Pregnancy & Prenatal Testing: Genetic Testing for Inherited …
There are hundreds of diseases that are related to changes in our genetic code, but most of them are extremely rare. or alterations in specific may prevent the genes from creating vital or cause alterations in the proteins that they produce. These changes can affect the way that the body functions and cause specific diseases. Some of the disease-related gene mutations are , while others are . Some are or sex-linked, associated with the X or Y that determines our sex, and are found only in males. Some mutations have arisen and been passed down in specific families, and some are more prevalent in individuals of certain ethnic descent.
Genetic testing is a personal choice. Blood tests for some of the more common genetic diseases may be performed on a woman and her partner before a pregnancy if they wish to know if they are . Many times, genetic testing is done first on the woman and only done on the partner if the woman is a carrier. Couples should talk to a genetic counselor about their ethnicity and family medical history to determine which tests are the most appropriate and to help them make an informed decision. For more information on genetics and genetic testing, see The Universe of Genetic Testing.
Individuals of Ashkinazi (East European) Jewish descent, for example, are at increased risk of carrying the genes for Tay-Sachs, Gaucher, Canavan disease, and familial dysautonomia. These genetic diseases can occur when both parents have an abnormal gene and their child inherits two copies of the abnormal gene, one from each parent. In both Tay-Sachs and Canavan diseases, there is a buildup of a substance in the childs brain that prevents normal development. There is no known cure for either disease. Children with Tay-Sachs rarely live past five years of age; children with Canavan disease may live to early adolescence. There are three types of Gaucher disease, each causing too much fatty substance to be stored in the bone marrow, spleen, and liver. Although one type of Gaucher disease is fatal, the most common type is not. Treatments are available for individuals with Gaucher disease. Familial dysautonomia is caused by incomplete development of nerve fibers in the autonomic and sensory nervous systems. There are a variety of symptoms (which range in severity), the most noticeable of which is the lack of tears during crying.
Links National Human Genome Research Institute: FAQ About Genetic Testing March of Dimes: Tay-Sachs and Sandhoff diseases Genetic and Rare Diseases Information Center
Excerpt from:
Pregnancy & Prenatal Testing: Genetic Testing for Inherited ...
Recommendation and review posted by simmons
Bone Marrow and Blood Stem Cell Transplants – City of Hope
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.
Read more from the original source:
Bone Marrow and Blood Stem Cell Transplants - City of Hope
Recommendation and review posted by Bethany Smith
Resident c-kit+ cells in the heart are not cardiac stem …
c-kit is expressed in the developing and adult mouse heart
We first generated a knock-in mouse model, c-kitH2B-tdTomato/+, by gene targeting (Fig. 1a and Supplementary Fig. 1). In this animal, the H2B-tdTomato cassette was inserted into the c-kit start codon without deleting any genomic sequences, thereby expressing tdTomato under the control of the full complement of endogenous c-kit regulatory elements. Since tdTomato is fused to histone H2B gene24, its expression is localized to the nucleus.
(a) Diagram of the c-kitH2B-tdTomato/+knock-in allele. (bi) Sections of c-kitH2B-tdTomato/+hearts at embryonic days (E) 8.5, 9.5, 12.5 and 14.5 (be) and at postnatal (P) days 1, 30, 60 and 120 (fi). c2, e2, g2 and i2 are high-magnification images (without DAPI) of the areas outlined in c1, e1, g1 and i1, respectively. c-kitH2B-tdTomato cells are denoted by arrows. LA, left atria; LV, left ventricle; OFT, outflow tract; RA, right atria; RV, right ventricle; VS, ventricular septum. n=3 for each stage. Scale bar, 100m.
To confirm the fidelity of the c-kitH2B-tdTomato signal to the endogenous c-kit expression pattern, we performed whole-mount RNA in situ hybridization on the wild-type mice from embryonic day (E) 9.5 to E13.5. By comparing c-kitH2B-tdTomato signals to c-kit mRNA expression, we found that the signals overlapped in all known regions of c-kit expression25, 26, such as the pharyngeal arches, liver, umbilical cord and melanocytes (Supplementary Fig. 2ac). Furthermore, H2B-tdTomato expression was detected in other organs, including the lung, stomach, intestine and spleen (Supplementary Fig. 2e), as well as the neural tube and yolk sac during embryogenesis. This finding is consistent with previous reports of c-kit expression in these organs25, 26. Immunostaining of sectioned c-kitH2B-tdTomato/+ mouse tissues revealed that the c-kitH2B-tdTomato-positive cells co-localized with c-kit antibody in the liver, lung and melanocytes (Supplementary Fig. 3). Further support for the sensitivity and fidelity of this reporter is the observation that cells with low c-kit expression detected by antibody exhibited bright H2B-tdTomato fluorescence (Supplementary Fig. 3b,c).
Next, we examined the location of c-kit+ cells in the hearts of c-kitH2B-tdTomato/+mice (Fig. 1). Endocardial cells with nuclear tdTomato expression were observed as early as E8.5 and 9.5 (Fig. 1b,c). Starting from E12.5, cells with strong c-kitH2B-tdTomato expression were dispersed throughout the heart, with the highest density in the inner layers of the atrial and ventricular chambers at all embryonic stages tested (Fig. 1d,e). At postnatal day (P) 1, P30, P60 and P120, c-kitH2B-tdTomatoexpressing cells were consistently detected in all chambers of the heart (Fig. 1fi). The broad distribution of c-kitH2B-tdTomato-positive cells in the heart from embryonic stages to adulthood is inconsistent with previous studies reporting that c-kit+ cells represent a small population of CSCs in the mammalian heart7, 12, 13, 14, 15, 27.
In the initial characterization of cardiac resident c-kit+ cells in the adult rat, c-kit+ cells were shown to contain a mixed population of cells exhibiting early stages of myogenic differentiation as demonstrated by the active expression of the early cardiac transcription factors Nkx2.5, Gata4 and Mef2c in the nucleus and of sarcomeric proteins in the cytoplasm of these cells7, 15. To determine whether c-kitH2B-tdTomato-positive cells express the cardiac progenitor marker Nkx2.5, we crossed Nkx2.5H2B-GFP/+knock-in mice28 with c-kitH2B-tdTomato/+mice to obtain compound heterozygotes (c-kitH2B-tdTomato/+;Nkx2.5H2B-GFP/+). H2BGFP expression in Nkx2.5H2B-GFP/+mice faithfully recapitulates the endogenous Nkx2.5 pattern28. We examined cardiac tissues throughout the embryonic (E9.518.5) and postnatal (P1120) stages (Supplementary Fig. 4). All histological sections from E9.5 to 13.5 hearts and more than 30 sections from E14.5 to P120 hearts were inspected (n=3 for each stage). However, no c-kitH2B-tdTomato and Nkx2.5H2B-GFP double-positive cells were found (Supplementary Fig. 4b,dg), except at E12.5, wherein only 11 double-positive cells were detected in the ventricular septum (Supplementary Fig. 4c, ~0.007% of total Nkx2.5H2B-GFP-positive cells).
To determine whether any c-kit+ cells produce sarcomeric or myocardial proteins7, 15, we applied a cTnTH2B-GFP/+ knock-in mouse model with insertion of an H2BGFP cassette into the start codon of cTnT (Tnnt2; Supplementary Fig. 5a). On examining heart sections from c-kitH2B-tdTomato/+;cTnTH2B-GFP/+ compound heterozygous animals at embryonic and postnatal stages (E8.5P120), we did not detect any cells in which both markers were co-localized (Supplementary Fig. 5), with the exception of E13.5, where an average of 15 double-positive cells were found within the ventricular septum (Supplementary Fig. 5d, ~0.009% of total cTnTH2B-GFP-positive cells). These observations reveal that c-kit+ cells in c-kitH2B-tdTomato/+mice very rarely co-express either Nkx2.5 or cTnT in the embryonic heart and do not co-express these markers in foetal or adult hearts.
To further determine the identity of c-kit+ cells, we performed immunostaining with antibodies against the endothelial marker PECAM (CD31) and the smooth muscle marker, -SMA. Surprisingly, at all the stages examined (E8.5P120), c-kitH2B-tdTomato-positive cells were PECAM+(Fig. 2a-f) but -SMA (Fig. 2g,h). This finding suggests that cardiac c-kitH2B-tdTomato-positive cells are endothelial cells. Quantitative flow cytometric analysis of 4-month-old hearts demonstrated that ~43% PECAM+ cells in the ventricles were also c-kit+ (Supplementary Fig. 6). Thus, our results indicate that c-kitH2B-tdTomato-positive cells represent a subset of cardiac endothelial cells.
(a,b) At E8.5 and E9.5, c-kitH2B-tdTomato cells are endocardial (PECAM+). (cf) c-kitH2B-tdTomato cells express PECAM at E16.5 (c) and at P1120 (df). Arrows indicate PECAM+ and tdTomato+ double-positive cells. Arrowheads indicate PECAM+ and tdTomato cells. (g,h) Cardiac smooth muscle cells (-SMA+) are tdTomato at P120 (arrowheads). a2h2 are high-magnification images of the areas outlined in a1h1 (without DAPI), respectively. n=3 for each stage. Scale bar, 100m.
tdTomato is a bright fluorescent protein29, 30. We were concerned that the long stability of tdTomato could complicate the detection of transient c-kit expression. To confirm the identity of c-kit+ cells identified by c-kitH2B-tdTomato/+, we generated another reporter line, c-kitnlacZ-H2B-GFP/+, by inserting a LoxP-nlacZ-4XPolyA-LoxP-H2BGFP cassette into the c-kit start codon (Fig. 3a and Supplementary Fig. 7). H2BGFP is not detected in this line unless the nlacZ-4XPolyA stop cassette is removed by Cre-mediated recombination. We performed whole-mount X-gal staining on c-kitnlacZ-H2B-GFP/+ embryos and found that the c-kitnlacZ signal was not only reliably recapitulated by c-kit mRNA expression, but also consistent with the H2BtdTomato expression patterns in c-kitH2B-tdTomato/+mice (Supplementary Fig. 2). Furthermore, X-gal staining of whole-mount and sectioned hearts at E15.5P90 readily detected a broad distribution of c-kitnlacZ-positive cells throughout the heart (Fig. 3b,d,f,h, and j), including the endocardium (Fig. 3b,h), similar to the pattern observed in c-kitH2B-tdTomato/+mice. X-gal staining of compound heterozygous littermate hearts bearing an endothelial-specific Tie2-Cre allele (c-kitnlacZ-H2B-GFP/+;Tie2Cre) could not detect c-kitnlacZ-positive cells (Fig. 3c,e,g,i and k; less than 10 randomly distributed c-kitnlacZ-positive cells were found in the adult heart, representing ~0.0002% of total c-kit+ cells). Consistent with the endothelial nature of c-kit+ cells in the heart, c-kitH2B-GFP-positive cells generated by Tie2Cre excision were all co-stained with anti-PECAM antibody (Supplementary Fig. 8). Thus, the c-kitnlacZ-H2B-GFP/+ reporter line confirms the endothelial identity of cardiac c-kit+ cells.
(a) Diagram of the c-kitnlacZ-H2B-GFP/+reporter allele (a1). The c-kitH2B-GFP/+ allele is generated when the nlacZ cassette is removed by Cre excision (a2). (bk) X-gal staining of c-kitnlacZ-H2B-GFP/+ and c-kitnlacZ-H2B-GFP/+;Tie2Cre littermate hearts at E15.5 (b,c, sections) and at P190 (dk). Arrows indicate comparable regions to X-gal+ or X-gal staining. Arrowheads indicate rare X-gal+ cells on c-kitnlacZ-H2B-GFP/+;Tie2Cre hearts, suggesting that most c-kit+ cells lose the nlacZ gene because they are in the Tie2Cre lineage. f2k2 are high-magnification images of the areas outlined in f1k1, respectively. n=35 for each stage. Scale bar, 400m (black) and 200m (white).
To further address the issue of stability of both H2BtdTomato and nlacZ proteins, we analysed cardiac c-kit cells with the third reporter allele c-kitMerCreMer/+, in which an inducible MerCreMer cassette was inserted into the c-kit start codon (Fig. 4a and Supplementary Fig. 9). c-kitMerCreMer/+;ROSA26RtdTomato/+mice were subsequently generated by crossing with ROSA26RtdTomato/+ mice. In the absence of tamoxifen treatment, no tdTomato-expressing cells were detected in the adult hearts. To confirm whether c-kit is actively expressed in the postnatal heart, we injected tamoxifen at P30, P60 or P90 for 3 consecutive days (days 1, 2 and 3), and immediately collected cardiac tissues for analysis at day 4 (P3034, P6064) or 14 (P90104). This treatment consistently resulted in tdTomato labelling of a large number of cells in the heart (Fig. 4b,d,e) that also expressed PECAM (Fig. 4c). This result further confirms that cardiac c-kit+ cells are endothelial (Figs 2 and 3), and supports the previous observation that cardiac c-kit+ cell progeny are endothelial19.
(a) Diagram of the c-kitMerCreMer/+ allele. c-kitMerCreMer/+ animals were crossed to the ROSA26RtdTomato reporter line to obtain c-kitMerCreMer/+;ROSA26RtdTomato/+. (be) Cre activity was transiently induced in c-kitMerCreMer/+;ROSA26RtdTomato/+ animals at P30, P60 and P90 by tamoxifen injection on days 13. Hearts were harvested on days 4 and 14. Many tdTomato+ cells (arrows in b2, d2 and e2) were detected in hearts at P34 (b1), P64 (d1) and P104 (e1). These tdTomato+ cells were PECAM+ (c2, arrows, P3034). b2, d2 and e2 are high-magnification florescent images of the areas outlined in b1, d1 and e1 (bright field), respectively. (f) Diagram of the cTnTnlacZ-H2B-GFP/+allele and lineage tracing using c-kitMerCreMer/+;cTnTnlacZ-H2B-GFP/+mice. Cre activity was transiently induced by tamoxifen injection for 4 days on days 1, 2, 3 and 5 (days 1 and 2 for E11.5). Samples were collected on day 7 (day 3 for E11.5). (g) cTnTH2B-GFP cells were detected at E13.5, P37, P67 and P97 (arrows), with the total number in the whole heart noted at the upper right corner. Scale bar, 1 mm (black) and 100m (white).
c-kitH2B-tdTomato/+, c-kitnlacZ-H2B-GFP/+ and c-kitMerCreMer/+ animals are heterozygous null for c-kit (c-kit+/). Haploinsufficiency of c-kit could affect c-kit regulation in vivo20, 31, 32, 33, possibly leading to ectopic cardiac expression. To determine whether ectopic c-kit expression occurs in the reporter mouse hearts, we performed immunostaining at embryonic (E11.515.5) and postnatal stages (P160) using c-kit antibody on mice of four different genotypes: wild type, c-kitH2B-tdTomato/+ (c-kit+/), c-kitH2B-tdTomato/MerCreMer(c-kit/) and c-kitMerCreMer/MerCreMer(c-kit/). Using c-kit antibody, we frequently detected cells in wild-type hearts that were dually labelled with c-kit and PECAM (Supplementary Fig. 10a4,d4,g2 and Supplementary Fig. 11a,f,h,i). In c-kitH2B-tdTomato/+ animals, c-kit antibody immunoreactivity co-localized with c-kitH2B-tdTomato (Supplementary Fig. 10b2, e2,h2 and Supplementary Fig. 11b,c), although the immunofluorescence was decreased compared with that in wild-type animals. Reduced c-kit immunoreactivity in c-kitH2B-tdTomato/+ tissues is consistent with the c-kit+/ genetic background (theoretically 50% c-kit protein reduction in c-kit+/). Importantly, c-kit antibody staining was completely undetectable in c-kit/mutant hearts or lungs, even with Tyramide Signal Amplification (TSA) amplification (Supplementary Figs 10c,f and 11d,e), demonstrating the specificity of the antibody staining. Therefore, immunostaining with c-kit antibody also reveals that cardiac c-kit+ cells are endothelial and indicates that no ectopic cardiac c-kit expression occurs in the new knock-in mouse models employed.
To further determine the myogenic potential of c-kit+ cells during heart formation, we applied cTnTnlacZ-H2B-GFP/+ cardiomyocyte-specific reporter mice with the LoxP-nlacZ-4XPolyA-LoxP-H2B-GFP cassette targeted into cTnT start codon. cTnTH2B-GFP expression is detected in cardiomyocytes when Cre is expressed in the myocardium or myogenic precursor cells (Fig. 4f). We crossed c-kitMerCreMer/+ mice with cTnTnlacZ-H2B-GFP/+mice and injected tamoxifen in c-kitMerCreMer/+;cTnTnlacZ-H2B-GFP/+ animals. After two doses of tamoxifen administration (days 1 and 2) to pregnant mice (E11.5 embryos) or four doses (days 1, 2, 3 and 5) to P30, P60 and P90 mice, we collected hearts for analysis at E13.5 or at P37, P67 and P97, respectively. All cardiac sections were assessed for cTnTH2B-GFP-positive cells. On average, approximately 50, 324, 156 and 66 cells were found in hearts (n=3 for each group) at E13.5, P37, P67 and P97, respectively (Fig. 4g), representing <0.04% of cardiomyocytes at corresponding stages (<0.007% after P90). This finding demonstrates that the myogenic potential of c-kit+ cells, if any, is extremely low in both embryonic and postnatal hearts.
Previous studies have reported that within 4 weeks of myocardial infarction in adult mouse hearts, the number of c-kit/Nkx2.5 double-positive myogenic precursors significantly increased in the injured region, and some of these myogenic precursors transformed into proliferative cardiomyocytes7, 15. To directly investigate the differentiation potential of cardiac c-kit+ cells post myocardial infarction, we ligated the left anterior descending (LAD) coronary artery of c-kitH2B-tdTomato/+;Nkx2.5H2B-GFP/+ mice (25 months old, n=12, Fig. 5a,b). Examination of cardiac sections at 1, 3, 7, 21, 30 and 60 days post-surgery (dps) revealed many c-kitH2B-tdTomato-positive cells in the infarcted region (Fig. 5cf). However, no c-kitH2B-tdTomato and Nkx2.5H2B-GFP double-positive cells were found in the injured area at any stage tested (Fig. 5c1f1). To further determine the cell identity of these c-kit+ cells, we performed LAD ligation on Tie2Cre;c-kitnlacZ-H2B-GFP/+ mice (24 months old, n=3). c-kitH2B-GFP-positive cells were readily detected in the infarcted region, demonstrating that they retained their endothelial nature after injury (Fig. 6a).
(a) Diagram of LAD ligation. (b) Masson trichrome staining shows the infarcted region of a c-kitH2B-tdTomato/+;Nkx2.5H2B-GFP/+heart at 60 days post-surgery (dps). b1 and b2 are high-magnification images of the numbered outlined areas in b. (cf) No c-kitH2B-tdTomato/Nkx2.5H2B-GFP double-positive cells were found in the infarcted regions at 3 (c), 21 (d), 30 (e) and 60dps (f). c1/c2, d1/d2, e1/e2, and f1/f2 are high-magnification images of the numbered outlined areas in c, d, e and f, respectively. Scale bar, 500m (black) and 50m (white).
(a) c-kitH2B-GFP-positive cells were present in the infarcted region of Tie2Cre;c-kitnlacZ-H2B-GFP/+ hearts at 30dps. a2 is green channel of a1, and a3 is high-magnification image of the area outlined in a2. (b) Masson trichrome staining of cTnTMerCreMer/+;c-kitnlacZ-H2B-GFP/+;ROSA26RtdTomato/+ hearts at 60dps shows the infarcted region. (c) Adjacent section of b. ROSA26RtdTomato signal indicates myocardial cells after tamoxifen induction (c1). No c-kitH2B-GFP cells were observed in the infarcted zone (arrows). c2 is green channel of c1. (d) Masson trichrome staining of c-kitMerCreMer/+;cTnTnlacZ-H2B-GFP/+ hearts at 60dps. (e) Adjacent section of d shows a few cTnTH2B-GFP cells (<20) that were found in the infarcted zone (e1, arrowhead). cTnTH2B-GFP cells were also present in a remote, uninjured region (e2, arrowhead). Scale bar, 100m.
