antibodies and ANA in peripheral blood (Figures 4D-F), S-BMMSCs showed a superior therapeutic effect in reducing anti-dsDNA IgG antibody and ANA levels when compared to BMMSCs (Figures 4D and 4F). Additionally, decreased serum albumin levels in MRL/lpr mice were recovered by S-BMMSC and BMMSC infusion (Figure 4G) but S-BMMSC treatment resulted in a more significant recovery than BMMSC treatment (Figure 4G). Next, flow cytometric analysis revealed that S-BMMSC showed more effectiveness in recovering the decreased level of CD4+CD25+Foxp3+ Tregs and increased the number of CD4+IL17+IFN- T-lymphocytes (Th17 cells) in peripheral blood when compared to BMMSCs (Figures 4H, 4I). In addition, highly passaged mouse S-BMMSCs failed to inhibit Th17 differentiation in vitro (data not shown) suggesting that mouse S-BMMSCs probably lose their immunomodulation property under long culture expansion.

Figure 4. S-BMMSCs showed superior therapeutic effect on SLE-like MRL/lpr mice. (A) Schema of BMMSC transplantation into MRL/lpr mice. (B) S-BMMSC and BMMSC treatment recover basal membrane disorder and mesangium cell over-growth in glomerular (G) (H&E staining). (C) S-BMMSC and BMMSC transplantation could reduce urine protein levels at two weeks post transplantation compared to the MRL/lpr group. S-BMMSCs offered a more significant reduction compared to BMMSCs. (D, E) The serum levels of anti-dsDNA IgG and IgM antibodies were significantly increased in MRL/lpr mice compared to controls (C3H). S-BMMSC and BMMSC treatments could reduce antibody levels but S-BMMSCs showed a superior treatment effect than BMMSC in reducing anti-dsDNA IgG antibody (D). (F) S-BMMSC and BMMSC treatments could reduce increased levels of anti nuclear antibody (ANA) in MRL/lpr mice. S-BMMSC showed a better effect in ANA reduction compared to BMMSC. (G) S-BMMSC and BMMSC treatments could increase the albumin level in MRL/lpr mice, which was decreased in controls. S-BMMSC treatments were more effective in elevating the albumin level compared to BMMSC treatment. (H) Flow cytometric analysis showed a reduced number of Tregs in MRL/lpr peripheral blood compared to control. BMMSC and S-BMMSC treatments elevated the number of Tregs. S-BMMSCs induced a more significant elevation of the Tregs level than BMMSCs. (I) Flow cytometric analysis showed an increased number of Th17 in MRL/lpr mice peripheral blood compared to control. Th17 were markedly decreased in BMMSC and S-BMMSC treated groups. S-BMMSC treatment induced a more significant reduction of Th17 cells than treatment with BMMSCs. *P <0.05; ** P <0.01; ***P <0.001. The graph bar represents mean SD. BMMSCs, bone marrow mesenchymal stem cells; Ig, immunoglobulin; S-BMMSCs, BMMSCs in suspension; SD, standard deviation; SLE, systemic lupus erythematosus; Tregs, regulatory T cells.

Furthermore, we showed that S-BMMSCs were superior to BMMSCs in terms of reducing increased numbers of tartrate-resistant acid phosphatase (TRAP) positive osteoclasts in the distal femur epiphysis of MRL/lpr mice [see Additional file 1, Figure S4A], elevated serum levels of sRANKL, a critical factor for osteoclastogenesis [see Additional file 1, Figure S4B] and bone resorption marker CTX [see Additional file 1, Figure S4C]. These data suggest that S-BMMSCs exhibit a superior therapeutic effect for SLE disorders compared to regular BMMSCs.

