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.

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Isolation and Expansion of Adult Cardiac Stem Cells From ...

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