A recent study reported that a subpopulation of endothelial cells yields progeny with CSC characteristics in the adult mouse heart34. This subpopulation purportedly arises from endothelialmesenchymal transition and gives rise to cardiomyocytes that contribute to heart renewal34. To determine whether c-kit+ endothelial cells produce CSCs that further differentiate into cardiomyocytes following cardiac injury, we performed LAD ligation on cTnTMerCreMer/+;c-kitnlacZ-H2B-GFP/+;ROSA26RtdTomato/+ mice (24 months old, n=4, Fig. 6b). cTnTMerCreMer/+ mediates specific and effective myocardial recombination after tamoxifen induction35. If c-kitnlacZ-H2B-GFP/+ cells become cardiomyocytes and if c-kit expression is maintained in these cells, then c-kitH2B-GFP-positive cells would be detected. However, after tamoxifen was injected at 37dps and 3135dps (three tamoxifen treatments for each period), we detected no c-kitH2B-GFP-positive cells in the infarcted region (Fig. 6c), although myocardial recombination was widely detected in and adjacent to the infarcted region (as revealed by ROSA26RtdTomato staining, Fig. 6c). Furthermore, examination of adult c-kitMerCreMer/+;cTnTnlacZ-H2B-GFP/+ mice after LAD ligation (35 months old, n=3, Fig. 6d) revealed <20 cTnTH2B-GFP-positive cells per heart (~0.002% of total myocardial cells) throughout the injured region (Fig. 6e). cTnTH2B-GFP-positive cells could also be detected in remote uninjured regions (~30 cells, ~0.003% of total myocardial cells, Fig. 6e), suggesting that the cTnTH2B-GFP-positive cells found in the injured region are likely not a response to cardiac injury. These cardiac injury mouse models revealed that the myocardial potential of c-kit+ endothelial cells, if any, is extremely low. However, these data do not preclude the possibility that c-kit cardiac endothelial cells may have the potential for endothelialmesenchymal transition and myocardial differentiation.
In the lineage tracing experiments used to determine the myocardial potential of c-kit+ cells during development and after cardiac injury in c-kitMerCreMer/+;cTnTnlacZ-H2B-GFP/+ animal models, very small number of cTnTH2B-GFP-positive cells was detected (Fig. 4g, ~66156 cells; and Fig. 6e, ~20 cells). In all cases, the number was extremely low when compared with the total number of c-kitH2B-tdTomato-positive cells (<0.005%) or myocardial cells (<0.015%) in whole hearts. The origin of these rare cells is unknown. These cells may be derived from uncommitted cells originally expressing c-kit, or they could be cardiomyocytes that express both c-kit and cTnT due to a rare stochastic event. To explore these possibilities, we examined cTnTMerCreMer/+;c-kitnlacZ-H2B-GFP/+ adult mouse hearts (24 months old, uninjured) after tamoxifen injection for 2 consecutive days (days 1 and 2). At days 3, 7 and 30, we detected ~2030 c-kitH2B-GFP-positive cells per heart after examining all the heart sections (n=3, Supplementary Fig. 12). This result suggests that a very small number of resident c-kit cells are cardiomyocytes (~0.005% of total c-kit+ cells and ~0.002% of total myocardial cells in the heart). Notably, the number of c-kitH2B-GFP-positive cells detected in cTnTMerCreMer/+;c-kitnlacZ-H2B-GFP/+ hearts (~2030, Supplementary Fig. 12) is less than the number of cTnTH2B-GFP-positive cells in c-kitMerCreMer/+;cTnTnlacZ-H2B-GFP/+ hearts (~66156, Fig. 4g3). This is probably due to much higher levels of cTnT expression than c-kit expression and/or to differential sensitivity of the reporters to Cre-mediated recombination.
View post:
Resident c-kit+ cells in the heart are not cardiac stem ...
Recommendation and review posted by Bethany Smith
Mobilization of hematopoietic stem cells from the bone …
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.
Read the original:
Mobilization of hematopoietic stem cells from the bone ...
Recommendation and review posted by Bethany Smith
Comparison Between Bone Marrow or Peripheral Blood Stem …
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.
Link:
Comparison Between Bone Marrow or Peripheral Blood Stem ...
Recommendation and review posted by Bethany Smith
STEM CELL THERAPY FOR ATHLETES – Mississippi Sports Law Review
vp`Dx #Bc+RG? V&yP-ir endstream endobj 5 0 obj 3609 endobj 2 0 obj > endobj 6 0 obj > /Font > >> endobj 12 0 obj > stream xwTS7" %z ;HQIP&vDF)VdTG"cEb PQDEk 5Yg}PtX4XXffGD=H.d,P&s"7C$ E63p H.Hi@A> A1vjpzN6pW pG@ K0iABZyCAP8C@&*CP=#t] 4}a ;GDxJ>,_@FXDBX$!k"EHqaYbVabJ0cVL6f3bX'?v 6-V``[a;p~2n5 &x*sb|! ' Zk! $l$T4QOt"yb)AI&NI$R$)TIj"]&=&!:dGrY@^O$ _%?P(&OJEBN9J@y@yCR nXZOD}J}/G3k{%Ow_.'_!JQ@SVF=IEbbbb5Q%O@%!ByM:e0G7 e%e[(R0`3R46i^)*n*|"fLUomO0j&jajj.w_4zj=U45n4hZZZ^0Tf%9->=cXgN].[7ASwBOK/X/_Q>QG[ `Aaac#*Z;8cq>[&IIMST`kh&45YYF9:>>>v}/avO8 FV>2 u/_$BCv{$vrK .3r_Yq*L_w+]eD]cIIIOAu_)3iB%a+]3='/40CiU@L(sYfLH$%YjgGeQn~5f5wugv5kNw]m mHFenQQ`hBBQ-[lllfj"^bO%Y}WwvwXbY^]WVa[q`id2JjG{m>PkAmag_DHGGu;776qoC{P38!9^rUg9];}}_~imp}]/}.{^=}^?z8hc' O*?f`gC/O+FFGGz)~wgbk?J9mdwi?cOO?w| x&mf endstream endobj 13 0 obj 2612 endobj 7 0 obj [ /ICCBased 12 0 R ] endobj 15 0 obj > stream x)p3swwey9/OCQ{g&@3Ggr_e_@f>&!?|?Kks|a]/7M Z{V@ r5W(npX&6n}yl>v&f]%Fsl ~eX"d77u]YD#1yCP60dy8u.X6Ojoxcyfj ;_sAVo #mi#I`1qmr=C$atw -w~SSL2[$/.n0Dn:1[I)sre4JM 4u{K W@qy#*21c.eJ M,wo:x(SOvQC2IK"h (2"'ANB.TjlX13-*Tg%7/7]>LE0uhc >BU0:5n:e#$~zfKt-%qAbr/&4=o|/1uq]l,mm"SH dBS])[*'k7W#,Wk8"bG C(4GKqgcPPtW.XDR;@pD0;TNc~nB!1uO6,"fAi,u+' #_w bC!m&&m0yRled~|hN>RXcl*4es1uUcyF]C$@VED]4^o!obxoqu}d Imw{@d?)'B&%iNo]V#ptcCHB7,U>:)nXumbPm?a#t4[O.wB.xo QoX}/hJI?>nAKB@Kb%1@yLFr$naXr {6N$H2*-qx|b -Sk_OV^.#Am,rz
!#h69T'BP(#FNx` MBd`70`8|yS a AAwFvUITX]NZXzzXvl!5"Tg?#0Oz>]F9|3 4xSL?8AGk@t9qb:cq2oPl53tgL*w=NkLEl6k_L^Hemvp~ a>U]4P/)BBGjwk%5/vg@7x?B7f]X@m!}O9*|G"?RC!PdoQUg ;$wik7;,9${sNhDB:8~~?Zy)tM{|.};B l )F2]CW #>E`LhOD ] m69zlV8hK3t#N.W} @EJZsVie_t4T*;^*|GGh!0w2/E5LUV{H.5:`"KP^K;bp)N]3_J>q>O!fmU&O^B+]#hj8w;gju~Rdh;
nX0]X TVyFxq~1^|F =yvD(0J+c*^;1CUUxM~9tfN9 S>"0Pc8 GhyZMxhA%;1ne !x-uQ6AOOk&F0SG H^U)E1`x?eGfI KaqYU+S|)*1E/9A6c%YpT0uj=T&x|"2&1&W|PO1{M7iX`,XV{D%zPSG?htfQFzmr$'%CnyQ~wL+d2XlNN/[~KE}**Qpts^dP(vYQz%8 $A#/!+2#v=F]_3Hen=ONSu&)[|vmo`/y"_.|!h>!N>2fs-re|Z>*(aPmn& c`W 2C{Biv9d3F@s |KNA 34"J,j*hD8HQ MSl[$B[; :DboPR^'{rU%YA}Xdb|CNp&+m1) Qv`3=/Y!2f`;i#@ePg'7[$-.VV=k+T gDv]mZb^rb){JF_x^D|*-|EJX0m(F a's%eZ{XtD';v!rf?lUA];Ou`nM(:Ush'E:R^z^vl*],n&[Tr)9z9^XTXIZ{8?0%7JA;}-dj#)JCO#&|:XM>0Ub$_8#~/y'&F^(B#TTkTz-fbVJrj[ ;J%*:YpHwvyD{x]QgnSY1tAt`I) SzON`iv7>"yC5q[_rp5:ZN#gQPEqV+gu*F='0,j]>#V"Itr|1j09#?#+8Q 8+)7>0 gT^UzYHm$}W|CjKV2Nbq3^3be`lAMp]?Y&>Kwe BAMyNm3xpX:R@D 1Q1Q K]he]uy5iWG'x_M-@tIU8hO]p=@5=Genpq's!Vi.!lyIQvdnLtx!7 j }pW-JRj rQ):H"St8/SdZ7&,PUW7r*#!dcO}dN=B D-a^6e ^v',"pd/!!Uc?C)sqzu TD& f?`;!A!DE[ tH$|dN L.:t,HILN~C._IY= W[@?fU=?$xgMuU^z.-Y? 5eMLS/;!oV=5Vf8t|*YP]cAS/oq"m_r`;A> S}v endstream endobj 20 0 obj 6929 endobj 18 0 obj > endobj 21 0 obj > /Font > >> endobj 23 0 obj > stream xm*!#svpI~FQG~wmtW__}rVwUe@Swvx~sCm/7o_}MV7_UvlW7]Wc?]XGc{f~(KN_W%l?VHy[wFuT@P}f~-|%WXiWmwK36r~cCV4N?Q`|RG!@lUL:K&O|~h}|ua@fj)XXx Fw*cg7 9{P>4 QM!f.OdA`R"Sw*uz7/xL _rI JI"M $$0>d}, vq1d7-?bTZaJgX ~y5aL_lpe8[tC'W`^b2 Z=&1}'nuZ3_0o`4sQNgh6B&lxG!Qi C)naBomm `#A_ZjCCaC4k{X6+qh` 8y-2sL}'_p>a}|.K_Bm,s]hW7H;gZhXd#h :aHp-DR}:u"gLCH8EB;Jfcv^8! Eo#cUj{"P"7Q;CM1b6u3X=w=tYs gn0nb$@/TqbkI!&3=1P7Rf {UN0(2}D"$d&HQ.z}ssLqq&2g=6Kqv_i2_~) hH K Gj]-c%a#vc5N-bR_kKuS3t=;~%`T_S!gwb9 j *2lG+Q1; kW]@dZfYX_2Gtjs?fP9s(8u; 91(N}O}1@vHNRGoe/m~a*nykN!!#_K=Z1#a8,8d9'qVDCqDzp6$buR6(&l?QMm_>P5i&,[mhz@^Tdlj:9L`Z7$L"}l.gj:Gnk #&VsKB0t%R'i1&5exj[xdPnbSL1,:0i_V&Zw+HQm0>l?r>!.8J8:l[JoO36CdO: xFI&Sy2RSa6V;h `QpNq2a#pq06'-0:$#x&du3_i"%>hl_~;BXH]x?)6|/)2K/Of3I-x[I|OF0f~!Cq `OqDKh q:=AbKT[vf&!.!izG.ba+eZee(.-Kml7(3H $7
sQF !-#'ggmMB~aP?E7(AjMI S}Mji@X1 cV =;eI_ c[u0Q?pTTIMq=*//}uvUU)ODq('Eog $Y6ym`C>~HNf8C=o{CS` Qr}}~"+]m7bl0{P2dk1)_7&=wjx>.c''lj"%p_b{I0C>6r]jX&JlW,Qw,B{P5#+xu20c1"b:3=}9Sbe$w CIotu|r KDZ/7ubQX6n)]h}KV/`W(xzmt|p^dD9=b'u_IS}Om+le.Ay]CWrGi%,__;JKu)~A[FG>@wjYk}vm5% MA5v.ZN`7[k(G~T.Ej 5 !Bvf3y:C ?_/,({a[H/f)r.!eP5GR6kfPKEW,^JK~!2u /m}VibU?&)u2hEK#ZB(*isL5P.4n[uc -cucRLh Gfh^xiK[rA| =s/9,{v[~hJZ@,p,cjf*OrR)$UNSM@I?` :ov9FTv2cm9;lg*]|xu;Zf]:N:kR gZuK/m]o].3)d,Tr PYI4x(+RE$ '{>u9^zjVH(Di>X^5ev "p/; ZR9KG2*Y0vE,>rH"o[dejE [i+f')FK]G4psu%:uLa]:>jsqqO@%6 endstream endobj 24 0 obj 7495 endobj 22 0 obj > endobj 25 0 obj > /Font > >> endobj 27 0 obj > stream xIsuX6Dsw/_ee~,&LJ>fsm4&2@Z/Xlve7T{+XnCOMyT[4}_j{_ l`nySn~*oQ2mvs0exmfDm-P'jn^}*rsUUy|rnR;Oy{1Z'{U%Ho P| ~ )eOWo"B=j~TVjWc2 pPL-e}A,fzQ(:lM[f {B>CAQ EhtXOZQ*r'xO@Mr/G{Ks"P9 X8@G_D$^1>gv}W c/?2 ,gy oaA5H1d.zDmUG^~b/@|QVql /c3q4}Gsw0tS :BMU > nIPd:6eS0b['QVn>)| 7]9)#M 42, xTPlp7YXbE}*{*+lex-RQRjf+Rw8%"w:5G):I%BVmjsAa x> &kkU J)l2!+MBR@e#/#%>qm `3y`N:F_|+B T0yW&,}y8l}uEGX QOI#BID2rh1`);`"kS>cQu|#DR-MLjqJj|tFdNc~H>C$?wI}5US%zseD-fP.IoTZ$i+3 9I*!) 4QFEwOXGRc1 ,`M@
TTnGRoYcl-U5Gpg#m.x| m k"?qH?ED6^#d ID#)R[BToWv>.BdZoQ5 Y5|`db@~07MK/94.ol drle'eE1 q4S|HXXVrb Js51AmB.CFxAJ-w.F{p3,o,jw.re/Yq-' s%brP=IE}4Ib[ i/lH[`}$&(ebD)j/ UJS*:0, '0i#hD8ZP 4(+l lJbFM/[>EqwI bmsn1QlfH( Xak|359yJ=B_{40=x A(A)gl^J>LX -&D5'@Xy,Kv~VK,[mPb,o&~A8KCrL"JlbvsVC9gYYk'sI7s%gA|edb+BZHH6&{Sluq=y_F9,3dRFX9-{9hAVu[0{sYAm;).X%s2mKKvv%29t=SM&ZyQ1O:7K?Sd&0RjCG,@acLPhB')*;d=gk,7_q-.(Pv& LD+VSP?dY@B.G=[>h[v0'kG/,l@qdh+Q`F@6 M cT`m>aZX/4Q$0l f) rn}3gK65G/E}*-+Y/n?g Cz8)$@"e[S2 ag}Ab1Oo't#fyk1PU:7'gnUb2_I!FYRcYkF;U2Fw"b[0(lY|70r.^c~1>,17rEc)"^kAASyn: "3Cj4>r=+ 0)tkzB:"jXk(XJzTaCB*+1]Zl,&L2wrl+wN $h6TY5q+Z"/ g?%cU;g _I[9u_#UHou#g,#psl4Oz"0kqs %e5 t1 gOAB"e@9n ,lSXDCVV>wz/*OB>1IC*] ;Q$B#i: ccE0"IvU@I8$&xm,'7`vO,oh9Q!E2@gA[9VnK;4u9I#9[GgW:5y}8F8X'q UuZ{^W{n$o`#dP%ASMw F8b z}~vH7@@/]mBeWj&:VjG~gN = OBB4DA/|a6 -L7 K-()6D4y^Fr8}qpLL {I01"Xf2T}aIr4^5b=8l/Yq ]^HL z4qV>A' "Dus#2O:?db:rC?~";Yy-7fN0t59=: l$x~*TU{9XiWIeI4qXY~U/m^,v;VSC_z;TDprivX11./'v.xfX$ME`G`Fh`-`~v"N2%)p%-)pnT[hQ[bQ;h|ejG+R_ZXjiK`ch=; ) `ogZLZW.7QnR)t;S)Lzy]2be[$]Ap-w{~O!`V2!GA1rK3jo KL#|{T !T9uOP pViJC!+g}s- 84{,ftLe?0[lYVhhX}5Iu4!A/r}V4WI%!/wa L8sO q:e I-6T+xmvQEey9bHHCt/Kb6e> endobj 29 0 obj > /Font > >> endobj 31 0 obj > stream x)pYAKuE$%)i49>JT5%IF"[.UWnOw:MkwO~T/^4}_]kvCu_wUS^mQ}|otL)U28;qsj/wet?{uyINLKZj3@x$zgx; kjgUf_3aH# _fC= ^$O]TMN*HeI]| ~]W!~#TzS4TxVO$x[B}h}}>U m@pmu])9Qm9wF]u*R Qn.C_^ m{2b6lnUiGGL)X}tWvD}7;L54l[aYSovjWa%rA4dYte1GlOJb3y[tI'/8haQ,.C=5,]`qL5*L,HXHLYR&&F)mm"Z###,|9]J?:K23mf6-6M(wE0A84/G -cTnR-m_4C $ys2%;("W,@$}4havb;6&9JBP^Qn LzfD0v]W sI7KVyHw3`[aAzf+{"08w2cxgFm:Y+3Nv5M"GDAIa+{ [Fp%YTa0V^8hz8.n9'eSB8f>,&l++!C}~_c_:sUbE X,rWPw# hK+SYg|m[PYSbGrXWCeqA(eiIMGs[*(Cz _tURWCPm@% EG))l`!S =YJN$D[5aJSwYU_NB @eC,l(pREt8 FY{Br)#Y-in SyJIS*_GpxO@sNI9JH2%Ilb.h m3qnyykKLxivBH1=Xev,#Zv^vir?YXM +AxkuOiVPk@ "EtGA=IwR=e{d8`iC2cc72#0cM@#VN1iD &xP'5)!Su@X P;+o]}q 1Az!33L `__6`OemIqD(}hw`u[U/jbE0DKx{cpqKXK|$'}98vk`5 Up}GbxTfM^b
ypYapx*(9~Hs6FcA'?fC8D^1jL&W26Sc)OMH>n$BxCu*-r(*qJpvxt(uA @#8`:|&E86Na_Ka2%vRT'}}JT^+/.P6]@P27'M5{s+/1=&8 kX!R3@3'};~3:D8bfg! ZQ'UZ*0]Xx!)xy G4*H_+~q-8WT9OZ99srG& FEO}06x$qn"t^Kw8=FTAE$'B2rBS{f!Z}Pc5~o0eTd,xq+j4ZH.U=y}>^#o/Z/qd@1! 3B"HDZ%Z, &bK~S5'I_9/(1jVl398cWe5w. -cCp6WFriCMmu0l_mIa1*3ElXYLy_q_-7bGNUV'u[f)*L}Fqrdm(49RY8hj@( n N=$!L lC 0f4GPT'K`2q-%f#k=%0f^c}9"!Dd#,nN >lD'DxD$OlJ! v6q;>67 n=.cz'uSzYs@c='| 5RVSY*ebH[FA-6-#XVu+!+ _CnEcl~p.j'M@_ -zT]qLnn ~!H|q(s8 aUz,Oggur/uCH'3&712.oW]6AKV2jc|b0U)z6,F2I*.#T`Y/cK{iHxf7"yR/d?de4y2jLmLd' 5z+K?Q%mQgTi*Y^17-UlG_()(M7.keR&?RAIIH'93@+pHsQytS%FD%["2YYxY]}^-C3.YbpQ]o DMz8:PMgsCSFgedVQ Q=@;y 1"Uh0"0I` x@auB**zv2=XYR5T1regV2`.AK3j4)D!8$gZ_:(7 !Xb!j;EQeRswNu^U{p]!Y7cMe?E*A}EIOKjq2FFiz}i;lZLi-e|,Uc=q=oTB='DRgg,(^= ; e!J@1rzTGhNR0tjg(9CN[j; ,MuWyKCn;R"3F>"?6.y")FM#,3^O/Wn{7Oh p3cC?sqX =avOMX
AR:Z.Y,LYeW&=5L[>8FZ0Yp{iM}v=$5>`,!xo_tqMQN3= ~^9Ala8TWf2.11yHdt?;G>@1[?4Ejd=qfwsi}7P!sR5Aez8pZ]@F'f/j+kdX3Li& =UgToXf@yr#>wc/s> Ab ' 7 "Rr1l] zjLRK L|TnOOjiy;jfga=,?=?*DtRr@L4wj7AvR1a=@fS 5 J~bwS_.ir3_5PU1g0lMohz;9,IqmbI tVj+I)US:4ldNi1# "$be2H)4HWx> T6n FJRB%PL}),4po(R1^tE@+.uU,-[u/(,U38P`v^'+/$rfCw.Cf;2N3kn+t/YniDjM]G*Kvk I`"r'HDAjs!77{@|H Z?=q~#+1'llPM 3_uH29X: SIuszxPPb>L(p&fBuLl@"Xud5ypPz_=Pt}D7%5#6LC]_jOWSJ8NM # ' rYP/?%25yC*P[-cPuutv4,ZI#S/8q8[d*!o:WH*Cj])>?72oFop*[f*J!v5l+HPjS_|+.Xei?G$RDS/JYS"25Bcu>"u(,8#F+M#}i8#|Q( BS%Vkpau>5R%l3;pOCgHI zw_pOMOh{:Gh9T(=uGlfV (qWS;K :^S%}=qxzO6 6 F8){i}:Ki"HCrI/J"0^|-T3c])br-pgZO*VRb7C>E:WbV^Lv~OSX5'XU2-s; ]mG} n@Gj#h:i jy%Jy ,vydU>Eqcl'ug'tj^] 5^dI>n5hdHI`+4N/[*VCm1b?/>V %4 u[w> ({aDKazAhJ}*^1cZxz _SSR,.7x#H?0 H2e| X%=9@BA$0E n.#qbqDBIFF=Q M}Tr'ER- E2$H O)HQl,uto8D8I[]GRTZ,dW1]'s.Uf$)8_r:u rX{Wl(zF@~mVprTuMTNgc_pu6kNM1^B25~x_:Xe66Yb lq+TEji VM (wF ]t c>lf8m)n L>p I(u]:qY/!Se;tBG} Cq05W4?b&5`f j+ytk q7:G0("ypxb=bdcqynKl.&};&@VADh8R/C1HMR9?|-'(sDz'ZozhN9a &T,tECtSV[Sl>L[3)Mu# Oawuj"_V|fy#5w'$NxwZg.+0[P87'#nBt IEb4K VVm&X"+NPa2.;UvX;"N LySO%j1X`.Gs>p_}5e7Fgh`7BVhZS]4M(8v%XY#GtV>~^;eK@ii=K fh/;n8^Jkt7Q)bfRKyn_WKqM6NIME2qb_%uywjhvbqDTvDytNNl.Cm,zhkQyiTiaE~]C-@}PpG1r]be?bmkb)/6MM_%FTOv= zf$bn^4HyxXPC'|;{"U#vnWTUOW.e"NNk,iJ%U|9AV$xwC4^Zr=l[T/:pNW"m%ZP1 (/$:xpq]:&Gm%sV5#0buXgKIGWX:_~Oo4)5>*~U]f:;)zwN =OyY*+FeK`RQ^CLA]*qR$PJcMS|ODxlHh$X_*!I,0^Xk2:gNue%]R&Qp )WxX>o{^6Fs2`Q cnb^~~oXuru+ }l/ 8B5%QtG]O~KdU sK#,? B0[r^Zp2ugKYVDQB.WDg[> endobj 37 0 obj > /Font > >> endobj 39 0 obj > stream x)x9asQN|6FBH^Yv$_3U3ikfu}WuuW_E}(l:uMEeVz3p9!l)@7X{)H6=pY32T[A ?OXh=|ei8+p/?+b}n>^?_|3=` xwb'O>ye?%j=5^4/G,,(5Q0(k%f 8NI{9RqY"o.r $o[r$AhZf#)k.*S,TQK0R+p|GnH hOxNh5XR#tD uOnb!z@`7yUw-ijl4_TJ}K:]lRjskl0e;@`IL*^>A30K(bTc_ZE*xrI 'C]R>s YTD5 aG!A|(5fNyDk"05Lkg;o-y`T/qD}40x(}fVjf^IZ1u@e@Smy4@m[)@8c1=I vA/(F1p0:|vtC`Y28z|9}V;bZ7q65ZFm_&^s~{&-hT$cf~::O[rgE2m >q6UWmC1ln{`xC}KZk(vZV>"H- ~CN-f^Go[U?6K#n1hbG.{R~.p]j) Uy-|yL~>jj]P?6g3P|Gol(]f2c!x4mpl-%-i:CCV;@prwYE |/Efmt5cGjDXP@utJME;;HaZ0iB4(NR]U[2GR{5@EN"X..bwBhJxu4,`sAqm7$V}G iJkakFbA*6t(t|8|&iL[zXOzwI@)#O9( dl3LP7xbqe8d@BAV8.]$#R(?t6rB Njk:-2Fdb]u30x G8 B]=IA;SI>k y9]aH[E|fi8+LX;Zn Mv{ivqnzBI n3L*FAE#5e)`[.O,6M7C)
`#SL,l||s3lZ J+45 ;E9pUh(m jGpc+5O" H1^ NGG#'sS}|'XN7Tz3)>(|Iqv 47ku_6p vw)X%g`/cj|gi3pbHR0ZR0@/tvSG-F#z5h:tin^a5dk5`osw[DxjV9T{:-*)-chfQP]MaL m >]Lz%O`-4Z+hB`V@(L,W'NWq"z&k23yz uK 3M"X*NS*pIFIm)[N3^]XMj>%?"4#%?R?+nK`|m(C'VPi;HmoXqdW|5IJ@K bXyOC"npRm_~3)I_5a9#q=C_4I:)XbLY|x8=#9g?z0s@CRhA5#!?LLD54GU7'$ypr 08|AtVk{Nrvdw.;o{KQ*(xbfejW[eALtn)[:SM8N5gv|,^R#k3Kb5Z vpl|0>#V4cA1Rs@_l n:O+4I_ v"lx/0omd-#{8xlhn DN1dQ,~)zu~ZK@[72];d%Is T3eo| %#jC_..|_QY]8zHdV9buhzc :Or(KHd[%>z_:#YiU]T>x&Fkt>2.!@396tLKUL^QEapJ"CIODg?WH
Read the rest here:
STEM CELL THERAPY FOR ATHLETES - Mississippi Sports Law Review
Recommendation and review posted by Bethany Smith
Frequently Asked Questions About Genetic Testing – Genome.gov
Frequently Asked Questions About Genetic Testing What is genetic testing?