Recently, immunomodulatory properties were identified as an important stem cell characteristic of BMMSCs, leading to the utilization of systemic infused BMMSCs to treat a variety of immune diseases [19-21]. Here, we found that S-BMMSCs exhibited a significantly increased capacity for NO production compared to regular BMMSCs when treated with IFN and IL-1 (Figure 5A). It is known that NO plays a critical role in BMMSC-mediated immunosuppression [see Additional file 1, Figures S5A-F] [29]. Therefore, we assessed the functional role of high NO production in S-BMMSC-associated immunomodulatory properties. Spleen (SP) cells were activated by anti-CD3 and anti-CD28 antibodies for three days and then co-cultured with S-BMMSCs or regular BMMSCs in the presence of the general NOS inhibitor, L-NMMA or the iNOS inhibitor, 1400 W, using a Transwell culture system. The efficacy of L-NMMA and 1400 W to inhibit NO production in BMMSCs was verified [see Additional file 1, Figures S6A and 6B]. Although both S-BMMSCs and regular BMMSCs were capable of inhibiting cell viability of activated SP cells, S-BMMSCs showed a marked inhibition of SP cell viability over that of regular BMMSCs (Figure 5B). Moreover, both BMMSCs and S-BMMSCs induced SP cell apoptosis (Figure 5C). However, S-BMMSCs showed an elevated capacity in inducing activated SP cell apoptosis compared to regular BMMSCs (Figure 5C). Interestingly, when L-NMMA and 1400 W were added to the cultures, the number of apoptotic SP cells was significantly reduced in both S-BMMSC and regular BMMSC groups (Figure 5D and 5E). These in vitro experimental data suggested that NO production is an essential factor for BMMSC-mediated immunomodulation.

Figure 5. S-BMMSCs show up-regulated immunomodulatory properties through nitric oxide (NO) production. (A) NO levels in the supernatant of S-BMMSC and BMMSC culture were significantly higher in the INF-/IL-1 treated S-BMMSC group than in BMMSCs. (B-C) S-BMMSCs showed a significant reduction in the cell viability of activated SP cells compared to the cells cultured without BMMSCs (SP cell) and with BMMSCs (B). Both BMMSCs and S-BMMSCs showed a significantly increased rate of SP cell apoptosis compared to the SP cell only group but S-BMMSCs could induce higher SP cell apoptosis (C). (D-E) The induction of SP cell apoptosis by BMMSCs or S-BMMSCs was abolished in general NOS inhibitor L-NMMA-treated (D) and iNOS specific inhibitor 1400 W-treated (E) group. (F-H) Activated CD4+CD25- T-cells and S-BMMSCs or BMMSCs were co-cultured in the presence of TGF1 and IL-2 with or without NOS inhibitor for three days. The floating cells were stained for CD4+CD25+FoxP3+ regulatory T cells (Tregs). Both BMMSCs and S-BMMSC up-regulated Tregs but S-BMMSCs showed a significant effect in up-regulating Tregs. (F). Interestingly, L-NMMA and 1400 W treatments resulted in an abolishing of S-BMMSC-induced up-regulation of Tregs (G, H). (I) BMMSCs and S-BMMSCs could inhibit Th17 differentiation in vitro. S-BMMSC could inhibit it more effectively. (J, K) L-NMMA (J) or 1400 W (K) could abolish the inhibition of Th17 differentiation by BMMSCs or S-BMMSCs. The results are representative of at least three independent experiments. *P <0.05; **P <0.01; ***P <0.001. The graph bar represents mean SD. BMMSCs, bone marrow mesenchymal stem cells; iNOS, inducible nitric oxide synthase; L-NMMA, L-NG-monomethyl-arginine; NOS, nitric oxide synthase; S-BMMSCs, BMMSCs in suspension; SD, standard deviation; SP, spleen; Tregs, regulatory T cells.

Since up-regulation of CD4+CD25+Foxp3+ Tregs is required for immunotolerance [30], we tested Tregs up-regulation property of S-BMMSCs and BMMSCs in an in vitro co-culture system. When nave-T-cells were co-cultured with S-BMMSCs or regular BMMSCs in the presence of IL-2 and TGF-1, S-BMMSCs showed a significant up-regulation of Treg levels compared to regular BMMSCs (Figure 5F). Both L-NMMA and 1400 W were able to inhibit BMMSC- and S-BMMSC-induced up-regulation of Tregs, as shown by flow cytometric analysis (Figures 5G and 5H). Interestingly, the regulation effect on Tregs was more significant in the S-BMMSC group compared to the BMMSC group (Figure 5G and 5H). Moreover, both BMMSCs and S-BMMSCs could inhibit differentiation of Th17 in vitro, with a more prominent effect observed with S-BMMSC (Figure 5I). These inhibitions of Th17 differentiation were abolished by L-NMMA (Figure 5J) and 1400 W (Figure 5K). These data further verified the functional role of NO in S-BMMSC-induced immunomodulatory effect.

In order to identify whether there are functional endogenous S-BMMSCs, we used fluorescence activated cell sorting (FACS) to isolate CD34 and CD73 double-positive cells from bone marrow ANCs which resulted in the recovery of 3.77% double-positive cells [see Additional file 1, Figure S7A]. These CD34 and CD73 double-positive cells exhibited mesenchymal stem cell characteristics, including the capacity to form single colony clusters of fibroblast-like cells [see Additional file 1, Figure S7B], which could differentiate into osteogenic cells in vitro [see Additional file 1, Figure S7C]. These data indicated the feasibility of this approach to isolate S-BMMSC-like cells directly from bone marrow. We found that CD34+/CD73+ BMMSCs were analogous to S-BMMSCs in terms of having higher levels of NO production when compared to regular BMMSCs [see Additional file 1, Figure S7D] and reducing levels of urine protein, serum anti-dsDNA IgG and IgM antibodies in MRL/lpr mice (data not shown). These data indicate that endogenous S-BMMSCs could be isolated from bone marrow using CD34 and CD73 antibodies double sorting.

Additionally, we used the same BMMSC-ECM isolation approach to reveal the existence of human S-BMMSCs (hS-BMMSC) that possess stem cell properties including multipotent differentiation and self-renewal but lack expression of CD34 (data not shown). hS-BMMSCs showed elevated NO and kynurenine production which indicate high indoleamine 2,3-dioxygenase (IDO) activity when compared to regular BMMSCs [see Additional file 1, Figures S8A-C]. Thus, when activated T cells were co-cultured with hS-BMMSCs, AnnexinV-7 aminoactinomycinD (7AAD) double positive apoptotic SP cells were significantly elevated compared to BMMSCs [see Additional file 1, Figure S8D].

Adherent BMMSCs are able to proliferate and undergo osteogenic differentiation, providing the first evidence of CFU-F as precursors for osteoblastic lineage [25]. For over a few decades, the adherent CFU-F assay has been used as an effective approach to identify and select BMMSCs. In the current study, we showed that the adherent CFU-F assay collects the majority of clonogenic BMMSCs, but a subpopulation of BMMSCs is sustained in the culture suspension. This newly identified subpopulation of BMMSCs may be lost in the standard CFU-F assay for BMMSC isolation.

Due to the heterogeneity of the BMMSCs, there is no single, unique marker allowing for BMMSC isolation, rather an array of cell molecules are utilized to profile BMMSCs. It is widely accepted that BMMSCs express SH2 (CD105), SH3/SH4 (CD73), integrin 1 (CD29), CD44, Thy-1 (CD90), CD71, vascular cell adhesion molecule-1 (CD106), activated leukocyte cell adhesion molecule (CD166), STRO-1, GD2, and melanoma cell adhesion molecule (CD146) [5,7-13,31,32]. Nevertheless, it is believed that BMMSCs lack expression of hematopoietic surface molecules including CD34, integrin M (CD11b) and CD14. However, recent studies have implied that mouse BMMSCs might express the hematopoietic surface molecules, CD45 [28] and CD34 [33]. To ensure purity of S-BMMSCs, we used immune FACS to collect SSEA4+ S-BMMSCs for proliferation and differentiation assays in this study. Interestingly, previous experimental evidence appeared to support a notion that HSCs are capable of differentiating into mesenchymal cells [34] and osteoblastic lineage in vivo [35]. Thus, it is critical to clarify whether BMMSCs express hematopoietic associated surface molecules.