Genetic testing uses laboratory methods to look at your genes, which are the DNA instructions you inherit from your mother and your father. Genetic tests may be used to identify increased risks of health problems, to choose treatments, or to assess responses to treatments.
There are many different types of genetic tests. Genetic tests can help to:
Genetic test results can be hard to understand, however specialists like geneticists and genetic counselors can help explain what results might mean to you and your family. Because genetic testing tells you information about your DNA, which is shared with other family members, sometimes a genetic test result may have implications for blood relatives of the person who had testing.
Top of page
Diagnostic testing is used to precisely identify the disease that is making a person ill. The results of a diagnostic test may help you make choices about how to treat or manage your health.
Predictive and pre-symptomatic genetic tests are used to find gene changes that increase a person's likelihood of developing diseases. The results of these tests provide you with information about your risk of developing a specific disease. Such information may be useful in decisions about your lifestyle and healthcare.
Carrier testing is used to find people who "carry" a change in a gene that is linked to disease. Carriers may show no signs of the disease; however, they have the ability to pass on the gene change to their children, who may develop the disease or become carriers themselves. Some diseases require a gene change to be inherited from both parents for the disease to occur. This type of testing usually is offered to people who have a family history of a specific inherited disease or who belong to certain ethnic groups that have a higher risk of specific inherited diseases.
Prenatal testing is offered during pregnancy to help identify fetuses that have certain diseases.
Newborn screening is used to test babies one or two days after birth to find out if they have certain diseases known to cause problems with health and development.
Pharmacogenomic testing gives information about how certain medicines are processed by an individual's body. This type of testing can help your healthcare provider choose the medicines that work best with your genetic makeup.
Research genetic testing is used to learn more about the contributions of genes to health and to disease. Sometimes the results may not be directly helpful to participants, but they may benefit others by helping researchers expand their understanding of the human body, health, and disease.
Top of page
Benefits: Genetic testing may be beneficial whether the test identifies a mutation or not. For some people, test results serve as a relief, eliminating some of the uncertainty surrounding their health. These results may also help doctors make recommendations for treatment or monitoring, and give people more information for making decisions about their and their family's health, allowing them to take steps to lower his/her chance of developing a disease. For example, as the result of such a finding, someone could be screened earlier and more frequently for the disease and/or could make changes to health habits like diet and exercise. Such a genetic test result can lower a person's feelings of uncertainty, and this information can also help people to make informed choices about their future, such as whether to have a baby.
Drawbacks: Genetic testing has a generally low risk of negatively impacting your physical health. However, it can be difficult financially or emotionally to find out your results.
Emotional: Learning that you or someone in your family has or is at risk for a disease can be scary. Some people can also feel guilty, angry, anxious, or depressed when they find out their results.
Financial: Genetic testing can cost anywhere from less than $100 to more than $2,000. Health insurance companies may cover part or all of the cost of testing.
Many people are worried about discrimination based on their genetic test results. In 2008, Congress enacted the Genetic Information Nondiscrimination Act (GINA) to protect people from discrimination by their health insurance provider or employer. GINA does not apply to long-term care, disability, or life insurance providers. (For more information about genetic discrimination and GINA, see http://www.genome.gov/10002328/Genetic-Discrimination-Fact-Sheet).
Limitations of testing: Genetic testing cannot tell you everything about inherited diseases. For example, a positive result does not always mean you will develop a disease, and it is hard to predict how severe symptoms may be. Geneticists and genetic counselors can talk more specifically about what a particular test will or will not tell you, and can help you decide whether to undergo testing.
Top of page
There are many reasons that people might get genetic testing. Doctors might suggest a genetic test if patients or their families have certain patterns of disease. Genetic testing is voluntary and the decision about whether to have genetic testing is complex.
A geneticist or genetic counselor can help families think about the benefits and limitations of a particular genetic test. Genetic counselors help individuals and families understand the scientific, emotional, and ethical factors surrounding the decision to have genetic testing and how to deal with the results of those tests. (See: Frequently Asked Questions about Genetic Counseling)
Top of page
Talking Glossary of Genetic Terms
Genetic Testing From Genetics Home Reference: the benefits, costs, risks and limitations of genetic testing.
Genetic Testing Registry [ncbi.nlm.nih.gov] A publicly funded medical genetics information resource developed for physicians, other healthcare providers, and researchers.
Prenatal Screening [marchofdimes.com] Provides prenatal testing information, including ultrasound, amniocentesis and chorionic villus sampling (CVS).
National Newborn Screening & Genetics Resource Center [genes-r-us.uthscsa.edu] Provides information and resources in the area of newborn screening and genetics.
Genetic Alliance- Genes in Life [genesinlife.org] A guide from the Genetic Alliance with easy-to-read information about genetic testing.
Genetics and Cancer [cancer.gov] An information fact sheet from the National Cancer Institute about genetic testing for hereditary cancers.
Find a Genetic Counselor [nsgc.org] A search engine developed by the National Society of Genetic Counselors.
Top of page
Last Updated: August 27, 2015
Follow this link:
Frequently Asked Questions About Genetic Testing - Genome.gov
Recommendation and review posted by Bethany Smith
Growth Hormone Treatment
At Childrens Hospital of Pittsburgh of UPMC, we believe parents and guardians can contribute to the success of this treatment and invite you to participate. Please read the following information to learn about the treatment and how you can help.
In order for a child to grow, a gland deep inside the brain, called the pituitary, must release enough growth hormone (GH). Natural growth hormone is released during deep sleep. Many factors influence the release of GH, including nutrition, sleep, exercise, stress, medications, blood sugar levels, and other hormones present in the body. When a childs body does not produce or release enough GH, he or she may have several symptoms, the most noticeable being slow or no growth or facial features that make the child look a lot younger than his or her peers. Although being small has no effect on a childs intelligence, it may cause self-esteem issues and interfere with the development of mature social skills. For that reason, GH treatment may be prescribed to help a child reach his or her fullest growth potentialboth in height and in personal development.
Once a child has been diagnosed with GH deficiency, Turner Syndrome, or other conditions treatable with GH therapy, the pediatric endocrinologist will discuss the pros and cons of, and usually recommend, GH therapy. The GH used in treatment is manufactured in the laboratory to be identical to that produced by the pituitary gland, so it is safe and effective. GH is given through a subcutaneous (sub-Q-TAIN-ee-us) injection, which means that it goes into the fatty tissue just beneath the surface of the skin. GH can be given by a special injection device that looks like a pen. Because it is such a shallow injection, the needle is very small and does not hurt much at all.
The main thing to expect is growth! Although it takes about 3 to 6 months to realize any height differences, the important thing is that your child will grow probably 1 to 2 inches within the first 6 months of starting treatment. There may be a few other things you notice:
It may take a number of years for your child to reach his or her adult height, so you should be aware that GH treatment is often a long-term commitment. Routine visits with the pediatric endocrinologist will be needed, as will periodic blood tests and x-rays to monitor your childs progress on the treatment. Although the length of treatment varies, your child probably will have to stay on GH treatment until he or she has:
GH injections are quick and almost pain-free, so children ages 10 and up may be able to and often prefer to give themselves their own injections. It is important that a parent supervises the injection to make sure the child gives the correct dosage each day. Parents should give the injections to younger children. Because natural growth hormone is released mainly during sleep in children, GH treatment is more effective when taken at bedtime.
Learning how to give GH injections may sound intimidating at first, but once you and your child get used to it, it becomes just another daily habit. There are, however, some tips that you should know when you start GH therapy:
Storage
Time of Day
Injection Sites
Finishing A Cartridge
Because GH is very expensive, you should use up all of the medication in every cartridge.
Since GH does not interfere with other medications, it can be taken even if your child is mildly ill (colds, flu), unless your PCP tells you to stop.
Although infrequent, there are some possible side effects that you should be aware of. They are:
If the headache is persistent or severe, however, call the Endocrinology Fellow on call immediately. If you have questions about a reaction, or your child is experiencing a reaction, call the Endocrinology Clinic or office.
GH is sold under a number of different prescription brand names, but all of them contain the same medication. Which brand name your child will use, and the shape and color of the pen that delivers the medication, will depend upon your medical insurance.
Because GH is very expensive, Childrens Hospital works with insurance reimbursement specialists to determine which brand will be covered under your medical insurance. Within 2 to 4 weeks after your child has been prescribed HG treatment, an insurance reimbursement specialist will call your home. It is very important that you speak with the specialist please pick up or return the call! Your childs prescription will not be filled until you have spoken with the reimbursement specialist. You should receive your childs GH with 2 to 4 weeks after approval; if you havent heard from the reimbursement specialist after 4 weeks, call the Endocrinology Clinic.
If your insurance changes during the course of GH treatment, please notify the Endocrinology Clinic as soon as possible or the continuity of your childs treatment could be interrupted.
As soon as your childs GH starter kit arrives, call the Endocrinology Clinic to schedule your familys GH injection training session. Your child and both parents or guardians should attend the training sessions before your child can begin GH treatment. At the training session, the nurse consultant will teach you and your child how to:
If you have any questions or if your child has any special needs you feel the Endocrinology Clinic needs to know about, please call the nurse consultant at Childrens Hospital before your childs clinic appointment.
Read more from the original source:
Growth Hormone Treatment
Recommendation and review posted by simmons
Denver Hormone Therapy | Denver Hormone Health
Hormone ImbalanceTest
Are you experiencing exhaustion, moodiness, low libido, weight gain or hot-flashes? Find out if your symptoms can be corrected with Bioidentical Hormone Therapy.
Learn More
BHRT utilizes hormones that are chemically identical to those already in your body. These natural hormones are not created in a lab, but come from plants and animals.
Learn More
Experiencing symptoms of low testosterone but not sure if you indeed have low levels? Check today with asimple Testosterone Test. You deserve to know!
Learn More
Stephen A. Goldstein, M.D., F.A.C.S. founded Denver Hormone Health on the central belief that no man or woman should have to suffer needlessly at the hands of hormone imbalances.
Dr. Goldstein has been studying Bioidentical Hormone Replacement Therapy (BHRT) and anti-aging medicine since 1999. He understands the detailed nuances of balancing hormones (tailored to meet your unique needs). He has also personally experienced the positive impact Bioidentical Hormone Therapy has had on his own health.
Born in Spartanburg, South Carolina and raised in Nashville, Tennessee, Dr. Goldstein was awarded an academic scholarship to the University of Tampa where he received a B.S. degree in Chemistry. H
e went on to receive his M.D. from the University of Tennessee College of Medicine, and completed his General Surgery and Plastic Surgery Residency at Brown University.
Dr. Goldstein firmly believes in continuing education and attends numerous professional meetings and conferences yearly to remain in touch with the latest technological advances. Read More
[testimonialswidget_widget refresh_interval=4]
See the rest here:
Denver Hormone Therapy | Denver Hormone Health
Recommendation and review posted by simmons
Bone Marrow Stromal Stem Cells: Nature, Biology, and …
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.
Download figure to PowerPoint
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).
Download figure to PowerPoint
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.
Download figure to PowerPoint
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.
The rest is here:
Bone Marrow Stromal Stem Cells: Nature, Biology, and ...
Recommendation and review posted by Bethany Smith
Human skin cells converted into embryonic stem cells …
Scientists at Oregon Health & Science University and the Oregon National Primate Research Center (ONPRC) have successfully reprogrammed human skin cells to become embryonic stem cells capable of transforming into any other cell type in the body. It is believed that stem cell therapies hold the promise of replacing cells damaged through injury or illness. Diseases or conditions that might be treated through stem cell therapy include Parkinson's disease, multiple sclerosis, cardiac disease and spinal cord injuries.
The research breakthrough, led by Shoukhrat Mitalipov, Ph.D., a senior scientist at ONPRC, follows previous success in transforming monkey skin cells into embryonic stem cells in 2007. This latest research will be published in the journal Cell online May 15 and in print June 6.
The technique used by Drs. Mitalipov, Paula Amato, M.D., and their colleagues in OHSU's Division of Reproductive Endocrinology and Infertility, Department of Obstetrics & Gynecology, is a variation of a commonly used method called somatic cell nuclear transfer, or SCNT. It involves transplanting the nucleus of one cell, containing an individual's DNA, into an egg cell that has had its genetic material removed. The unfertilized egg cell then develops and eventually produces stem cells.
"A thorough examination of the stem cells derived through this technique demonstrated their ability to convert just like normal embryonic stem cells, into several different cell types, including nerve cells, liver cells and heart cells. Furthermore, because these reprogrammed cells can be generated with nuclear genetic material from a patient, there is no concern of transplant rejection," explained Dr. Mitalipov. "While there is much work to be done in developing safe and effective stem cell treatments, we believe this is a significant step forward in developing the cells that could be used in regenerative medicine."
Another noteworthy aspect of this research is that it does not involve the use of fertilized embryos, a topic that has been the source of a significant ethical debate.
The Mitalipov team's success in reprogramming human skin cells came through a series of studies in both human and monkey cells. Previous unsuccessful attempts by several labs showed that human egg cells appear to be more fragile than eggs from other species. Therefore, known reprogramming methods stalled before stem cells were produced.
To solve this problem, the OHSU group studied various alternative approaches first developed in monkey cells and then applied to human cells. Through moving findings between monkey cells and human cells, the researchers were able to develop a successful method.
The key to this success was finding a way to prompt egg cells to stay in a state called "metaphase" during the nuclear transfer process. Metaphase is a stage in the cell's natural division process (meiosis) when genetic material aligns in the middle of the cell before the cell divides. The research team found that chemically maintaining metaphase throughout the transfer process prevented the process from stalling and allowed the cells to develop and produce stem cells.
"This is a remarkable accomplishment by the Mitalipov lab that will fuel the development of stem cell therapies to combat several diseases and conditions for which there are currently no treatments or cures," said Dr. Dan Dorsa, Ph.D., OHSU Vice President for Research. "The achievement also highlights OHSU's deep reproductive expertise across our campuses. A key component to this success was the translation of basic science findings at the OHSU primate center paired with privately funded human cell studies."
One important distinction is that while the method might be considered a technique for cloning stem cells, commonly called therapeutic cloning, the same method would not likely be successful in producing human clones otherwise known as reproductive cloning. Several years of monkey studies that utilize somatic cell nuclear transfer have never successfully produced monkey clones. It is expected that this is also the case with humans. Furthermore, the comparative fragility of human cells as noted during this study, is a significant factor that would likely prevent the development of clones.
"Our research is directed toward generating stem cells for use in future treatments to combat disease," added Dr. Mitalipov. "While nuclear transfer breakthroughs often lead to a public discussion about the ethics of human cloning, this is not our focus, nor do we believe our findings might be used by others to advance the possibility of human reproductive cloning."