In this study, we have identified a novel subset of S-BMMSCs that failed to form adherent CFU-F in regular culture dishes, but were capable of adhering on mesenchymal stem cell-produced ECM and differentiating into osteoblasts, adipocytes and chondrocytes from both C3H/HeJ and C57BL/6J mice. S-BMMSCs co-expressed the HSC marker CD34 with the MSC markers CD73 and Oct4, excluding the potential of HSC contamination. Furthermore, S-BMMSCs were found to be distinct from HSC because they lacked the ability to differentiate into hematopoietic cell lineages in vitro and failed to rescue lethally-irradiated mice. The mechanism that may contribute to the up-regulated immunomodulatory function was associated with high NO production in S-BMMSCs and a NO-driven high Tregs level [36]. NO is a gaseous biological mediator with important roles in affecting T cell function [37].

This is the reason that S-BMMSCs showed a superior therapeutic effect in treating SLE mice.

One successful approach is to isolate cells that express specific molecules on their cell surfaces using monoclonal antibodies and cell sorting technologies. Enriched populations of BMMSCs have been isolated from human bone marrow aspirates using a STRO-1 monoclonal antibody in conjunction with antibodies against VCAM-1/CD106 [32], CD146 [11], low affinity nerve growth factor receptor/CD271, PDGR-R, EGF-R and IGF-1-R [38], fibroblast cell marker/D7-Fib [39] and integrin alpha 1/CD49a [40]. A more recent study has also identified molecules co-expressed by a CD271+ mesenchymal stem cell population including platelet derived growth factor receptor- (CD140b), human epidermal growth factor 2/ErbB2 (CD340) and frizzled-9 (CD349) [41]. Further cell separation based upon multi-parameter FACS identified a population of proposed mouse mesenchymal precursors with the composite phenotype Lin-CD45-CD31-Sca-1+[42]. Another recent study also identified and characterized an alternate population of primitive mesenchymal cells derived from adult mouse bone marrow, based upon their expression of the SSEA-1 [43]. All approaches used for BMMSC purification and isolation will undergo ex vivo expansion to enrich cell numbers for tissue regeneration or systemic therapies by plastic adherent assay. In addition to identifying a novel sub-population of BMMSCs that possess enhanced immunomodulatory properties when compared to regular BMMSCs, we showed that CD34+/CD73+ BMMSCs could be isolated directly from whole bone marrow and that CD34+/CD73+ BMMSCs are endogenous S-BMMSCs with higher NO production, and are superior in treating SLE-like mice when compared to regular BMMSCs.

Recently, non-adherent bone marrow cells (NA-BMCs) were identified [44,45]. The NA-BMSCs could be expanded in suspension and gave rise to multiple mesenchymal phenotypes, including osteoblasts, chondrocytes, and adipocytes in vitro, suggesting the presence of non-adherent BMMSCs in primary CFU-F cultures [45]. Although it has been reported that the NA-BMCs can rescue lethally-irradiated mouse recipients, our data indicated that S-BMMSCs only showed improved survival lifespan without a complete rescue of lethally-irradiated mice, compared to whole bone marrow transplantation. While the mechanism of S-BMMSC-mediated lifespan extension in lethally-irradiated mice is unknown, it is possible that S-BMMSCs have a more active interplay with hematopoietic cells than regular BMMSCs. It has been reported that granulocyte colony stimulating factor might promote BMMSCs into the circulation in humans [46], suggesting that non-attached BMMSCs may exist in vivo for specific functional needs. Added evidence indicated that osteocalcin-positive cells in circulation were able to differentiate into osteoblastic cells when cultured in the presence of TGF [47]. However, it is unknown whether S-BMMSCs are associated with circulating mesenchymal stem cells initially identified in mice, and this is very rare in humans.