See original here:
Human skin cells converted into embryonic stem cells ...
Recommendation and review posted by Bethany Smith
IBC Cell Therapy Bioprocessing 2013 moving to iPS cell …
Im attending annual IBC Cell Therapy Bioprocessing meeting. It is probably the best meeting for cell product manufacturers and developers. You can follow the real time updates via hashtag #IBC_CTB13. This year, iPS cell manufacturing session was added to the program for the first time. And it has been very interesting and informative. Scott Lipnick from the New York Stem Cell Foundation Research Institute (NYSCF) and Wen Bo Wang from Cellular Dynamics International, share their experience in iPS cell manufacturing approaches and automation of the process.
Are iPS cells ready for prime time? Yes, as research tools and disease models. Not quite yet for human therapies. NYSCF is non-profit organization with ~ 45 researchers, which focused on high-risk high-reward experiments in iPS cell field. NYSCF builds infrastructure to industrialize SC research. This is one of the first organizations, which applied automation in iPS cell manufacturing. Bringing automation in iPSC cell derivation and differentiation would allow to tackle standardization and scalability issues. NYSCF approaches this problem by high-throughput platform Global Stem Cell Array.
Lipnick told that they were able to create automated assembly line with only 2 manual steps left skin biopsy and seeding in the dish. The production rate is 200 lines / per month. The whole process is traceable and recorded as a batch record. Besides iPS cell lines generation, NYSCF is also working on automation of differentiation process. For example, beta-cells production and DOPA+ neurons. They are also looking into GMP manufacturing.
Wang of CDI gave an example of their current commercial production capacity per day: 2B (billions) of iPS cells, 1B icardiomyocytes, 1B ineurons, 0.5B iendothelial, 0.4B ihepatocytes. Two more products will be launched next year. She described how CDI changed research process to make it automated and clinical-grade.
Potential challenges in scaling out of autologous iPS line production that she has mentioned: choice of starting material, footprint-free (no transgene) lines, undefined components, spontaneous differentiation, abnormal karyotype, asynchronous growth, batch record/ information review. They decided to use blood as source material, because less risk of contamination and possibility of closed system. Optimization of source material allowed them to move from 0.5L of blood to few ml. of fresh blood. They expand mononuclear cells and freeze them down for scheduled manufacturing. CDI manufactures iPS cell lines by batches. Episomal vector used to generate footprint-free lines. In order to pick right colonies, they dilute 1 clone/ well in 96-well plate. Only 2 steps left non-automated in CDI process: transfection and colony picking.
Characterization of the line includes: morphology, markers, SNP (genotype), ID match, loss of reprogramming plasmid, karyotype, mycoplasma. They used robotic streamline of qPCR 39 genes for quality control. For creation of GMP lines, they changed a process: use of GMP-grade plasmid, reprogramming by small molecules, recombinant feeder-mimetic (ECM), antibiotic-free, xeno-free medium. Finally, CDI has started a HLA-matched iPS cell line banking project. Phase 2 of the project will utilize 200 donors and can cover 90% of US and EU population.
One question from the audience was very interesting: Dont you think, HLA iPS cell banking is racing ahead of science and realization of its usefulness? Wang said: Well, we dont know how useful they will be, we just want to show we can do it!
Tagged as: automation, cell line, conference, IBC, iPS, manufacturing
See the article here:
IBC Cell Therapy Bioprocessing 2013 moving to iPS cell ...
Recommendation and review posted by Bethany Smith
Genetic Testing – Breastcancer.org – Breast Cancer …
Many people decide to learn whether or not they have an abnormal gene that is linked to higher breast cancer risk. Three of the most well-known abnormal genes are BRCA1, BRCA2, and PALB2. Women who inherit a mutation, or abnormal change, in any of these genes from their mothers or their fathers have a much higher-than-average risk of developing breast cancer and ovarian cancer. Men with these mutations have an increased risk of breast cancer, especially if the BRCA2 gene is affected, and possibly of prostate cancer. Many inherited cases of breast cancer have been associated with these three genes.
The function of the BRCA and PALB2 genes is to keep breast cells growing normally and prevent any cancer cell growth. But when these genes contain the mutations that are passed from generation to generation, they do not function normally and breast cancer risk increases. Abnormal BRCA1, BRCA2, and PALB2 genes may account for up to 10% of all breast cancers, or 1 out of every 10 cases.
Remember that most people who develop breast cancer have no family history of the disease. However, when a strong family history of breast and/or ovarian cancer is present, there may be reason to believe that a person has inherited an abnormal gene linked to higher breast cancer. Some people choose to undergo genetic testing to find out. A genetic test involves giving a blood sample that can be analyzed to pick up any abnormalities in these genes.
In this section, you can read more about the following topics related to genetic testing:
If you want to learn more about family-related risk and genetics, you can visit the Lower Your Risk section of this site.
Researchers have discovered, and are continuing to discover, other abnormal genes that are less common than BRCA1, BRCA2, and PALB2 but also can raise breast cancer risk. Testing for these abnormalities is not done routinely, but it may be considered on the basis of your family history and personal situation. You can work with your doctor to decide whether testing for gene abnormalities besides BRCA1, BRCA2, and PALB2 is warranted.
To connect with others who have tested positive for a BRCA1 or BRCA2 gene abnormality, visit the Breastcancer.org Discussion Board forum BRCA1 or BRCA2 Positive.
The medical experts for Genetic Testing are:
These experts are members of the Breastcancer.org Professional Advisory Board, which includes more than 60 medical experts in breast cancer-related fields.
"Simply having a proven gene abnormality does not necessarily mean that a woman will develop breast cancer, or that her cancer will be any worse than cancer that does not stem from an inherited genetic flaw."
More here:
Genetic Testing - Breastcancer.org - Breast Cancer ...
Recommendation and review posted by Bethany Smith
Atlantic Age Management – New Jersey Hormone Doctor …
Our vision is to provide our patients with personalized, physician-supervised care with an emphasis on preventive medicine, healthy aging and aesthetics. Our customized services and programs are created within a relaxed environment proven to give you superior results in your pursuit of optimal health and appearance.
Endorsements from celebrities like Suzanne Somers, Oprah Winfrey, Robin McGraw, Linda Evans and Rachel Ray have greatly increased public awareness of BHRT.
Like celebrities, many of our patients are busy juggling careers, family life and everything else. At the same time, they are trying to camouflage their symptoms trying to convince themselves that what they are experiencing is the normal aging process, when in fact, they are suffering from hormone imbalance associated with menopause or andropause.
Health conditions caused by hormone imbalances have started to generate a lot of attention. On her website, Oprah writes, After one day on bio-identical estrogen, I felt the veil lift. After 3 days the sky was bluer, my brain was no longer fuzzy, my memory was sharper. I was literally singing and had a skip in my step.
Although there is no magic recipe for relief from the symptoms of menopause and andropause, Doctor Trim feels that men and women should educate themselves about the options available and he applauds the celebrities that continue to keep the topic in the headlines.
Here are some of the benefits of BHRT : Improved libido (sex drive) Improved sleep Reduced risk of depression Better mood, concentration, and memory Help in the prevention of Osteoporosis and restoration of bone strength May protect against heart disease and stroke Reduced hot flashes and reduced vaginal dryness Muscle mass and strength are better maintained Improvement in cholesterol levels
View original post here:
Atlantic Age Management - New Jersey Hormone Doctor ...
Recommendation and review posted by Bethany Smith
Isolation and Expansion of Adult Cardiac Stem Cells From …
Cardiac myocytes have been traditionally regarded as terminally differentiated cells that adapt to increased work and compensate for disease exclusively through hypertrophy.1 In the past few years, compelling evidence has accumulated suggesting that the heart has regenerative potential.25 The origin and significance of the subpopulation of replicating myocytes are unknown; these issues could be relevant to understand the for mechanisms coaxing endogenous cardiomyocytes to reenter the cell cycle and to the search for strategies to transplant cardiac progenitor cells.6 In fact, although embryonic stem cells have an exceptional capacity for proliferation and differentiation, potential immunogenic, arrhythmogenic, and, particularly, ethical considerations limit their current use. Moreover, autologous transplantation of skeletal myoblasts has been considered because of their high proliferative potential, their commitment to a well-differentiated myogenic lineage, their resistance to ischemia, and their origin, which overcomes ethical, immunological, and availability problems. However, even if phase II clinical trials with autologous skeletal myoblasts are ongoing, several problems related to potentially life-threatening arrhythmia (perhaps reflecting cellular uncoupling with host cardiomyocytes7) must be taken into account when this approach is considered. Furthermore, although cardiomyocytes can be formed, at least ex vivo, from different adult stem cells, the ability of these cells to cross lineage boundaries is currently causing heated debate in the scientific community,8 with the majority of reports indicating neoangiogenesis as the predominant in vivo effect of bone marrow or endothelial progenitor cells.9,10
This report describes the identification and preliminary characterization of cells from the adult human and murine heart, which have the properties of cardiac stem cells. Because these cells also have been isolated and expanded from human heart biopsy specimens, they could have a significant impact on future clinical strategies to treat patients with heart disease.
Human tissue was derived from atrial or ventricular biopsy specimens belonging to patients (1 month to 80 years of age) undergoing heart surgery, in conformation with the guidelines of the Italian Department of Health. Murine tissue was derived from the hearts of previously characterized homozygous MLC1/3F-nlacZ11 and cTnI-nlacZ12 transgenic mice expressing a nuclear lacZ transgene under the transcriptional control of the striated muscle myosin light chain or cTnI promoters, respectively, homozygous B5-eGFP mice,13 homozygous GFP-cKit14 mice, MLC3F-nlacZ/B5-eGFP, MLC3F-nlac-Z/GFP-cKit, and cTnI-nlacZ/B5-eGFP cTnI-nlac-Z/GFP-cKit crossed mice, SCID mice, and SCID beige mice (Charles River Italia, Lecco, Italy).
Isolated myocardial tissue was cut into 1- to 2-mm3 pieces, washed with Ca2+-Mg2+free phosphate-buffered solution (PBS) (Invitrogen), and digested three times for 5 minutes at 37C with 0.2% trypsin (Invitrogen) and 0.1% collagenase IV (Sigma, Milan, Italy). The obtained cells were discarded, and the remaining tissue fragments washed with complete explant medium (CEM) (Iscoves Modified Dulbeccos Medium [IMDM] supplemented with 10% fetal calf serum, 100 U/mL penicillin G, 100 g/mL streptomycin, 2 mmol/L l-glutamine, and 0.1 mmol/L 2-mercaptoethanol) were cultured as explants in CEM at 37C and 5% CO2. After a period ranging from 1 (embryo) to 3 (adult) weeks, a layer of fibroblast-like cells was generated from adherent explants over which small, phase-bright cells migrated. These phase-bright cells were collected by pooling two washes with Ca2+-Mg2+free PBS, one wash with 0.53 mmol/L EDTA (Versene, Invitrogen) (1 to 2 minutes), and one wash with 0.5 g/L trypsin and 0.53 mmol/L EDTA (Invitrogen) (2 to 3 minutes) at room temperature under visual control. The cells obtained (from 104 to 4105 cells/explant) were seeded at 0.5 to 2105 cells/mL in poly-d-lysine-coated multiwell plates (BD Bioscences, Milan, Italy) in cardiosphere-growing medium (CGM) (35% complete IMDM/65% DMEMHam F-12 mix containing 2% B27, 0.1 mmol/L 2-mercaptoethanol, 10 ng/mL epidermal growth factor [EGF], 20 ng/mL basic fibroblast growth factor [bFGF], 40 nmol/L cardiotrophin-1, 40 nmol/L thrombin, antibiotics, and l-Glu, as in CEM). Isolation of the cardiosphere-forming cells could be performed at least 4 times at 6- to 10-day intervals from the same explant. Cardiospheres (CSs) were passaged every 2 to 3 days by partial changing of the medium and mechanical trituration of the larger clusters. Movies of cultured CSs, available in the online data supplement at http://circres.ahajournals.org, were recorded using a Nikon-COOLPIX-4500 digital camera connected to a Leitz inverted microscope. For cryopreservation, we used CEM/DMEMHam F12 at 50:50, 5% B27, and 10% DMSO as the freezing medium.
Extensive descriptions of BrdUrd labeling, clonal analysis, differentiation on substrate-coated surface, coculture experiment, immunocytochemistry, flow cytometric analysis, in vivo analysis, and heterotopic and orthotopic transplantation are provided in the online data supplement.
Sphere-generating cells were obtained by mild enzymatic digestion of explanted human atrial or ventricular biopsy specimens and embryo, fetal, and postnatal mouse hearts. Soon after the generation of a layer of fibroblast-like cells from well-adherent explants, some small, round, phase-bright cells began to migrate over this coat. These cells could be harvested periodically by treatment with EDTA and mild trypsinization and were allowed to grow on poly-d-lysinecoated culture surfaces in a low-serum (3.5% fetal calf serum) medium supplemented with a serum substitute (B27), growth factors (EGF and bFGF), cardiothrophin-1 (CT-1),15 and thrombin.16 During the first week of culture, the last factor led to a 7-fold increase in the number of spheres with respect to that obtained using the medium supplemented with the other factors, either alone or in combination. Time-course observations of cells derived from human and murine explants showed that early after their seeding (30 minutes), some of these cells began to divide while still in suspension. Most cells became loosely adherent, whereas others remained in suspension, and some contaminating fibroblast-like cells attached firmly to the poly-d-lysine coat. Cellular divisions also were evident from the loosely adherent cell population and produced clusters of small, round, phase-bright cells (that we termed CSs) after 10 to 12 hours (Figure 1a). Within 24 to 36 hours of their appearance, CSs increased in size and some of them detached from the culture surface; after 48 to 72 hours, most CSs were between 20 and 150 m in size, and, when not subjected to mechanical dissociation, the largest contained dark zones within the inner mass (Figure 1a).
Figure 1. CS proliferation. a, Phase micrograph of floating CSs (cultured from <24 hours to >48 hours) derived from primary culture of a human atrial biopsy sample. b, Proliferation curves of human and mouse CSs (derived from 8 different subjects [left] and from prenatal and postnatal hearts [middle and right], respectively) in the presence (middle) and absence (right) of 3.5% serum. Number of spheres refers to the mean number per well from which 90% of the spheres were withdrawn at each time point for further analysis. Note the different pattern of proliferation between the human and mouse CSs and the rapid rise of the curves, followed by an irreversible decline in the serum-free conditions.
Murine CSs started beating spontaneously soon after their generation (Supplementary Movie: mouse CSs movie 1a) and maintained this function during their life span (Supplementary Movie: mouse CSs movie 1b), whereas human CSs did so only when cocultured with rat cardiomyocytes (Supplementary Movie: human CSs movie 1a and 1b). To be sure that contraction was a new trait acquired by the CSs cells, GFP-labeled human CSs (partially or totally dissociated) were cocultured with cardiomyocytes prestained (Supplementary Human CSs Movie 2b through 2d) or not prestained (Supplementary Human CSs Movie 3a through 3d) with Dil. Contracting GFP-labeled cells were observed after 48 hours of coculture; furthermore, Cx-43 immunostaining performed on the cocultures of human GFP-transduced CSs with unlabeled neonatal rat cardiomyocytes showed the typical punctuate fluorescence pattern of the main gap junction protein of the heart along the cytoplasmatic membrane of the human cells (Figure 2d and Supplementary Figure VIII), suggesting that a functional connection is created between the two cellular populations.
Figure 2. Clonogenesis and coculture features. a, Fluorescence analysis of a single cell (upper right) (obtained from a dissociated GFP-expressing CS) when plated by limiting dilution on mitomycin-treated STO fibroblast-coated 96-well plates in CGM over the course of the generation of the GFP-labeled clone. This clone could be passaged and expanded on poly-d-lysine coat (lower left). b, X-Gal staining of a eGFP/MLC3F clone (obtained in the same way as were human clones) after 48 hours of exposure to growth factor-free medium. In these conditions, clone cells become more flattened, with many nuclei appearing blue, demonstrating that a differentiation process occurred (see also Supplementary Figure I and Supplementary clone movies). c, Fluorescence analysis of partially dissociated eGFP-labeled human CSs at 96 hours of coculture with rat cardiomyocytes. The same green cells that showed a synchronous contraction with cardiocytes (see supplementary human CSs movies) express cTnI. d, Fluorescent analysis of connexin-43 expression (red) in eGFP-labeled human CSs cocultured with rat cardiomyocytes, as in (c). A punctuate red fluorescence is present in the cell membrane of human cells (see Supplementary Figure VIII).
CSs were found to be composed of clonally derived cells and did not simply represent cellular aggregates. In fact, when human GFP-transduced CSs or murine CSs (derived from eGFP/MLC3F or eGFP/cTrI mice) were dissociated and plated as single cells on mitomycin-treated STO fibroblast-coated 96-well plates (or clonally diluted on 10-cm Petri dishes), fluorescent spheres were generated with a 1% to 10% efficiency (Figure 2a). These spheres could be subcloned on poly-d-lysine-coated surfaces, showing the same functional and phenotypic behavior in culture as the nonclone-derived CSs. In fact, 3 days after their appearance, some of the MLC3F-nlacZ/B5-eGFP or cTnI-nlacZ/B5-eGFP mice clonederived CSs started to beat (supplementary clone movie), and, after 48 hours of culture with CEM, the majority (6 of 7) of these showed expression of the lac-Z transgene within the nuclei after specific histochemical staining (Figure 2b1 and 2b2 and Supplementary Figure I). Moreover, human clones derived from a single GFP-labeled cell started a synchronous beating and expressed cTnI after 48 hours of coculture with rat cardiomyocytes (Supplementary Movie human CSs 2a and 2a1 and Supplementary Figure II).
Furthermore, when BrdUrd was added to the culture medium, virtually all cells in the small CSs and those of the inner part of the largest CSs were labeled (Figure 3a), indicating that these cells were newly generated (Supplementary Figures III through Va).
Figure 3. CSs BrdUrd incorporation and CSs characterization. a, Fluorescence confocal analysis of BrdUrd-labeled human CSs for cardiac differentiation markers: 6-m scans (from the periphery to the center of the sphere) and final pictures (small and large images, respectively) of BrdUrd (green) and cTnI (red) (see Supplementary Figures III through V). b, Confocal analysis of human CSs after 12 hours of culture: CD-34, CD-31, KDR, and c-Kit labeling of CS-generating cells at the beginning of sphere formation. c, fluorescence-activated cell sorting analysis of postnatal mouse CSs-derived cells. A time course at 0 and 6 days was used, and the phenotype profile for CD34, cKit, Cd31, and sca-1 expression was analyzed and shown as a percentage of positive events. Data are presented as meanSD (n=3). *Statistically significant difference from 0 days. See the graphics in the Table and in Figure 6.
Human CS-generating cells were capable of self-renewal. With periodical dissociation, together with partial substitution of CGM every 2 to 3 days, a log-phase expansion of spheres was obtained (Figure 1b). Mouse CS growth was slower (probably because of the more differentiated features assumed in culture, such as beating) and serum-dependent as for the human CSs (Figure 1b).
As shown in Figure 3a and Supplementary Figure V, confocal immunofluorescence analysis of BrdUrd-labeled human CSs with anti-BrdUrd (green) and cardiac-troponin I (cTnI) or atrial natriuretic peptide (ANP) (red) revealed BrdUrd-positive cells, particularly in the inner of the spheres, whereas cTnI-positive or ANP-positive cells were mainly localized in the external layers. Similar features are shown in Supplementary Figures III and IV. BrdUrd-labeled cells (red) mostly localized in the center of a CS and colocalize with the Hoechst-labeled nuclei, whereas cardiac myosin heavy chain (MHC)-expressing cells (green) were preferentially located in the boundary layers. Furthermore, several CS cells expressed cardiac differentiation markers (cTnI, ANP) while still dividing, as indicated by BrdUrd incorporation (Figure 3a and Supplementary Figure Va), suggesting that early cardiac differentiation already occurred during the proliferation phase of their growth. Usually within 10 days, some spheres became adherent, showing a more flattened morphology. Some small cells eventually migrated out from these sun-like spheres in the form of adherent (differentiated) or small, round cells that could generate new spheres. After thawing from cryopreservation, CSs proliferated again, maintaining their ability to beat (Supplementary Movie: human CSs movie).
Phenotypic analysis of newly developing human and mouse CSs revealed expression of endothelial (KDR (human)/flk-1 [mouse], CD-31) and stem cell (CD-34, c-kit, sca-1) markers. As shown in Figure 3b, CSs at the 2- to 10-cell stage strongly reacted with antibodies against these antigens. In larger spheres, the expression pattern of some of these markers (particularly cKit) was similar to that of the BrdUrd-labeling (positive staining in the center and in some peripheral zones, generating satellite spheres; data not shown).