A new subset of BMMSCs (S-BMMSCs) which failed to adhere to culture dishes possesses similar stem cell properties as those seen in BMMSCs, including CFU-F, stem cell markers, osto-, adipo-, and chondro-genic differentiation. However, S-BMMSC showed distinct features including expression of CD34 and a superior immunomodulation property through high NO production. These findings suggest that it is feasible to improve immunotherapy by identifying new subset BMMSCs.

7AAD: 7aminoactinomycineD; ALP: alkaline phosphatase; ANCs: all nucleated cells; BMMSCs: bone marrow mesenchymal stem cells; BrdU: bromodeoxyuridine; CFU-F: colony forming unit fibroblastic; CTX: C-terminal telopeptides of type I collagen; DAPI: 4', 6-diamidino-2-phenylindole; (D)MEM: (Dulbecco's) modified Eagle's medium; ECM: extracellular cell matrix; ELISA: enzyme-linked immunosorbent assay; EPO: erythropoietin; FACS: fluorescence-activated cell sorting; FBS: fetal bovine serums; FITC: fluorescein isothiocyanate; H & E: hematoxylin and eosin; HA/TCP: hydroxyapatite/tricalcium phosphate; HSC: hematopoietic stem cell; IDO: indoleamine 2,3-dioxygenase; IFN: interferon gamma; IgG: immunoglobulin G; IL-1: interleukin-1 beta; iNOS: inducible NOS; L-NMMA: L-NG-monomethyl-arginine; lpl: lipoprotein lipase; NF-B: nuclear factor-kappa B; NOS: nitric oxide synthase; PBMNCs: peripheral blood mononuclear cells; PBS: phosphate-buffered saline; PE: phycoerythrin; PFA: paraformaldehyde; ppar2: peroxisome proliferator-activated receptor gamma 2; RT-PCR: reverse transcriptase polymerase chain reaction; S-BMMSC: BMMSCs in suspension; SLE: systemic lupus erythematosus; SP: spleen; sRANKL: soluble runt-related NF-B ligand; SSEA: stage-specific embryonic antigen; TGF: transforming growth factor beta; Th17: T helper 17 cells; TRAP: tartrate-resistant acid phosphatase; Tregs: regulatory T cells.

The authors declare that they have no competing interests.

KA and YY: contributions to conception and design of experiments, acquisition of data, analysis and interpretation of data. TY, CC, LT, and YJ: contributions to acquisition of data, analysis and interpretation of data. XC and SG: contributions to drafting the manuscript and revising critically. SS: contributions to conception and design, drafting the manuscript, and giving final approval of the version to be published. All authors have read and approved the manuscript for publication.

We thank Dr. Tao Cai from NIH for discussions and critical reading of the manuscript. This work was supported by grants from the National Institute of Dental and Craniofacial Research, National Institutes of Health, Department of Health and Human Services (R01DE017449 and R01 DE019932 to S.S.).

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Characterization of bone marrow derived mesenchymal stem ...

People usually volunteer to donate stem cells for an allogeneic transplant either because they have a loved one or friend who needs a match or because they want to help people. Some people give their stem cells so they can get them back later for an autologous transplant.

People who want to donate stem cells or join a volunteer registry can speak with their doctors or contact the National Marrow Donor Program to find the nearest donor center. Potential donors are asked questions to make sure they are healthy enough to donate and dont pose a risk of infection to the recipient. For more information about donor eligibility guidelines, contact the National Marrow Donor Program or the donor center in your area (see the To learn more section for contact information).

A simple blood test is done to learn the potential donors HLA type. There may be a one-time, tax-deductible fee of about $75 to $100 for this test. People who join a volunteer donor registry will most likely have their tissue type kept on file until they reach age 60.

Pregnant women who want to donate their babys cord blood should make arrangements for it early in the pregnancy, at least before the third trimester. Donation is safe, free, and does not affect the birth process. For more, see the section called How umbilical cord blood is collected.

If a possible stem cell donor is a good match for a recipient, steps are taken to teach the donor about the transplant process and make sure he or she is making an informed decision. If a person decides to donate, a consent form must be signed after the risks of donating are fully discussed. The donor is not pressured take part. Its always a choice.

If a person decides to donate, a medical exam and blood tests will be done to make sure the donor is in good health.

This process is often called bone marrow harvest, and its done in an operating room. The donor is put under general anesthesia (given medicine to put them into a deep sleep so they dont feel pain) while bone marrow is taken. The marrow cells are taken from the back of the pelvic (hip) bone. A large needle is put through the skin and into the back of the hip bone. Its pushed through the bone to the center and the thick, liquid marrow is pulled out through the needle. This is repeated several times until enough marrow has been taken out (harvested). The amount taken depends on the donors weight. Often, about 10% of the donors marrow, or about 2 pints, are collected. This takes about 1 to 2 hours. The body will replace these cells within 4 to 6 weeks. If blood was taken from the donor before the marrow donation, its often given back to the donor at this time.

After the bone marrow is harvested, the donor is taken to the recovery room while the anesthesia wears off. The donor may then be taken to a hospital room and watched until fully alert and able to eat and drink. In most cases, the donor is free to leave the hospital within a few hours or by the next morning.

The donor may have soreness, bruising, and aching at the back of the hips and lower back for a few days. Over-the-counter acetaminophen (Tylenol) or non-steroidal anti-inflammatory drugs (such as aspirin, ibuprofen, or naproxen) are helpful. Some people may feel tired or weak, and have trouble walking for a few days. The donor might be told to take iron supplements until the number of red blood cells returns to normal. Most donors are back to their usual schedule in 2 to 3 days. But it could take 2 or 3 weeks before they feel completely back to normal.

There are few risks for donors and serious complications are rare. But bone marrow donation is a surgical procedure. Rare complications could include anesthesia reactions, infection, transfusion reactions (if a blood transfusion of someone elses blood is needed this doesnt happen if you get your own blood), or injury at the needle insertion sites. Problems such as sore throat or nausea may be caused by anesthesia.

Allogeneic stem cell donors do not have to pay for the harvesting because the recipients insurance company usually covers the cost.

Once the cells are collected, they are filtered through fine mesh screens. This prevents bone or fat particles from being given to the recipient. For an allogeneic or syngeneic transplant, the cells may be given to the recipient through a vein soon after they are harvested. Sometimes they are frozen, such as when the donor lives far away from the recipient.

For several days before starting the donation process, the donor is given a daily injection (shot) of filgrastim (Neupogen). This is a growth-factor drug that causes the bone marrow to make and release stem cells into the blood. Filgrastim can cause some side effects, the most common being bone pain and headaches. These may be helped by over-the-counter acetaminophen (Tylenol) or nonsteroidal anti-inflammatory drugs (like aspirin or ibuprofen). Nausea, sleeping problems, low-grade (mild) fevers, and tiredness are other possible effects. These go away once the injections are finished and collection is completed.

Blood is removed through a catheter (a thin, flexible plastic tube) that is put in a large vein in the arm or chest. Its then cycled through a machine that separates the stem cells from the other blood cells. The stem cells are kept while the rest of the blood is returned to the donor through the same catheter. This process is called apheresis (a-fur-REE-sis). It takes about 2 to 4 hours and is done as an outpatient procedure. Often the process needs to be repeated daily for a few days, until enough stem cells have been collected.