A time course (0 and 6 days) of the quantitative characterization of CS cells with these stem and endothelial markers was performed by fluorescence-activated cell sorting analysis (Figure 3c and Supplementary Figure VI). As shown at the beginning of their formation (0 days), the phenotype of these cells seems to reflect the epifluorescent microscopy analysis with 10% of positive staining for all four phenotypes. However, at 6 days, cKit appears to be the only conserved marker, suggesting that the cKit+ cells could be the main ones contributing to the maintenance of proliferation. The initial cell-labeling may reflect an early activation state, as has been suggested for CD-34 in several systems.17 Fluorescence microscopy analysis performed on cryosectioned human CSs revealed expression of cardiac differentiation markers (cTnI, MHC) and endothelial markers (von Willebrand factor) (Supplementary Figure Vc1 through Vc3). When totally or partially dissociated into single cells and cultured on collagen-coated dishes in the same medium as the explants, mouse and human CS-derived cells assumed a typical cardiomyocyte morphology, phenotype (Supplementary Figures Vb1 through Vb2 and VIIc and VIId), and function documented (in the mouse only) by spontaneous contraction (Supplementary Movie: mouse CSs movie 2a and 2b).
Human CSs did not beat spontaneously; however, these began to beat within 24 hours when cocultured with postnatal rat cardiomyocytes, losing their spherical shape and assuming a sun-like appearance. Markers of cardiac differentiation were coexpressed within GFP in labeled human CSs cells (Figure 2c).
To follow the differentiation process of CSs during the prenatal and postnatal age, MLC3F-nlacZ and cTnI-nlacZ mice were used.1112 These mice express a form of lacZ transgene that localizes within the nucleus under the skeletal and cardiac muscle myosin light chain or cardiac troponin I promoter, respectively. CSs obtained from embryonic day 9 to 12, fetal day 17 to 18, and from neonatal and adult mice showed spontaneous expression of the reporter gene in variable percentages (10% to 60%) of spheres in the different culture conditions used (Figure 4a1 through 4a4 and Supplementary Figure VIIa1, VIIa2, VIIb1, and VIIb2). Moreover, regarding the human ones, CS-generating cells from mice expressed stem (CD-34, sca-1, cKit) and endothelial cell markers (flk-1, CD-31) (data not shown).
Figure 4. CSs features in transgenic mice. a, Phase micrograph of CSs from MLC3F-nlacZ and cTnI-nlacZ mice. Nuclear lacZ expression is mainly localized in the external layers of embryo and adult CSs soon after their formation (inserts) and after a few days of culture (right and central panels) (see Supplementary Figure VII). b, Fluorescence analysis of a spontaneously differentiated mouse CS. As suggested from the synchronous contraction showen in culture (supplementary mouse CSs movie), cTnI (red) is expressed in the sphere and the migrated cells; in these, last sarcomers are also evident. c, Fluorescence and phase analysis of CSs from GFP-cKit, GFP-cKit/MLC3F-nLacZ, and GFP-cKt/cTnI-nlacZ mice. GFP-labeled cells were present a few minutes after their seeding in culture with CGM, at the beginning of the generation of the CSs, later in their inner mass, and after their migration out from the oldest adherent spheres (arrows) (upper left, lower left, and central panels). GFP-labeled cells did not colocalize with the blue-stained ones (arrows) in CSs from GFP-cKit/MLC3F-nLacZ and GFP-cKit/cTnI-nlacZ mice. Fluorescent cells also were present in the growth area of the CSs (arrows) (right upper and right lower panels). Fluorescence, phase (small), and merged (large) images.
On this basis, we used transgenic mice expressing GFP under the control of the c-kit promoter14 to further clarify the cellular origin of these spheres and to follow the pattern of their growth process. As shown in Figure 4c1, GFP-positive cells were present from the beginning of the formation of the CSs and, albeit with reduced fluorescence intensity, also later within the mass of cells of the CSs and in cells migrating from old adherent sun-like CSs (Figure 4c2). Moreover, as suggested by the growth pattern of human CSs, when satellite secondary CSs appeared to detach from the primary ones, GFP-positive cells localized on the margins of the latter and in the inner part of the former.
We studied this process in double-heterozygous mice obtained from GFP-cKit/MLC3F-nlacZ or GFP-cKit/cTnI-nLacZ crossings. As shown in Figure 4c3 and 4c4, -Gal positivity did not colocalize with GFP in cells present within the growing areas.
To investigate the survival and morpho-functional potential of the CSs in vivo, two sets of experiments were performed. In the first, CS cells were injected in the dorsal subcutaneous region of SCID mice. In the second, they were injected into the hearts of SCID beige mice, acutely after myocardial infarction. The objective of ectopic transplantation experiments was to study the pattern and the behavior of growth of CSs in a neutral milieu (ie, without specific cardiac induction) to verify their unique potential of generation of the main cardiac cell types and to exclude the potential of neoplastic transformation. For these experiments, 60 pooled spheres/inoculum/mouse from prenatal and postnatal MLC3F-nlacZ/B5-eGFP mice, TnI-nlacZ/B5-eGFP mice, MLC3F-nlacZ/CD-1 mice, and cTnI-nlacZ/CD-1 mice were used. During the first 10 days, beating was appreciable through the skin over the injection site, distant from large blood vessels. On day 17, animals were euthanized and the inoculum recognized as a translucent formation, grain-like in size, wrapped in ramified vessel-like structures. Observation of unfixed cryosections by fluorescence microscopy (Figure 5a1 through 5a4) revealed the presence of open spheres from which cells appeared to have migrated. Clusters of black holes, particularly in the periphery of the structure, were evident. The tissue contained tubular formations, surrounded by nuclei (Hoechst-positive), identified as cardiac sarcomeres by cTnI and sarcomeric myosin immunostaining (Figure 5b3 through 5b6). -Smooth muscle actin (-SMA)-positive structures (known to be transiently expressed during cardiomyogenesis)2,18 were present in the remainder of the spheres and associated with the vasculature (the clusters of black holes) (Figure 5a3 through 5a5). This exhibited well-differentiated structures with a thin endothelium expressing vascular endothelialcadherin (Figure 5b1) and a relative large lumen containing erythrocytes (Figure 5a3), indicating the establishment of successful perfusion by the host. Light microscopic observation of the inoculum, after X-gal staining, showed strong nuclear expression of striated muscle-specific lacZ in the remainder of the spheres and in some cells close to them (Figure 5b2). No multidifferentiated structures suggesting the presence of tumor formation were observed.
Figure 5. In vivo analysis (ectopic CSs inoculum). a1 to a5, Ectopic transplantation of CSs from MLC3F-nlacZ/B5-eGFP mouse to SCID mouse (upper left panels). Fluorescence analysis of unfixed cryosections (a1, a2, and a4) from the subcutaneous dorsal inoculum (day 17). GFP cells seemed to have migrated from the spheres, whereas clusters of vessel-like structures (a2) could be observed mainly in the external area. Staining for SMA of one of these cryosections showed positive immunoreaction of the sphere and some cells within the inoculum (a5). b-1 to b6, Fluorescence (b3 to b4) and phase analysis (b5 to b6) of fixed and immunostained cryosections from dorsal inoculum of CSs from MLC3F-nlacZ/CD-1 and cTnI-lacZ/CD-1 mice. Tubular structures were stained for sarcomeric myosin (b3 to b5) and cTnI (b4 through b6). X-Gal staining labeled the cells within and those migrating from CS (b2). Endothelial markers (SMA and vascular endothelialcadherin) stained the vasculature (black holes) (a3 and b1).
To test the acquisition of functional competence and the cardiac regenerative potential of the CSs when challenged into an infarcted myocardium, orthotopic transplantation experiments with human CSs were performed. To perform these, thawed (cryopreserved) adult human CSs from three atrial (one male and two female) and one ventricular (one female) biopsy specimens were injected into the viable myocardium bordering a freshly produced infarct. Each mouse received CSs from a single passage of an explant (derived from a single subject). Four control infarcted animals were injected with an equal volume of PBS. Eighteen days after the intervention, the animals were euthanized and infarct size was determined. Infarct size was 34.97.1 (SEM, 3.6) and 31.96.9 (SEM, 3.5) in the CS-treated group and PBS-injected group, respectively (P=NS). However, echocardiography showed better preservation of the infarcted anterior wall thickness in the CS-treated group compared with the PBS-injected group (0.800.29 [SEM, 0.15] versus 0.600.20 [SEM, 0.08]) (P=NS), particularly of percent fractional shortening (36.8516.43 [SEM, 8.21] versus 17.875.95 [SEM, 2.43]) (P<0.05) (Figure 6 and the Table).
Figure 6. In vivo analysis (orthotopic transplantation of human CSs). Orthotopic transplantation performed in a SCID-beige mouse. Cryopreserved human CSs were transplanted into the viable myocardium bordering a freshly produced infarct. Confocal analysis of cryosectioned left ventricular heart 18 days after the coronary ligature shows that (a) cardiomyocytes expressing MHC (red) in the regenerating myocardium (particularly those indicated by the two central arrows) also stain positive for lamin A/C (green) (a specific human nuclear marker). In these cells, MHC expression is evident mainly in the perinuclear area (see Supplementary Figure X). Lamin A/C-labeled cells (red) are present in newly generated capillaries staining for -SMA (b1 through d), and platelet endothelial cell adhesion molecule (c). d, Confocal analysis of colocalization of lamin A/C-labeled cells (red) with the newly generated capillaries staining for -smooth muscle actin. e, Low-magnification image shows viable lamin A/C-expressing cells (green) in regenerating myocardium expressing MHC (red).
Myocardial Repair (Echocardiography)
At the time of evaluation, bands of regenerating myocardium were present (with different degrees of organization and thickness) throughout most of the infarcted areas, as evaluated with hematoxylineosin histochemistry (data not shown) and MHC immunofluorescence (Supplementary Figure IXa1 and IXa2). In the regenerating myocardium, cells expressing lamin A/C (a specific human nuclear marker) also colocalize with cardiomyocytes stained positive for MHC (Figure 6a and 6e and Supplementary Figures IXb1, IXb2, and X), newly generated capillaries stained for -SMA (Figure 6b1, 6b2, and 6d) and platelet endothelial cell adhesion molecule (Figure 6c), and with connexin-43expressing cells (data not shown).
CSs appear to be a mixture of cardiac stem cells, differentiating progenitors, and even spontaneously differentiated cardiomyocytes. Vascular cells were also present, depending on the size of the sphere and time in culture. It is possible that, as for neurospheres,19 differentiating/differentiated cells stop dividing and/or die, whereas stem cells continue to proliferate in an apparently asymmetric way, giving rise to many secondary spheres and to exponential growth in vitro. Mechanical dissociation favors this process. Death, differentiation, and responsiveness to growth factors of the different cells within the CSs could depend on the three-dimensional architecture and on localization within the CSs.20 The spontaneous formation of spheres is a known prerogative of neural stem cells, some tumor cell lines (LIM),21 endothelial cells,22 and fetal chicken cardiomyocytes.23 All these models (ours included) that mimic the true three-dimensional architecture of tissues consist of spheroids of aggregated cells that develop a two-compartment system composed of a surface layer of differentiated cells and a core of unorganized cells that first proliferate and then disappear over time (perhaps through apoptotic cell death). As well-documented in fetal chick cardiomyocytes and endothelial cell spheroid culture, three-dimensional structure affects the sensitivity of cells to survival and growth factors.21,22 In particular, central spheroid cells do not differentiate and are dependent on survival factors to prevent apoptosis, whereas the cells of the surface layer seem to differentiate beyond the degree that can be obtained in two-dimensional culture and become independent of the activity of survival factors.23 Furthermore, cellcell contact and membrane-associated factors, known to be important for the division of neural precursor cells,24 could be involved in our system. This is in accordance with the notion that stem cells (or cells with stem cell function) will only retain their pluripotency within an appropriate environment, as suggested by the niche hypothesis.25
Thus CSs can be considered clones of adult stem cells, maintaining their functional properties in vitro and in vivo after cryopreservation.
While the experiments performed for this article were ongoing, two articles were published concerning the isolation of cardiac stem cells or progenitor cells from adult mammalian hearts.26,27 Isolation of these cells was based exclusively on the expression of a stem cell-related surface antigen: c-kit in the first article and Sca-1 in the second one. In the first study,26 freshly isolated c-kit+ Lin cells from rat hearts were found to be self-renewing, clonogenic, and multi-potent, exhibiting biochemical differentiation into the myogenic cell, smooth muscle cell, or endothelial cell lineage but failing to contract spontaneously. When injected into an ischemic heart, these cells regenerated functional myocardium. In the second study,27 Sca-1+ cKit cells from mice hearts were induced in vitro to differentiate toward the cardiac myogenic lineage in response to 5-azacytidine. When given intravenously after ischemia/reperfusion, these cells targeted injured myocardium and differentiated into cardiomyocytes, with and without fusion with the host cells. Our data obtained on GFP-cKit transgenic mice also suggest that the adult cardiac stem cell is cKit+. It is possible that CSs enclose a mixed population of cells that, as in the niche, could promote the viability of cKit progenitors and contribute to their proliferation. The data obtained in the present article confirm the existence of adult cardiac stem cells/progenitor cells. More importantly, they demonstrate for the first time to our knowledge that it is possible to isolate cells from very small fragments of human myocardium and expand these cells in vitro many-fold (reaching numbers that would be appropriate for in vivo transplantation in patients) without losing their differentiation potential. Previously unforeseen opportunities for myocardial repair could now be identified.
Excerpt from:
Isolation and Expansion of Adult Cardiac Stem Cells From ...
Recommendation and review posted by simmons
Treatment Options for Hypogonadism Healthline
The sex glands, also known as gonads, primarily consist of the testes in men and the ovaries in women. These glands produce sex hormones such as testosterone and estrogen. Sex hormones help control secondary sex characteristics, including breast development in women, testicular development in men, and pubic hair growth in both sexes. They also play a role in the menstrual cycle and sperm production.
Hypogonadism develops when the sex glands produce little or no sex hormones, resulting in reduced function of the testes in males and ovaries in females. The condition can be present at birth, but it may also develop after infection or injury.
There are two types of hypogonadism.Primary hypogonadismoriginates from a problem with the testes or the ovaries, causing the sex glands to produce fewer sex hormones.
Secondary hypogonadism, also known as hypogonadotropic hypogonadism, is caused by a problem with the pituitary gland or hypothalamus. The hypothalamus and pituitary gland are located in the brain and help regulate various body functions, including the production of sex hormones. Disorders affecting these parts of the brain can result in lowered function of the sex glands and insufficient amounts of sex hormones.
The complications of hypogonadism in newborns may include abnormal genitals. In pubescent boys, a lack of treatment can lead to impaired genital growth, the absence of body hair, and enlarged breasts.
The complications of hypogonadism in untreated adult males include:
The complications of hypogonadism in untreated females include:
Hypogonadism is usually treated with hormone replacement therapy (HRT). However, your course of treatment may differ depending on the exact cause of your condition. The symptoms of hypogonadism often improve significantly with the proper treatment.
In most cases, hypogonadism can be treated effectively with HRT. This treatment consists of taking medications containing the hormone that your body is lacking, such as testosterone, estrogen and progesterone, or pituitary hormones to replace the ones that the body no longer produces.
Adult males can be treated with testosterone replacement therapy if their condition is caused by testicular failure. This treatment can:
In young boys and adolescent males, low doses of testosterone over time can be used to replace naturally occurring testosterone during puberty. This also lowers the risk of negative side effects from taking hormones. Aside from stimulating puberty, testosterone replacement therapy for young males can:
Women who have a decreased sex drive may also benefit from low-dose testosterone.
Testosterone replacement therapy can be administered is several ways, including the following:
You or a healthcare provider can inject testosterone into a muscle, usually at two-week intervals.
You can rub a clear gel containing testosterone onto the skin of your shoulder, upper arm, or lower abdomen. After applying the gel, you must avoid bathing for several hours so your skin has time to absorb the testosterone properly. The gel can also be transferred to someone else through direct contact, so make sure you refrain from skin-to-skin contact until the gel has dried.
You can place a skin patch containing testosterone on your body at night. The patch should be switched to a different area of the body every few weeks. This helps to reduce the risk of having an adverse site reaction. You may want to consider rotating where you place the patch. You can place the patch on your:
You can take testosterone in a pill form. Over time, however, oral testosterone can cause cholesterol levels to rise and can increase your risk of heart and liver problems. For these reasons, it usually isnt considered for long-term use.
You can apply a small patch containing testosterone to your upper gum, above your front teeth. This is called a buccal patch. The patch softens and releases the hormone gradually. Its generally applied every 12 hours on alternating sides. The gum looks like a tablet, but you should never chew or swallow it.
You and your doctor can discuss which method would be best for you.
For females, treatment for hypogonadism mostly consists of increasing the amount of female sex hormones in the body. Increasing levels of estrogen and progesterone can help strengthen bones, improve cholesterol levels, and support sex drive.
If youre a premenopausal female, you can benefit from estrogen that comes in pill or patch form. Estrogen and progesterone are sometimes combined to lower the chances of developing endometrial cancer.
Pituitary hormones can help treat hypogonadism that was caused by a problem with the pituitary gland. In adults, pituitary hormone replacement administered in pill form can increase sperm production. In boys and adolescent males, it can help promote growth of the testicles.
If a tumor is found on the pituitary gland, it can be treated with surgery, medication, or radiation therapy.
Testosterone replacement tends to increase the risk of urinary problems. It may also increase the risk of edema, or water retention, in people who have heart, liver, or kidney problems. Testosterone therapy may even aggravate sleep apnea or interfere with male fertility.
When used for an extended period, oral testosterone can increase your risk of liver problems, heart disease, and high cholesterol.
Your doctor will monitor your blood counts and hormone levels during treatment, and they can make adjustments if necessary. This will help reduce the risks associated with HRT.
If youre a male, then your doctor will also perform prostate screening tests to check prostate-specific antigen levels for signs of serious medical conditions. These tests will need to be done every three, six, and 12 months while you receive HRT.
Hypogonadism can take an emotional toll, but there are things you can do to minimize stress, including:
Here is the original post:
Treatment Options for Hypogonadism Healthline
Recommendation and review posted by simmons
Hypogonadism – Medscape
Back
Morbidity for men and women with hypogonadism includes infertility and an increased risk of osteoporosis; there is no increase in mortality.
Hypogonadotropic hypogonadism (see the image below) is one of several types of hypogonadism.
History
Considerations in the evaluation of males with hypogonadism include the following:
Developmental anomalies associated with the genital system (eg, hypospadias, micropenis, and cryptorchidism)[1]
For postpubertal males, the rate of beard growth, libido and sexual function, muscle strength, and energy levels
Possible causes of acquired testicular failure (eg, mumps orchitis, trauma, radiation exposure of the head or testes, and chemotherapy)
Drugs that may interrupt testicular function: Including agents that interfere with testosterone synthesis, such as spironolactone, cyproterone, marijuana, heroin, and methadone
Considerations in the evaluation of females with hypogonadism include the following:
Signs associated with Turner syndrome (eg, lymphedema, cardiac or renal congenital anomalies, and short growth pattern)
Age of menarche
Physical examination
Considerations in the physical examination of males with hypogonadism include the following:
Evaluation of the testes: This is the most important feature of the physical examination; determine whether both testes are palpable, their position in the scrotum, and their consistency; testes size can be quantitated by comparison with testicular models (orchidometer), or their length and width may be measured
Examination of the genitalia for hypospadias
Examination of the scrotum to see if it is completely fused
Evaluation of the extent of virilization
Staging of puberty: Use the Tanner criteria for genitalia, pubic hair, and axillary hair
Examination for signs of Klinefelter syndrome (eg, tall stature, especially if the legs are disproportionately long, gynecomastia, small or soft testes, and a eunuchoid body habitus)
Considerations in the physical examination of females with hypogonadism include the following:
Examination of the genitalia is important
Determination of the extent of androgenization: May be adrenal or ovarian in origin and is demonstrated in pubic and axillary hair
Determination of the extent of estrogenization: As evidenced by breast development and maturation of the vaginal mucosa
Examination for signs of Turner syndrome (eg, short stature, webbing of the neck [such as pterygium colli], a highly arched palate, short fourth metacarpals, widely spaced nipples, or multiple pigmented nevi)
See Clinical Presentation for more detail.
The following studies may be indicated in males with hypogonadism:
Follicle-stimulating hormone (FSH) levels
Luteinizing hormone (LH) levels
Prolactin levels
Testosterone levels
Thyroid function
Seminal fluid examination
Karyotyping
Testicular biopsy
For males after puberty, the Guidelines of the Endocrine Society[2] require that the diagnosis of hypogonadism be based on symptoms and signs of hypogonadism plus the presence of a low testosterone level measured on at least 2 occasions.
The following studies may be indicated in females with hypogonadism:
Additional tests in the evaluation of patients with hypogonadism include the following:
Adrenocorticotropic hormone (ACTH) stimulation testing: In patients in whom a form of congenital adrenal hyperplasia is suspected, adrenal steroid synthesis is best evaluated by performing a cosyntropin (ACTH 1-24) stimulation test
Luteinizing-hormone releasing hormone (LHRH) stimulation testing: To distinguish between true hypogonadotropic hypogonadism and constitutional delay in growth and maturation
Testicular tissue testing: If the testes are not palpable and if it is not certain whether any testicular tissue is present, administering human chorionic gonadotropin (hCG) and measuring testosterone response may be helpful
See Workup for more detail.
Hormonal replacement
The simplest and most successful treatment for males and females with either hypergonadotropic or hypogonadotropic hypogonadism is replacement of sex steroids, but the therapy does not confer fertility or, in men, stimulate testicular growth.
When fertility is desired, an alternative therapy for men with hypogonadotropic hypogonadism is administration of pulsatile LHRH or injections of hCG and FSH. (In patients with hypergonadotropic hypogonadism, fertility is not possible.)
In a 6-year European study of men being treated for hypogonadism, long-term transdermal testosterone treatment did not increase prostate-specific antigen (PSA) levels or influence prostate cancer risk.[3, 4]
Investigators used data from a 5-year, open-label extension of a 1-year trial of a transdermal testosterone patch (Testopatch) in men with hypogonadism. Study subjects wore two 60 cm2 patches, each of which delivered 2.4 mg of testosterone per day. More than 90% of patients had PSA concentrations below 2 ng/mL during the 6-year study, and no prostate cancer was found in patients over the course of the trial.
See Treatment and Medication for more detail.
Hypogonadism manifests differently in males and in females before and after the onset of puberty.[5] If onset is in prepubertal males and testosterone replacement is not instituted, the individual has features of eunuchoidism, which include sparse body hair, poor development of skeletal muscles, and delay in epiphyseal closure, resulting in long arms and legs. When hypogonadism occurs in postpubertal males, lack of energy and decreased sexual function are the usual concerns. In females with hypogonadism before puberty, failure to progress through puberty or primary amenorrhea is the most common presenting feature. When hypogonadism occurs in postpubertal females, secondary amenorrhea is the usual concern.
The gonad (ovary or testis) functions as part of the hypothalamic-pituitary-gonadal axis. A hypothalamic pulse generator resides in the arcuate nucleus, which releases luteinizing hormone (LH)-releasing hormone (LHRH), which is also termed gonadotropin-releasing hormone (GnRH), into the hypothalamic-pituitary portal system. Data suggest that a gene named KISS is important in the development of the LHRH-secreting cells.[6, 7]
In response to these pulses of LHRH, the anterior pituitary secretes follicle-stimulating hormone (FSH) and LH, which, in turn, stimulate gonadal activity. The increase in gonadal hormones results in lowered FSH and LH secretion at the pituitary level, completing the feedback loop. In the testes, LH stimulates Leydig cells to secrete testosterone, whereas FSH is necessary for tubular growth. In the ovaries, LH acts on theca and interstitial cells to produce progestins and androgens, and FSH acts on granulosa cells to stimulate aromatization of these precursor steroids to estrogen.
Hypogonadism may occur if the hypothalamic-pituitary-gonadal axis is interrupted at any level. Hypergonadotropic hypogonadism (primary hypogonadism) results if the gonad does not produce the amount of sex steroid sufficient to suppress secretion of LH and FSH at normal levels. Hypogonadotropic hypogonadism may result from failure of the hypothalamic LHRH pulse generator or from inability of the pituitary to respond with secretion of LH and FSH. Hypogonadotropic hypogonadism is most commonly observed as one aspect of multiple pituitary hormone deficiencies resulting from malformations (eg, septooptic dysplasia, other midline defects) or lesions of the pituitary that are acquired postnatally. In 1944, Kallmann and colleagues first described familial isolated gonadotropin deficiency. Recently, many other genetic causes for hypogonadotropic hypogonadism have been identified.
Normosmic hypogonadotropic hypogonadism, in which the sense of smell is not disrupted, has been associated with mutations in GNRH1, KISS1R, and GNRHR genes. Although their exact functions are unclear, the genes TAC3 and TACR3 have also been associated with normosmic hypogonadotropic hypogonadism. Kallmann syndrome (anosmic hypogonadotropic hypogonadism) has been associated with mutations in KAL1, FGFR1, FGF8, PROK2, and PROKR2 genes. The relationship with Kallmann syndrome is thought to be because these genes are all related to the development and migration of GnRH neurons. Mutations of an additional gene, CHD7, which has been associated with CHARGE syndrome, has also been found in patients with both normosmic or anosmic hypogonadotropic hypogonadism.
In women with hypergonadotropic hypogonadism (ie, gonadal failure), the most common cause of hypogonadism is Turner syndrome, which has an incidence of 1 case per 2,500-10,000 live births. In men with hypergonadotropic hypogonadism, the most common cause is Klinefelter syndrome, which has an incidence of 1 case per 500-1000 live births. Hypogonadotropic hypogonadism is more rare.
No racial predilection has been described.
Hypergonadotropic hypogonadism is more common in males than in females because the incidence of Klinefelter syndrome (the most common cause of primary hypogonadism in males) is higher than the incidence of Turner syndrome (the most common cause of hypogonadism in females). Incidence of hypogonadotropic hypogonadism is equal in males and females.
Hypogonadism may occur at any age; however, consequences differ according to the age at onset. If hypogonadism occurs prenatally (even if incomplete), sexual ambiguity may result. If hypogonadism occurs before puberty, puberty does not progress. If hypogonadism occurs after puberty, infertility and sexual dysfunction result.
No increase in mortality is observed in patients with hypogonadism. Morbidity for men and women includes infertility and an increased risk ofosteoporosis. In women, an increased risk of severe osteoporosis is noted. In men, hypogonadism causes decreased muscle strength and sexual dysfunction.
Men and women with hypogonadism can lead a normal life with hormone replacement.
Approximately 20-25% of females with Turner syndrome have some spontaneous puberty. Spontaneous estrogenization occurs more commonly in women with mosaic karyotypes and those karyotypes with an abnormal second X chromosome, such as 46,XXiq or 46,XXip. Reports exist of women with mosaic Turner syndrome becoming pregnant without in vitro fertilization.
For patient education resources, see theMen's Health CenterandWomen's Health Center, as well asImpotence/Erectile DysfunctionandAmenorrhea.
For both males and females with hypogonadism, determining whether evidence of a genital abnormality is present at birth or determining the timing and extent of puberty is important. In addition, because Kallmann syndrome (hypogonadotropic hypogonadism and anosmia [ie, lack of a sense of smell]) is a common cause of hypogonadotropic hypogonadism, inquiring about the sense of smell is important.
Physical findings may include the following:
The following causes of hypogonadism are noted:
Hypogonadotropic hypogonadism
See the image below.
Causes of hyogonadotropic hypogonadism include the following:
CNS disorders
Hypergonadotropic hypogonadism in males
Hypergonadotropic hypogonadism in females
Genetics of hypogonadotropic hypogonadism
To date, numerous genes have been identified as causes of hypogonadotropic hypogonadism. The genes include the following:
KALis located on the X chromosome, just below the pseudoautosomal region. An abnormality in this gene results in Kallmann syndrome, which is characterized by anosmia and hypogonadotropic hypogonadism.FGFR1, FGF8, PROK2,andPROKR2have also been associated with Kallmann syndrome. The relationship with Kallmann syndrome is thought to be due to the relation of these genes to the development and migration of gonadotropin-releasing hormone (GnRH) neurons.
TheDAX1gene is associated with X-linked adrenal hypoplasia congenita (hypogonadotropic hypogonadism and adrenal insufficiency).
GNRHRis the gene associated with the GnRH (LHRH) receptor.
GNRH1, KISS1R,andGNRHRgenes have been associated with normosmic (sense of smell is not disrupted) hypogonadotropic hypogonadism.
TAC3andTACR3mutations have also been associated with normosmic hypogonadotropic hypogonadism, although their exact functions are unclear.
CHD7mutation, which has been associated with CHARGE syndrome, has also been found in patients with both normosmic and anosmic hypogonadotropic hypogonadism.
PC1is the gene for prohormone convertase 1. Abnormality of this gene causes hypogonadotropic hypogonadism and defects in prohormone processing.
In addition, mutations in thePROP1gene have resulted in absence of several pituitary hormones, including growth hormone, thyroid-stimulating hormone, prolactin, and gonadotropins.PROP1encodes a protein expressed in the embryonic pituitary, which is necessary for function ofPOU1F1(formerlyPIT1), which codes for a pituitary transcription factor.
In addition, mutation of the geneHESX1has been associated with septooptic dysplasia, which may include poor development of the pituitary.
The following studies may be indicated in hypogonadism:
Pelvic ultrasonography may be helpful in females.
Adrenocorticotropic hormone (ACTH) stimulation testing: In patients in whom a form of congenital adrenal hyperplasia is suspected, adrenal steroid synthesis is best evaluated by performing a cosyntropin (ACTH 1-24) stimulation test. Baseline serum adrenocortical hormone levels are measured, then 0.25 mg of cosyntropin is intravenously injected, and serum hormone levels are remeasured after 60 minutes. Precursor product ratios are compared with those in age-matched control subjects to determine whether a steroidogenic defect is involved in sex hormone synthesis.
Luteinizing-hormone releasing hormone (LHRH) stimulation testing: To distinguish between true hypogonadotropic hypogonadism and constitutional delay in growth and maturation, performing a stimulation test with LHRH may be helpful.
LHRH is intravenously injected, and LH and FSH levels are determined at 15-minute intervals following LHRH administration.
A shortened version of the study has been used, in which LHRH is subcutaneously injected, and the specimen for LH and FSH levels is taken at 30-40 minutes.
Obtaining LHRH for testing over the past several years has been difficult. Some centers have substituted testing LH response to aqueous leuprolide.
Testicular tissue testing: If testes are not palpable and whether any testicular tissue is present is unclear, administering human chorionic gonadotropin (hCG) and measuring testosterone response may be helpful.
Bone age may be helpful in distinguishing hypogonadism from constitutional delay in growth and maturation. Timing of onset of puberty is related more to bone age than to chronologic age. Distinguishing hypogonadism from constitutional delay in growth and maturation is often difficult until the bone age is at a point adequate for pubertal development.
Occasionally, testicular biopsy findings are helpful, particularly if azoospermia or oligospermia is present.
In prepubertal patients with hypogonadism, treatment is directed at initiating pubertal development at the appropriate age. All such treatment is hormonal replacement therapy. Although the simplest and most successful treatment for both males and females with either hypergonadotropic or hypogonadotrophic hypogonadism is replacement of sex steroids, in hypogonadotropic hypogonadism, the therapy does not confer fertility or, in men, stimulate testicular growth.
An alternative for men with hypogonadotropic hypogonadism has been treatment with pulsatile LHRH or hCG, either of which can stimulate testicular growth. Because such treatment is more complex than testosterone replacement, and because treatment with testosterone does not interfere with later therapy to induce fertility, most male patients with hypogonadotropic hypogonadism prefer to initiate and maintain virilization with testosterone.
At a time when fertility is desired, it may be induced with either pulsatile luteinizing hormone-releasing hormone (LHRH) or (more commonly) with a schedule of injections of human chorionic gonadotropin (hCG) and follicle-stimulating hormone (FSH).
Read more:
Hypogonadism - Medscape
Recommendation and review posted by Bethany Smith
Hypogonadism | Conditions & Treatments | UCSF Medical Center
Hypogonadism is a condition that causes decreased function of the gonads, which are the testis in males and the ovaries in females, and the production of hormones that play a role in sexual development during puberty. You may be born with the condition or it can develop later in life from injury or infection. Some types of hypogonadism can be treated with hormone replacement therapy.
There are two forms of the condition primary hypogonadism resulting from problems of the testis or ovary and central hypogonadism caused by problems with the pituitary or hypothalamic glands. Central hypogonadism leads to decreased levels of luteinizing hormone (LH) and follicle stimulating hormones (FSH), released by the pituitary gland.
The condition may have genetic, menopausal autoimmune and viral causes or may develop after cancer treatments such as radiation and chemotherapy.
Fasting, weight loss, eating disorders such as anorexia nervosa, and bulimia, and stressful conditions can cause the condition.
In children before puberty, hypogonadism causes no symptoms. In adolescents, it can delay or prevent exual development.
Adult women with the condition may stop menstruating or develop infertility, loss of libido, vaginal dryness and hot flashes. Prolonged periods of hypogonadism can cause osteoporosis.
Men with the condition may experience loss of libido, erectile dysfunction and infertility.
To diagnose hypogonadism, tests may be performed to check hormone levels estogren in females and testosterone in males. In addition, levels of luteinizing hormone (LH) and follicle stimulating hormones (FSH) will be tested. LH and FSH are pituitary hormones that are stimulated by the gonads.
Other tests may measure thyroid hormones, sperm count and prolactin, a hormone released by the pituitary gland that stimulates breast development and milk production Tests also may be performed to test for anemia and possible genetic causes of symptoms.
For women, your doctor may request a sonogram of your ovaries.
If pituitary disease is suspected, a magnetic resonance imaging (MRI) scan or computed tomography (CT) scan may be performed to examine the the pituitary gland.
Hormone replacement therapy has proven to be effective treatment for hypogonadism in men and pre-menopausal women.
Estrogen may be administered in the form of a patch or pill. Testosterone can be given by a patch, a product soaked in by the gums, a gel or by injection.
For women who have not had their uterus removed, a combination of estrogen and progesterone is often recommended to decrease the chance of developing endometrial cancer. Low-dose testosterone may be added for women with hypogonadism who have a low sex drive.
Other hormones may be prescribed to restore fertility in men and women.
Reviewed by health care specialists at UCSF Medical Center.
The rest is here:
Hypogonadism | Conditions & Treatments | UCSF Medical Center
Recommendation and review posted by Bethany Smith
Dr. Komer The Komer Clinics – Treatment, Education …
The CORNERSTONE of The Komer Method is the attitude that only OPTIMAL is acceptable.
This encompasses optimal levels for hormones, blood chemistry, nutrients, supplements, nutrition, exercise and behavior for each individual.
Many lab ranges are very wide and are accepted as normal. Large portions of these ranges include levels that are far from ideal. The Komer Method has developed its own set of optimal ranges, and strives to achieve these for each patient.
When physicians treat abnormal blood sugars or high cholesterol or high blood pressure, they choose the ideal standard as the levels for a healthy young adult. However, when the same physicians correct abnormal hormone levels, they do not follow this practice. In fact, they will accept aging and deterioration of hormone levels as a normal event. Dr. Komers belief, and what has worked so successfully in his practice, is that achieving these optimum levels at any age fine tunes the body to minimize the effects of age, time and stress. These levels result in men and women who are their healthiest. They lead to a reduction in long term illness and an increase in wellbeing.
These optimal values have been developed over years of experience, by research by Dr. Komer and others, and by patients reporting back what makes them feel their very best.
The Komer Method has developed its own protocols for diagnosis and for treatment for various conditions, and these have been tested and improved in thousands of individuals. Innovation and continuous improvement of all protocols is ongoing. Medical literature is reviewed daily to integrate new research into The Komer Method.
A major emphasis of the Komer Method has been dedicated to achieving ideal hormone levels in both genders, for such conditions as menopause in women and low testosterone in men.
Dr. Komer also treats other individuals, including both pro and amateur athletes, suffering from concussions or injuries. He has a particularly large percentage of men and women who are in the military, are police officers, correctional officers or firemen. These professions involve stress and long hours and sometimes trauma, which can lead to abnormal hormone levels. There is especially a need for understanding, assessment and treatment of these individuals.
Dr. Komer has been an innovator in bringing new techniques and new ideas to medicine and has, at times, stood alone in championing ideas that have turned out to be leading edge concepts in medicine. In his personal practice, he has over 12,000 women and 5,000 men in his program who are reaping the benefits of hormone restoration. They are feeling well and happy, functioning optimally and staying healthy.
Continue reading here:
Dr. Komer The Komer Clinics - Treatment, Education ...
Recommendation and review posted by simmons
Dr. Komer, MD – Your Temporary Index
The CORNERSTONE of The Komer Method is the attitude that only OPTIMAL is acceptable.
This encompasses optimal levels for hormones, blood chemistry, nutrients, supplements, nutrition, exercise and behavior for each individual.
Many lab ranges are very wide and are accepted as normal. Large portions of these ranges include levels that are far from ideal. The Komer Method has developed its own set of optimal ranges, and strives to achieve these for each patient.
When physicians treat abnormal blood sugars or high cholesterol or high blood pressure, they choose the ideal standard as the levels for a healthy young adult. However, when the same physicians correct abnormal hormone levels, they do not follow this practice. In fact, they will accept aging and deterioration of hormone levels as a normal event. Dr. Komers belief, and what has worked so successfully in his practice, is that achieving these optimum levels at any age fine tunes the body to minimize the effects of age, time and stress. These levels result in men and women who are their healthiest. They lead to a reduction in long term illness and an increase in wellbeing.
These optimal values have been developed over years of experience, by research by Dr. Komer and others, and by patients reporting back what makes them feel their very best.
The Komer Method has developed its own protocols for diagnosis and for treatment for various conditions, and these have been tested and improved in thousands of individuals. Innovation and continuous improvement of all protocols is ongoing. Medical literature is reviewed daily to integrate new research into The Komer Method.
A major emphasis of the Komer Method has been dedicated to achieving ideal hormone levels in both genders, for such conditions as menopause in women and low testosterone in men.
Dr. Komer also treats other individuals, including both pro and amateur athletes, suffering from concussions or injuries. He has a particularly large percentage of men and women who are in the military, are police officers, correctional officers or firemen. These professions involve stress and long hours and sometimes trauma, which can lead to abnormal hormone levels. There is especially a need for understanding, assessment and treatment of these individuals.
Dr. Komer has been an innovator in bringing new techniques and new ideas to medicine and has, at times, stood alone in championing ideas that have turned out to be leading edge concepts in medicine. In his personal practice, he has over 12,000 women and 5,000 men in his program who are reaping the benefits of hormone restoration. They are feeling well and happy, functioning optimally and staying healthy.
Read more here:
Dr. Komer, MD - Your Temporary Index
Recommendation and review posted by Bethany Smith
Newly identified stem cell population in skin’s epidermis …
Researchers at the Universit Libre de Bruxelles, ULB identify a new stem cell population in the skin epidermis responsible for tissue repair.
The skin, which is an essential barrier that protects our body against the external environment, undergoes constant turnover throughout life to replace dead cells that are constantly sloughed off from the skin surface. During adult life, the number of cells produced must exactly compensate the number of cells lost. Different theories have been proposed to explain how this delicate balance is achieved.
In a new study published in Nature, researchers lead by Pr. Cdric Blanpain, MD/PhD, FNRS/FRS researcher and WELBIO investigator at the IRIBHM, Universit libre de Bruxelles, Belgium, in collaboration with Pr. Benjamin Simons, University of Cambridge, UK, demonstrate the existence of a new population of stem cells that give rise to progenitor cells that ensure the daily maintenance of the epidermis and demonstrate the major contribution of epidermal stem cells during wound healing.
In this new study, Guilhem Mascr and colleagues used novel genetic lineage tracing experiments to fluorescently mark two distinct epidermal cell populations, and follow their survival and contribution to the maintenance of the epidermis overtime. Interestingly, in doing so, they uncover the existence of two types of dividing cells. One population of proliferative cells presented a very long term survival potential while the other population is progessively lost overtime. In collaboration with Pr. Benjamin D. Simons, the authors developed a mathematical model of their lineage tracing analysis. The authors proposed that the skin epidermis is hierarchically organized with slow cycling stem cells residing on the top of the cellular hierarchy that give rise to more rapidly cycling progenitor cells that ensure the daily maintenance of the skin epidermis. Analysis of cell proliferation confirms the existence of slow cycling stem cells and gene profiling experiments demonstrate that the stem and the progenitors cells are characterized by distinct gene expression.
Importantly, by assessing the contribution these two populations of cells during wound healing, they found that only stem cells are capable of extensive tissue regeneration and undergo major expansion during this repair process, while the progenitors did not expand significantly, and only provide a short-lived contribution to the wound healing response. As well as resolving the cellular hierarchy of epidermis, this is the first demonstration of a critical role of epidermal SC during wound healing. "It was amazing to see these long trails of cells coming from a single stem cell located at a very long distance from the wound to repair the epidermis" comments Cdric Blanpain, the senior author of this study.
In conclusion, this work demonstrates the existence of slow-cycling stem cells that promote tissue repair and more rapidly cycling progenitors that ensure the daily maintenance of the epidermis. A similar population of slow cycling stem cells that can be rapidly mobilized in case of sudden need has been observed in other tissues, such as the blood, muscle and hair follicle, and the partition between rapidly cycling progenitors and slow cycling stem cells could be relatively conserved across the different tissues. This study may have important implications in regenerative medicine in particular for skin repair in severely burnt patients or in chronic wounds.