Possible side effects of the catheter can include trouble placing the catheter in the vein, a collapsed lung from catheter placement, blockage of the catheter, or infection of the catheter or at the area where it enters the vein. Blood clots are another possible side effect. During the apheresis procedure donors may have problems caused by low calcium levels from the anti-coagulant drug used to keep the blood from clotting in the machine. These can include feeling lightheaded or tingly, and having chills or muscle cramps. These go away after donation is complete, but may be treated by giving the donor calcium supplements.

The process of donating cells for yourself (autologous stem cell donation) is pretty much the same as when someone donates them for someone else (allogeneic donation). Its just that in autologous stem cell donation the donor is also the recipient, giving stem cells for his or her own use later on. For some people, there are a few differences. For instance, sometimes chemotherapy (chemo) is given before the filgrastim is used to tell the body to make stem cells. Also, sometimes it can be hard to get enough stem cells from a person with cancer. Even after several days of apheresis, there may not be enough for the transplant. This is more likely to be a problem if the patient has had certain kinds of chemo in the past, or if they have an illness that affects their bone marrow.

Sometimes a second drug called plerixafor (Mozobil) is used along with filgrastim in people with non-Hodgkin lymphoma or multiple myeloma. This boosts the stem cell numbers in the blood, and helps reduce the number of apheresis sessions needed to get enough stem cells. It may cause nausea, diarrhea, and sometimes, vomiting. There are medicines to help if these symptoms become a problem. Rarely the spleen can enlarge and even rupture. This can cause severe internal bleeding and requires emergency medical care. The patient should tell the doctor right away if they have any pain in their left shoulder or under their left rib cage which can be symptoms of this emergency.

Parents can donate their newborns cord blood to volunteer or public cord blood banks at no cost. This process does not pose any health risk to the infant. Cord blood transplants use blood that would otherwise be thrown away.

After the umbilical cord is clamped and cut, the placenta and umbilical cord are cleaned. The cord blood is put into a sterile container, mixed with a preservative, and frozen until needed.

Remember that if you want to donate or bank (save) your childs cord blood, you will need to arrange it before the baby is born. Some banks require you to set it up before the 28th week of pregnancy, although others accept later setups. Among other things, you will be asked to answer health questions and sign a consent form.

Many hospitals collect cord blood for donation, which makes it easier for parents to donate. For more about donating your newborns cord blood, call 1-800-MARROW2 (1-800-627-7692) or visit Be the Match.

Privately storing a babys cord blood for future use is not the same as donating cord blood. Its covered in the section called Other transplant issues.

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Whats it like to donate stem cells?

Surgeon performs bone marrow harvest

The terms "Hodgkin's Disease," "Hodgkin's Lymphoma," and "Hodgkin Lymphoma" are used interchangeably throughout this site.

Bone Marrow Transplants (BMT) and Peripheral Blood Stem Cell Transplants (PBSCT) are emerging as mainstream treatment for many cancers, including Hodgkin's Disease and Medium/High grade aggressive)Non-Hodgkin's lymphoma.

BMTs have been used to treat lymphoma for more than 10 years, but until recently they were used mostly within clinical trials. Now BMTs are being used in conjunction with high doses of chemotherapy as a mainstream treatment.

When high doses of chemotherapy are planned, which can destroy the patients bone marrow, physicians will typically remove marrow from the patients bone before treatment and freeze it. After chemotherapy, the marrow is thawed and injected into a vein to replace destroyed marrow. This type of transplant is called an autologous transplant. If the transplanted marrow is from another person, it is called an allogeneic transplant.

In PBSCTs, another type of autologous transplant, the patient's blood is passed through a machine that removes the stem cells the immature cells from which all blood cells develop. This procedure is called apheresis and usually takes three or four hours over one or more days. After treatment to kill any cancer cells, the stem cells are frozen until they are transplanted back to the patient. Studies have shown that PBSCTs result in shorter hospital stays and are safer and more cost effective than BMTs.

Read more here:
Bone Marrow and Stem Cell Transplants Lymphoma Info