This work was supported by the FNRS, the " Brain back to Brussels " program from the Brussels Region, the program d'excellence CIBLES of the Wallonia Region, a research grant from the Fondation Contre le Cancer, the ULB foundation, the fond Gaston Ithier. Cdric Blanpain is an investigator of WELBIO and is supported by a starting grant of the European Research Council (ERC) and the EMBO Young Investigator Program.
Story Source:
The above post is reprinted from materials provided by Libre de Bruxelles, Universit. Note: Materials may be edited for content and length.
See the article here:
Newly identified stem cell population in skin's epidermis ...
Recommendation and review posted by simmons
Human cardiac stem cells – PNAS
Communicated by Eugene Braunwald, Harvard Medical School, Boston, MA, July 19, 2007 (received for review May 2, 2007)
The identification of cardiac progenitor cells in mammals raises the possibility that the human heart contains a population of stem cells capable of generating cardiomyocytes and coronary vessels. The characterization of human cardiac stem cells (hCSCs) would have important clinical implications for the management of the failing heart. We have established the conditions for the isolation and expansion of c-kit-positive hCSCs from small samples of myocardium. Additionally, we have tested whether these cells have the ability to form functionally competent human myocardium after infarction in immunocompromised animals. Here, we report the identification in vitro of a class of human c-kit-positive cardiac cells that possess the fundamental properties of stem cells: they are self-renewing, clonogenic, and multipotent. hCSCs differentiate predominantly into cardiomyocytes and, to a lesser extent, into smooth muscle cells and endothelial cells. When locally injected in the infarcted myocardium of immunodeficient mice and immunosuppressed rats, hCSCs generate a chimeric heart, which contains human myocardium composed of myocytes, coronary resistance arterioles, and capillaries. The human myocardium is structurally and functionally integrated with the rodent myocardium and contributes to the performance of the infarcted heart. Differentiated human cardiac cells possess only one set of human sex chromosomes excluding cell fusion. The lack of cell fusion was confirmed by the Cre-lox strategy. Thus, hCSCs can be isolated and expanded in vitro for subsequent autologous regeneration of dead myocardium in patients affected by heart failure of ischemic and nonischemic origin.
The recent identification of different classes of cardiac progenitor cells has suggested that the heart is not a terminally differentiated, postmitotic organ but an organ regulated by a stem cell compartment (1). The possibility has also been raised that stem cells are present in the normal and pathological human heart (2, 3). Together, these results point to a shift in paradigm concerning the biology of the heart and put forward potential therapeutic strategies for the failing heart. However, the actual existence of a human cardiac stem cell (hCSC) remains to be demonstrated. By definition, stem cells have to be self-renewing, clonogenic, and multipotent in vitro and in vivo (4, 5), and no studies to date have shown that the human heart contains primitive cells with these properties. Cells with limited growth and differentiation ability may acquire only the myocyte, endothelial cell (EC) or smooth muscle cell (SMC) lineage in vitro, and may not be capable of forming functionally competent myocardium in vivo. hCSCs have to be able to replace dead tissue with contracting myocardium composed of cardiomyocytes and vascular structures, independently from cell fusion. Heterokaryons divide poorly and have, at best, a transient positive impact on the age of the fused cells (6). Here, we report that these issues have been resolved, and hCSCs may represent a form of therapy for the diseased heart.
The documentation of hCSCs requires the identification of interstitial structures with the characteristics of stem cell niches and the recognition of the mechanisms of stem cell division that regulate niche homeostasis and the self-renewing properties of the human heart in vivo (7). We have found that the human heart contains clusters of hCSCs that are intimately connected by gap junctions and adherens junctions to myocytes and fibroblasts (Fig. 1 AC); myocytes and fibroblasts represent the supporting cells within the cardiac niches (7). Additionally, symmetric and asymmetric division of hCSCs was detected, respectively, by the uniform and nonuniform localization of the cell-fate determinants Numb and -adaptin (7) at one or both poles of hCSCs in mitosis (Fig. 1 D and E). The commitment to the myocyte lineage of hCSCs was also found within the niches. The coexpression of the stem cell antigen c-kit and myocyte transcription factors and sarcomeric proteins [see supporting information (SI) Fig. 6] is consistent with a lineage relationship between hCSCs and myocyte formation. C-kit POS cells expressing transcription factors for SMCs and ECs were also detected (data not shown). In the niches, hCSCs and committed cells were negative for hematopoietic markers and KDR (SI Table 1). These findings in the normal human heart, together with earlier observations in the diseased heart (3, 8), support the notion of a resident hCSC compartment that gives rise to the various cardiac cell progenies.
Cardiac niches and hCSC division. Sections of normal human myocardium. (AC) Cluster of c-kit POS cells (green). Arrows in A define the areas in B and C. Gap (connexin 43: Cx43, white; arrowheads) and adherens (N-cadherin: N-cadh, magenta; arrowheads) junctions are shown at higher magnification. Cx43 and N-cadh are present between c-kit POS cells and myocytes (-SA, red) and fibroblasts (procollagen, light blue); fibronectin, yellow. (D and E) Mitosis (phospho-H3, magenta; arrows) in c-kit POS cells; -adaptin (D, white) and Numb (E, yellow) show a uniform (D) and nonuniform (E) localization in the mitotic c-kit POS cells.
C-kit POS cells, i.e., hCSCs, were prepared with two methodologies. The first consisted of the enzymatic dissociation of myocardial samples from which c-kit POS cells were sorted with immunobeads and plated at low density (SI Fig. 7 AC ) to obtain multicellular clones from single founder cells. This procedure was dictated by the small size of the samples, 30 mg, which precluded FACS analysis at the outset. Successful isolation was obtained in 8 of 12 cases. The phenotype of the freshly isolated cells was characterized by FACS in 6 additional cases in which larger samples, 60 mg, were available. C-kit POS cells comprised 1.1 1.0% of the entire cell population. They were different from human bone marrow cells and were negative for markers of hematopoietic cells and KDR (Fig. 2 A and B; SI Table 2). Only small fractions of hCSCs expressed GATA4 and Nkx2.5, 0.5%.
Human CSCs. (A and B) Scatter plots of hCSCs (A) and human bone marrow cells (B). hCSCs do not express hematopoietic markers, KDR, GATA4, and Nkx2.5. (C) Nuclei (blue) of hCSCs were stained with a telomere probe (magenta). Lymphoma cells with short (7-kbp) and long (48-kbp) telomeres are shown for comparison. (D) Products of telomerase activity in hCSCs start at 50 bp and display a 6-bp periodicity. Samples treated with RNase and CHAPS buffer were used as negative controls, and HeLa cells were used as positive control. The band at 36 bp corresponds to an internal control for PCR efficiency. Optical density (arbitrary units, AU) is shown as mean SD.
With the second protocol, samples were cultured by the primary explant technique (SI Fig. 7 D and E ). Successful cell outgrowth was obtained in 46 of 70 cases. A monolayer of 6,000 cells was present at the periphery of each tissue aggregate, 3 weeks after seeding. C-kit POS cells accounted for 1.6 1.4%. Adherent cells at passage P0 were analyzed by immunocytochemistry and FACS (SI Fig. 8; SI Tables 1 and 2). In enzymatically dissociated cells, lineage negative (Lin) c-kit POS cells were 41 14%, and early committed cells (GATA4-positive) were 59 14%. Corresponding values with the primary explant technique were 52 12% and 48 12% (SI Fig. 9A ). In the presence of serum, hCSCs obtained with both protocols attached rapidly and continued to grow up to P8, undergoing 24 population doublings (PDs); the majority of experiments were concluded at P8. Cells maintained a stable phenotype and did not reach growth arrest. The percentage of c-kit POS cells did not vary from P1 to P8, averaging 71 8%. Undifferentiated cells constituted 63 6%. Ki67POS cycling-cells averaged 48 10%. p16INK4a, a cdk inhibitor, was present in 6 4% of the cells (SI Fig. 9 BD ). Thus, hCSCs are distinct from bone marrow cells and can be isolated and expanded in vitro.
To determine whether hCSCs reach senescence in culture, telomeric length was evaluated by Q-FISH (Fig. 2 C). From P3P4 (912 PDs) to P5P6 (1518 PDs) and P8P9 (2427 PDs), average telomere length in hCSCs decreased from 9.3 to 8.2 and 6.9 kbp, respectively (SI Fig. 10). From P3 to P9 there were 18 PDs with an average telomeric shortening of 130 bp per PD. This rate of telomere attrition is comparable with that of human bone marrow stem cells (9). Additionally, nearly 50% of the telomerase activity in hCSCs at P3P4 was still present at P8P9 (Fig. 2 D).
Critical telomere length associated with growth arrest and cellular senescence of hCSCs and human hematopoietic SCs varies from 2.0 to 1.5 kbp (3, 9). The fraction of hCSCs with critical telomeric shortening increased from 1% at P3P4 to 2% at P5P6 and 5% at P8P9. However, after 2427 PDs at P8P9, 69% hCSCs had telomere length 5.0 kbp (SI Fig. 10). It can be predicted that cells at P8P9 can undergo 23 additional PDs (52 = 3kbp/0.13kbp = 23 PDs) before irreversible growth arrest (10). In theory, 50 PDs can result in the formation of 1 1015 hCSCs before replicative senescence is reached. Thus, hCSCs can be extensively grown in vitro in the absence of a major loss in their expansion potential.
hCSCs obtained by enzymatic digestion and explant technique were plated at limiting dilution and in Terasaki plates, respectively. In the first case, 1,530 c-kit POS cells were seeded, and after 34 weeks, 11 clones were generated. In the second case, cells were placed in individual wells, and 53 clones were formed from 6,700 seeded cells. Thus, hCSCs had 0.70.8% cloning efficiency (Fig. 3 AC). Clones were expanded and characterized. Doubling time was 29 10 h, and 90 7% of cells after 5 days were BrdUPOS. Clonogenic hCSCs retained largely their primitive state and were negative for hematopoietic markers, KDR, and transcription factors and cytoplasmic proteins of cardiac cells (SI Fig. 11 A and B; SI Table 1).
In vitro properties of hCSCs. (AC) Clones formed by hCSCs (c-kit, green) isolated by enzymatic digestion (A and C) or primary explant (B). The number of cells increased with time (C). (D) hCSCs generate myocytes positive for cardiac myosin heavy-chain (MHC), -SA, and -cardiac-actinin (-actinin). Sarcomeres are apparent (Insets); phalloidin, green. (E) Myocyte shortening in cells derived from clonogenic hCSCs was recorded by two-photon microscopy and laser line-scan imaging (Left). The line scan is shown (Right), and arrowheads point to individual contractions. (F) Myocytes derived from EGFP-positive hCSCs, cocultured with neonatal myocytes. EGFP-positive human myocytes shorten (arrowheads) with electrical stimulation. (G) Calcium transients in EGFP-positive human myocytes and EGFP-negative rat myocytes (calcium indicator Rhod-2, red).
In differentiating medium, hCSCs gave rise to myocytes, SMCs, and ECs (Fig. 3 D; SI Fig. 11 C and D ). Developing myocytes had sarcomere striation (Fig. 3 D) and, after electrical stimulation at 1 Hz, showed contractile activity (Fig. 3 E). Moreover, hCSCs were infected with a lentivirus expressing EGFP and cocultured with neonatal rat myocytes. Two weeks later, cultures were stimulated, and 9% shortening of EGFP-positive human myocytes was detected (Fig. 3 F). In the presence of the calcium indicator Rhod-2, calcium transient was identified in EGFP-positive human myocytes and EGFP-negative rat myocytes (Fig. 3 G). Thus, hCSCs form multicellular clones and differentiate into contracting myocytes.
Nonclonogenic hCSCs, collected from eight patients, were injected in the infarcted mouse or rat heart to form chimeric organs containing human myocytes and coronary vessels. Cell treatment led to areas of myocardial regeneration that were located within the infarct and were positive for -sarcomeric actin (-SA) and human AluDNA sequences (Fig. 4 A). Human myocardium was found in 17 of 25 treated mice (68%), and 14 of 19 treated rats (74%). hCSCs were delivered with rhodamine-labeled microspheres for the recognition of the sites of injection and correct administration of cells (1). The absence of myocardial regeneration was due to technical failure to properly inject hCSCs in the rodent heart. Conversely, successful cell implantation was invariably associated with the presence of human myocardium.
hCSCs regenerate infarcted myocardium. (A) Mouse heart 21 days after infarction and injection of hCSCs. Human myocardium (arrowheads) is present within the infarct (MI). BZ, border zone. Areas in rectangles are shown at higher magnification below. Human myocytes are -SA- (red) and Alu- (green) positive. Asterisks indicate spared myocytes. (B) Expression of human (h) genes by real-time RT-PCR in treated infarcted rats at 511 and 1221 days. Clonogenic hCSCs were used for comparison of human transcripts. (C) Electrophoresis of real-time RT-PCR products (for sequences see SI Fig. 11J ).
The human myocardium comprised 1.3 0.9 mm3 in mice and 3.7 2.9 mm3 in rats. Accumulation of new cells was also determined by BrdU labeling because BrdU was given to the animals throughout the experiment (SI Fig. 12 A and B ). The human myocardium consisted of myocytes that occupied 84% of the tissue, whereas arterioles and capillaries accounted for 8%. The human origin of the myocardium was confirmed by the detection of human Alu and Mlc2v DNA by PCR in sections of regenerated infarcts (SI Fig. 12C ). PCR products had the expected molecular weight, and the nucleotide sequences confirmed the specificity of the assay (SI Fig. 12 DF ).
Three control groups were used: (i) unsuccessfully treated-animals (eight mice, five rats); (ii) immunodeficient infarcted mice (n = 12) and immunosuppressed infarcted rats (n = 9) injected with PBS; and (iii) immunosuppressed infarcted rats injected with c-kit-negative cells obtained from the unfractionated cell population at P1 (n = 16). Infarct size was similar in all groups: 48 9% in mice and 54 11% in rats. Myocardial regeneration was absent in control hearts with the exception of 3 of the 16 hearts treated with c-kit-negative cells. In one case, a few -SA and Alu-positive cells were found within the infarct, whereas, in the other two, a small band of human myocardium was identified near the border zone (SI Fig. 12 G and H ).
For completeness, clonogenic hCSCs were injected in infarcted rats shortly after coronary ligation to determine their multipotentiality in vivo (n = 6) and establish whether multipotentiality persisted when cell implantation was performed 5 days after coronary occlusion under the condition of a fully developed ischemic injury (n = 10). In both cases, clonogenic hCSCs regenerated the infarcted myocardium (SI Fig. 12I ) by forming human myocytes and coronary vessels (see below).
Real-time RT-PCR was used to demonstrate human transcripts for myocyte (MLC2v, connexin 43), SMC (smooth-muscle myosin heavy-chain 11 = Mhc 11) and EC (vWF) genes, and the housekeeping gene GAPDH in infarcted rat hearts treated with clonogenic hCSCs. Because there is no baseline in the rat myocardium for the analysis of human genes, clonogenic hCSCs were used for comparison (n = 4). With respect to clonogenic hCSCs, there was a substantial up-regulation of human myocardial transcripts for parenchymal and vascular cells in the infarcted heart (Fig. 4 B). The expression of human MLC2v, connexin 43, Mhc 11, and vWF increased from 511 days (n = 8) to 1221 days (n = 15) after infarction and cell implantation. RT-PCR products had the expected molecular weight (Fig. 4 C), and the nucleotide sequences confirmed the specificity of the assay (SI Fig. 12J ). Thus, hCSCs generate human myocardium.
After the identification of AluDNA, cardiac myosin heavy-chain (MHC), troponin I, and -SA were detected in human myocytes. Moreover, GATA4, MEF2C, connexin 43, and N-cadherin were identified (SI Figs. 13 and 14). Human myocytes varied in size from 100 to 2,900 m3 (SI Fig. 14). Human coronary arterioles and capillaries were also found (SI Figs. 13 and 14). The number of human arterioles and capillaries was comparable in rats and mice; there was one capillary/eight myocytes (SI Fig. 14), and the diffusion distance for oxygen averaged 18 m. These parameters are similar to those found in the late fetalneonatal human heart. Thus, hCSCs differentiate into human myocytes and coronary vessels, leading to the formation of a chimeric heart.
Two protocols were used to test whether the generation of human myocardium involved fusion events between hCSCs and rodent cells. hCSCs were infected with a lentivirus carrying Cre-recombinase (infection efficiency = 90%) and injected in the infarcted heart of mice expressing loxP-flanked EGFP (n = 6). If fusion were to occur, EGFP transcription would be activated in the recipient cells by Cre-mediated excision of the stop codon in the EGFP promoter (1). At 10 days after infarction and cell implantation, newly formed human myocardial cells showed a nuclear localization of Cre protein (Fig. 5 A; SI Fig. 15). However, human myocytes and vessels were negative for EGFP, indicating that the formation of heterokaryons was not involved in cardiac repair.
Integration of human myocardium. (A) Human myocytes are Cre-recombinase-positive (white) but EGFP negative. (B) Human myocytes and vessels show, at most, two human X-chromosomes (X-Chr, white dots; arrowheads). Mouse X-Chr (magenta dots; arrows) are present in myocytes of the border zone (BZ). (C) Transmural infarct in a treated rat; human myocardium (arrowheads) is present within the infarct. The area in the rectangle is shown at higher magnification (Bottom); human myocytes are -SA- (red) and Alu- (green) positive. Echocardiogram shows contraction in the infarcted wall (arrowheads). (D) Ventricular function. Results are mean SD. * and , Difference, P < 0.05, versus SO (sham-operated) and MI, respectively. (E) Calcium transient in EGFP-positive human myocytes and EGFP-negative mouse myocytes recorded by two-photon microscopy and laser line-scan imaging (calcium indicator Rhod-2, red). (F) Myocardial regeneration at 3 weeks. Connexin 43 (Cx43, yellow) is present between human-myocytes (-SA, red; Alu, green) and spared rat myocytes (-SA, red; Alu-negative). See Inset for higher magnification.
The second protocol consisted of the evaluation of the number of sex chromosomes by Q-FISH in human myocytes and coronary vessels. Because female human cells were injected in female mice and rats, human, rat and mouse X-chromosomes were measured. We never found a colocalization of a human X-chromosome with a mouse or rat X-chromosome in regenerated myocytes and vessels (Fig. 5 B). Human myocytes, SMCs, and ECs carried, at most, two human X-chromosomes. Thus, hCSCs form human myocardium independently from cell fusion.
Echocardiograms were examined retrospectively after the histological documentation of transmural infarcts and the presence of human myocardium (Fig. 5 C; SI Fig. 16 AC ). Tissue regeneration restored partly contractile function in the infarct, resulting in an increase of ejection fraction (SI Fig. 16D ), attenuation of chamber dilation (SI Fig. 16E ), and improvement of ventricular function (Fig. 5 D).
The interaction between human and rodent myocardium was determined by an ex vivo preparation and two-photon microscopy. EGFP-positive hCSCs were injected in infarcted mice, and the heart was studied 2 weeks later (n = 6). After the blockade of contraction and spontaneous activity, the heart was perfused with Rhod-2 and stimulated at 1 Hz. Calcium transient was recorded in EGFP-positive human myocytes and EGFP-negative mouse myocytes. The synchronicity in calcium tracings between these myocyte populations documented their functional integration (Fig. 5 E). hCSC-derived myocytes acquired the properties of the recipient rodent myocardium, indicating that primitive cells of human origin possess a high level of plasticity. Additionally, connexin 43 was found between human and rodent myocytes (Fig. 5 F) demonstrating their structural coupling. Thus, both myocardial components of the chimeric heart participate in the performance of the infarcted heart.
The current work demonstrates that the human heart possesses a pool of clonogenic hCSCs that can acquire the myocyte, SMC, and EC lineages in vitro and in vivo. The ability of hCSCs to create cardiomyocytes and coronary vessels in vivo provides strong evidence in favor of the role that hCSCs have in cardiac homeostasis and myocardial regeneration. Besides their therapeutic implications, these observations challenge the view of the heart as a postmitotic organ (11) and form the basis of a paradigm in which multipotent hCSCs modulate the physiological turnover of the heart. Understanding the mechanisms of cardiac homeostasis would offer the opportunity to potentiate this process and promote cardiac repair after injury.
Human cells with the ability to differentiate into cardiomyocytes have been obtained from myocardial biopsies and were claimed to possess the properties of stem cells (2). These cells express the typical markers of human circulating endothelial-progenitor cells (EPCs): CD34, CD31, and KDR, together with c-kit (12). The expression of CD34, CD31, and KDR does not compromise the ability of these circulating cells to acquire the myocyte lineage in vitro (13) and in vivo (14). The presence of these epitopes, however, suggests that these cells originate from the bone marrow and only subsequently accumulate within the heart. These early findings failed to provide evidence for the clonogenicity of these cells in vitro and their multilineage differentiation in vivo, which are critical for the recognition of a tissue-specific adult stem cell (1). The inability of these cells to generate a functional human myocardium in vivo is consistent with the role of EPCs in cardiac repair; they acquire, at low efficiency, the myocyte lineage and exert a paracrine effect on the infarcted heart (13).
Conversely, as demonstrated here, hCSCs are positive for the stem cell antigen c-kit but are negative for the hematopoietic and endothelial antigens CD45, CD34, CD31, and KDR; CD45 and KDR are typically expressed in a subset of bone marrow c-kit POS cells that have the ability to migrate to the heart after injury (12). Stem cell niches have been identified here in the normal human myocardium, and hCSCs divide symmetrically and asymmetrically and give rise to differentiating and lineage-negative cells. This provides evidence in favor of a linear relationship between hCSCs and myocyte formation. Additionally, these observations do not support the notion of dedifferentiation of mature myocytes with the acquisition of a stem cell phenotype. Importantly, clonogenic hCSCs have the inherent potential to form contracting myocardium integrated structurally and functionally with the recipient heart. Although CSCs with similar characteristics were shown in animal models (4, 5), the applicability of this information to humans was seriously questioned and considered a major limitation for the clinical implementation of CSCs (15).
In the current study, three possibilities were considered in the formation of human myocardium within the infarcted mouse and rat heart (1): (i ) Growth and differentiation of hCSCs; (ii ) Fusion of hCSCs with the surviving mouse or rat cardiac cells, followed by proliferation of the heterokaryons and generation of myocytes and coronary vessels; and (iii ) A combination of these two processes. The evaluation of human, mouse, and rat sex chromosomes together with the Cre-lox strategy has indicated that the generation of human myocardium involved only the commitment of hCSCs to cardiomyocytes, SMCs, and ECs. The unlikely involvement of cell fusion was supported by the size (1002,900 m3) of human myocytes. If fusion were to be implicated, the newly formed human myocytes should have had a volume of at least 25,000 m3 or larger, that is, the volume of adult mouse and rat cardiomyocytes. It is improbable that fusion of a primitive cell with a terminally differentiated myocyte can induce division of a highly specialized and rapidly contracting cell permanently withdrawn from the cell cycle (1, 6).
The identification of a resident hCSC pool in the human heart is apparently at variance with the small foci of myocardial regeneration present after acute and chronic infarcts or pressure overload in patients (3, 16). The limitation that resident hCSCs have in reconstituting myocardium after infarction has been interpreted as the unequivocal documentation of the inability of the adult heart to create cardiomyocytes (11). The inevitable evolution of ischemic injury is myocardial scarring with loss of mass and contractile function. A possible explanation of this apparent paradox has been obtained in animal models of the human disease (5). Stem cells are present throughout the infarcted myocardium but, despite the postulated resistance of these cells to death stimuli, they follow the same pathway of cardiomyocytes and die by apoptosis. The fate of hCSCs is comparable with that of the other cells, and myocyte formation is restricted to the viable portion of the human heart (3).
It might come as a surprise, but a similar phenomenon occurs in solid and nonsolid organs, including the skin, liver, intestine, and kidney. In all cases, occlusion of a supplying artery leads to scar formation mimicking cardiac pathology (1720). In the presence of polyarteritis nodosa and vasculitis, microinfarcts develop in the intestine and skin, and resident SCs do not repair the damaged organs (21). In nonsolid organs, infarcts of the bone marrow are seen with sickle cell anemia (21). Thus, the SC compartment appears to be properly equipped to modulate growth during postnatal development and regulate homeostasis in adulthood. However, SCs do not respond effectively to ischemic injury or, late in life, to aging and senescence of the organ and organism (22, 23).
Current knowledge supports the notion that primitive cells are present in the heart at the very beginning of embryonic life and regulate heart morphogenesis and postnatal development (24). By introducing the EGFP gene in the mouse embryo, at the stage of the morulablastocyst transition, the patterns of myocardial histogenesis have been defined and the presence of a common progenitor of cardiomyocytes in prenatal and postnatal life suggested (24). The documentation of myocardial specification of embryonic stem cells (25, 26), in particular c-kit POS Nkx2.5POS cells (26), supports the view that a pool of resident c-kit POS progenitors is implicated in cardiac morphogenesis. These findings are consistent with the existence of a pool of primitive cells in the adult human heart.
hCSCs have been isolated expanded and characterized in vitro and in vivo after implantation in the infarcted rodent heart. Protocols are described in SI Materials and Methods .
The lentivirus expressing Cre-recombinase was kindly provided by Drs. Chang and Terada (University of Florida, Gainesville, FL) and the lymphoma cells by Dr. Blasco (Spanish National Cancer Centre, Madrid, Spain). This work was supported by National Institutes of Health grants.
Author contributions: C.B., M.R., T.H., R.W.S., K.U., R.B., J.K., A.L., and P.A. designed research; C.B., M.R., T.H., J.T., A.N., A.D.A., S.Y.-A., I.T., R.W.S., N.L., S.C., A.P.B., D.A.D., E.Z., F.Q., K.U., R.E.M., J.K., and A.L. performed research; C.B., M.R., T.H., J.T., A.N., A.D.A., S.Y.-A., I.T., R.W.S., N.L., D.A.D., K.U., R.E.M., R.B., J.K., A.L., and P.A. analyzed data; and C.B., M.R., J.K., A.L., and P.A. wrote the paper.
Conflict of interest statement: P.A. has applied for a patent.
This article contains supporting information online at http://www.pnas.org/cgi/content/full/0706760104/DC1.
Read more here:
Human cardiac stem cells - PNAS
Recommendation and review posted by Bethany Smith
Network Of Doctors – Renew Man
Thanks to our nationwide network of qualified physicians, safe & effective andropause treatment is available in a city near you.
Most men come to our website because they want to make informed decisions about their healthcare as they age. The internet is an excellent place to start your healthy aging research, but information gained online isnt worth much unless you can actually act on it!
At Renew Man, we make it easy to both learn about your treatment options andwhen youre comfortableimplement your treatment plan. Get the big picture from our website, and then reach out to a Renew Man doctor near you to learn more. Our network of doctors covers dozens of cities across the country, making our safe and effective andropause treatments available to thousands of men right in their local areas.
Unfortunately, many regular physicians dont fully understand andropause. They may not recognize your symptoms as signs of hormonal decline, and if this is the case they certainly wont be able to prescribe the right treatments. At Renew Man, we understand healthy aging. You can trust our network of doctors to provide superior relief from andropause symptoms and support for healthy aging because:
The information on our website is just the beginning when it comes to your health and well-being during andropause. If youd like to learn more, please feel to give us a call. We wont try to sell you anything; but we will do our best to provide answers that are fact-based, direct, and relevant. When youre ready, well connect you with a Renew Man doctor near you to provide even more personalized information.
You can reach us at 800-859-7511, or request a consultation through our contact form.
See original here:
Network Of Doctors - Renew Man
Recommendation and review posted by simmons
Male Hypogonadism | Uroweb
3.1.Epidemiology
Definition: male hypogonadism is a clinical syndrome caused by androgen deficiency which may adversely affect multiple organ functions and quality of life (QoL) [1].
Androgen deficiency increases slightly with age also in healthy men [2,3]. In middle-aged men, the incidence of biochemical hypogonadism varies from 2.1-12.8% [4]. The incidence of low testosterone and symptoms of hypogonadism in men aged 40-79 varies form 2.1-5.7% [3,4]. Hypogonadism is more prevalent in older men, in men with obesity, those with co-morbidities, and in men with a poor health status.
Androgens, which are produced by the testis and by the adrenal glands, play a pivotal role in male reproductive and sexual function. Androgens are crucial for the development of male reproductive organs, such as the epididymis, vas deferens, seminal vesicle, prostate and penis. In addition, androgens are needed for puberty, male fertility, male sexual function, muscle formation, body composition, bone mineralisation, fat metabolism, and cognitive functions [5].
Male sexual development starts between the 7th and 12th week of gestation. The undifferentiated gonads develop into a foetal testis through expression of multiple genes located on the short arm of the Y chromosome, including the sex-determining region of the Y chromosome (SRY gene complex) and the SOX gene on chromosome 17 [6]. The foetal testis produces three hormones: testosterone, insulin-like peptide 3 (INSL3) and anti-Mllerian hormone (AMH). Testosterone is needed for the stabilisation of the Wolffian ducts, resulting in formation of the epididymis, vas deferens and seminal vesicle. AMH activity results in regression of the Mllerian ducts (Figure 1). INSL3 and AMH regulate testicular descent.
Under the influence of intratesticular testosterone, the number of gonocytes per tubule increases threefold during the foetal period [7]. In addition, testosterone is needed for development of the prostate, penis and scrotum. However, in these organs testosterone is converted into the more potent metabolite 5a-dihydrotestosterone (DHT) by the enzyme 5a-reductase. Testosterone and DHT are required for penile growth, both activating the androgen receptor [8].
Intratesticular testosterone is needed to maintain the spermatogenic process and to inhibit germ cell apoptosis [9]. The seminiferous tubules of the testes are exposed to concentrations of testosterone 25-100 times greater than circulating levels. Suppression of gonadotropins (e.g. through excessive testosterone abuse) results in a reduced number of spermatozoa in the ejaculate and hypospermatogenesis [10]. Complete inhibition of intratesticular testosterone results in full cessation of meiosis up to the level of round spermatids [11,12]. Testosterone does not appear to act directly on the germ cells, but functions through the Sertoli cells by expression of the androgen receptor (AR) and influencing the seminiferous tubular microenvironment [11]. Testosterone can also be metabolised into oestradiol by aromatase, present in fat tissue, the prostate, the testes and bone. Oestradiol is also essential for bone mineralisation in men [13]. The production of testosterone is controlled in the foetus by placental choriongonadotropin (hCG) and after birth by luteinising hormone (LH) from the pituitary gland. Immediately after birth, serum testosterone levels reach adult concentrations over several months (mini puberty). Thereafter and until puberty, testosterone levels are low, thus preventing male virilisation. Puberty starts with the production of gonadotropins, initiated by gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus and results in testosterone production, male sexual characteristics and spermatogenesis [14]. Figure 1 shows the development of the male reproductive system.
Testosterone exerts its action through the AR, located in the cytoplasm and nucleus of target cells. During the foetal period, testosterone increases the number of ARs by increasing the number of cells with the AR and by increasing the number of ARs in each individual cell [8,13]. The AR gene is located on the X chromosome (Xq 11-12): defects and mutations in the AR gene can result in male sexual maldevelopment, which may cause testicular feminisation or low virilisation (i.e. disorder of sexual development (DSD)). Less severe mutations in the AR gene may cause mild forms of androgen resistance and male infertility [15]. In exon 1 of the gene, the transactivation domain consists of a trinucleotide tract (cytosine-adenine-guanine (CAG) repeats)) of variable length. Androgen sensitivity may be influenced by the length of the CAG repeats in exon 1 of the AR gene [15]. The AR CAG repeat length is inversely correlated with serum total and bioavailable testosterone and oestradiol in men. Shorter repeats have been associated with an increased risk for prostate disease, and longer repeats with reduced androgen action in several tissues [16]. CAG repeat number may influence androgenic phenotypical effects, even in case of normal testosterone levels [17].
Summary of evidence
Testosterone is essential for normal male development.
Figure 1: Development of the male reproductive system
FSH=follicle-stimulating hormone; LH=luteinising hormone; SRY=sex determining region of the Y chromosome; INSL3=insulin-like peptide 3.
Hypogonadism results from testicular failure, or is due to the disruption of one or several levels of the hypothalamic-pituitary-gonadal axis (Figure 2).
Male hypogonadism can be classified in accordance with disturbances at the level of:
Primary testicular failure is the most frequent cause of hypogonadism and results in low testosterone levels, impairment of spermatogenesis and elevated gonadotropins. The most important clinical forms of primary hypogonadism are Klinefelter syndrome and testicular tumours.
The main reasons for primary testicular failure are summarised in Table 1.
Central defects of the hypothalamus or pituitary cause secondary testicular failure. Identifying secondary hypogonadism is of clinical importance, as it can be a consequence of pituitary pathology (including prolactinomas) and can cause infertility, which can be restored by hormonal stimulation in most patients with secondary hypogonadism.
The most relevant forms of secondary hypogonadism are:
These disorders are characterised by disturbed hypothalamic secretion or action of gonadatropin-releasing hormone (GnRH), as a pathophysiology common to the diseases, resulting in impairment of pituitary LH and FSH secretion. An additional inborn error of migration and homing of GnRH-secreting neurons results in Kallmann syndrome [23,24]. The most important symptom is the constitutional delay of puberty: it is the most common cause of delayed puberty (pubertas tarda) [25]. Other rare forms of secondary hypogonadism are listed in Table 2.
Combined primary and secondary testicular failure results in low testosterone levels and variable gonadotropin levels. Gonadotropin levels depend predominantly on primary or secondary failure. What has been labelled as late-onset hypogonadism and age-related hypogonadism is comprised of three types of hypogonadism and formally secondary hypogonadism is the most prevalent [26,27]. It should however be stated that low testosterone and low gonadotropin levels do not exclude a compromised gonadal response to LH stimulation as has been demonstrated in obesity, corticosteroid induced hypogonadism etc.
These forms are primarily rare defects and will not be further discussed in detail in these guidelines. There are AR defects with complete, partial and minimal androgen insensitivity syndrome; Reifenstein syndrome; bulbospinal muscular atrophy (Kennedy disease); as well as 5a-reductase deficiency (for a review, see Nieschlag et al. 2010) [28].
The classification of hypogonadism has therapeutic implications. In patients with secondary hypogonadism, hormonal stimulation with human chorionic gonadotropin (hCG) and FSH or alternatively pulsatile GnRH treatment can restore fertility in most cases [29,30]. Detailed evaluation may for example detect pituitary tumours, systemic disease, or testicular tumours. Combined forms of primary and secondary hypogonadism can be observed in ageing, mostly obese men, with a concomitant age-related decline in testosterone levels resulting from defects in testicular as well as hypothalamic-pituitary function.
Table 1: Most common forms of primary hypogonadism
Disease
Causes of deficiency
Maldescended or ectopic testes
Failure of testicular descent, maldevelopment of the testis
Testicular cancer
Testicular maldevelopment
Orchitis
Viral or unspecific orchitis
Acquired anorchia
Trauma, tumour, torsion, inflammation, iatrogenic, surgical removal
Secondary testicular dysfunction
Medication, drugs, toxins, systemic diseases
(Idiopathic) testicular atrophy
Male infertility (idiopathic or specific causes)
Congenital anorchia (bilateral in 1 in 20,000 males,
unilateral 4 times as often)
Intrauterine torsion is the most probable cause
Klinefelter syndrome 47,XXX
Sex-chromosomal non-disjunction in germ cells
46,XY disorders of sexual development (DSD)
(formerly male pseudohermaphroditism)
Disturbed testosterone synthesis due to enzymatic defects of steroid biosynthesis (17,20- lyase defect, 17-hydroxysteroid dehydrogenase defect)
Gonadal dysgenesis (synonym streak gonads)
XY gonadal dysgenesis can be caused by mutations in different genes
46,XX male syndrome (prevalence of 1 in 10,000-20,000)
Males with presence of genetic information from the Y chromosome after translocation of a DNA segment of the Y to the X chromosome during paternal meiosis
Noonan syndrome (prevalence of 1 in 1,000 to 1 in 5,000)
Short stature, congenital heart diseases, cryptorchidism
Inactivating LH receptor mutations, Leydig cell hypoplasia (prevalence of 1 in 1,000,000 to 1 in 20,000)
Leydig cells are unable to develop due to the mutation [31]
Table 2: Most common forms of secondary hypogonadism
Disease
Causes of deficiency
Hyperprolactinemia
Prolactin-secreting pituitary adenomas (prolactinomas) or drug-induced
Isolated hypogonadotropic hypogonadism (IHH) (formerly termed idiopathic hypogonadotrophic hypogonadism, IHH)
Specific (or unknown) mutations affecting GnRH synthesis or action
Kallmann syndrome (hypogonadotropic hypogonadism with anosmia, prevalence 1 in 10,000)
GnRH deficiency and anosmia, genetically determined
Secondary GnRH deficiency
Medication, drugs, toxins, systemic diseases.
Hypopituitarism
Radiotherapy, trauma, infections, haemochromatosis and vascular insufficiency or congenital
Pituitary adenomas
Hormone-secreting adenomas; hormone-inactive pituitary adenomas; metastases tothe pituitary or pituitary stalk
Prader-Willi syndrome (PWS) (formerly Prader-Labhart-Willi syndrome, prevalence 1 in 10,000 individuals)
Congenital disturbance of GnRH secretion
Congenital adrenal hypoplasia with hypogonadotropic hypogonadism (prevalence 1 in 12,500 individuals)
X-chromosomal recessive disease, in the majority of patients caused by mutations in the DAX1 gene
Pasqualini syndrome
Isolated LH deficiency
Recommendation
LE
GR
Differentiate the two forms of hypogonadism (primary and secondary) (LH levels), as this has implications for patient evaluation and treatment and makes it possible to identify patients with associated health problems and infertility.
1b
B
Figure 2: The hypothalamic-pituitary-testes axis
FSH=follicle-stimulating hormone; GnRH=Gonadotropin-releasing hormone; LH=luteinising hormone.
Hypogonadism is diagnosed on the basis of persistent signs and symptoms related to androgen deficiency and assessment of consistently low testosterone levels (on at least two occasions) with a reliable method [4,32-35].
Low levels of circulating androgens may be associated with signs and symptoms (Table 3) [4,36,37]
Table 3: Clinical symptoms and signs suggestive for androgen deficiency
Delayed puberty
Small testes
Male-factor infertility
Decreased body hair
Gynaecomastia
Decrease in lean body mass and muscle strength
Visceral obesity
Decrease in bone mineral density (osteoporosis) with low trauma fractures
Reduced sexual desire and sexual activity
Erectile dysfunction
Fewer and diminished nocturnal erections
Hot flushes
Changes in mood, fatigue and anger
Sleep disturbances
Metabolic syndrome
Read the original:
Male Hypogonadism | Uroweb
Recommendation and review posted by simmons
Local Doctor Leads Study Of Gene Therapy Treatment For …
February 18, 2016 5:55 PM By Dr. Maria Simbra
PITTSBURGH (KDKA) When part of the brain is no longer working properly, would it be possible to put something in to boost function?
Neurosurgeon Dr. Mark Richardson is trying to find out.
What were trying to do with this study is to replace an enzyme thats lost as cells degenerate in Parkinsons disease, Dr. Richardson said. The enzyme helps the brain make dopamine.
The brain chemical dopamine is important to keeping movements smooth. The problem in Parkinsons disease is the lack of dopamine because of worn out brain cells, and you end up with shaking, stiffness, and slowness of movement.
People can take medicine for Parkinsons disease, but there can be symptom fluctuations and at higher doses, side effects.
Typically in Parkinsons disease, these symptoms kind of go up and down like this, and they can be masked very well by medication, but unfortunately what tends to happen for all of these patients is progression to more of a roller coaster ride of ups and downs during the day, Dr. Richardson said.
Dr. Richardson is leading part of a study, first funded by the Michael J. Fox Foundation and now by a biotherapy company, to see whether inserting a gene into a specific part of the brain will be the on switch for more dopamine production.
The idea of brain surgery for a chronic disease is very different than continuing to take medication, Dr. Richardson said.
The gene is delivered into the brain through the skull by a thin tube and carried by a virus
The idea of a virus probably sounds very scary to some people. But, this virus cannot reproduce, Dr. Richardson said. It can insert itself into a cell, and it can only do one thing there. It can release the gene to allow this enzyme to be made.
Dr. Richardson and the lead investigator in San Francisco are looking for 20 patients to participate. They will be followed for three years, and their need for medication will be evaluated and compared before and after.
To qualify you have to be between 40 and 70 and on certain medicines for Parkinsons disease for at least three years with increasing fluctuations in movement.
Dr. Richardson hopes gene therapy leads to smoother days and fewer symptoms.
If we can show this is a small group of patients, the trial will be expanded, Dr. Richardson said. With a little bit of luck, within the next decade, we will see a gene therapy accepted as a proven and viable treatment option.
Join The Conversation On The KDKA Facebook Page Stay Up To Date, Follow KDKA On Twitter
Dr. Maria Simbra is a multi award-winning medical journalist, who brings a unique set of skills to her position as medical reporter on KDKA-TV. A member of the KDKA news team since May 2002, this physician and formally trained journalism professi...
Follow this link:
Local Doctor Leads Study Of Gene Therapy Treatment For ...
Recommendation and review posted by simmons