Early cardiovascular progenitors feature a unique microRNA signature.

Besides gene transcription, expression of proteins defining a specific cell fate is also regulated at the posttranscriptional level by small non-coding RNAs (miRNAs, miRs), and profiles of miRNAs often reflect a cell-specific signature. We thus used a genomic wide approach (30) to compare the profile of miRNAs in BMP2-induced SSEA-1⁺ cells and remaining SSEA-1^– or undifferentiated HUESCs. This broad approach showed that SSEA-1⁺ cells gained expression of specific miRNAs, belonging to the cluster miR-17-92. These include miR-20a and miR-20b, known to be turned on when ESCs acquire a cardiac fate (31), and miR-17-92, whose downregulation causes a cardiac defect. Furthermore, miR-106, whose deletion concomitant to the one of miR-17-92 results in severe cardiac defects (32), was also upregulated in SSEA-1⁺ cells. miR-513, another miRNA not expressed in undifferentiated ESCs, has still unknown functions that were highly upregulated in SSEA-1⁺ cells; its function is currently under investigation in the laboratory. The gain in expression of these miRNAs was marked in SSEA-1⁺ cells when compared with the one in SSEA-1^– cells and became even more prominent when compared with the profile of undifferentiated stem cells (Figure ​(Figure2).2). miR-302a, belonging to the cluster 302, whose expression is regulated by OCT4 and potentially SOX2 and NANOG, was also more expressed in SSEA-1⁺ cells versus SSEA-1^– cells or HUESCs. This phenomenon could be related to the dual role of this miRNA in both pluripotency and then in specification of HUESCs toward the mesendoderm (33). Another miRNA expressed in HUESCs was also upregulated in SSEA-1⁺ cells in agreement with its high expression when HUESCs differentiate within embryoid bodies (30). Expression of miR-615 and miR-135 connected to the pluripotency network in ESCs was unchanged (34). Of note, the miR-302bcd, also involved in the pluripotency transcriptional network (34, 35), was downregulated in SSEA-1⁺ cells when compared with SSEA-1^– cells, while it appeared as upregulated when compared with expression level in HUESCs (Figure ​(Figure2). 2).

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Figure 2

Heat map representative of the miRNA profile between SSEA-1⁺ and HUESCs and SSEA-1⁺ and SSEA-1^– cells.

Numbers beside the figure represent miRs. Total RNA was extracted from HUES, SSEA-1⁺, and SSEA-1^– cells and hybridized to a miRNA microarray. Experiments were performed 6 times and log2 ratio values are shown in the heat map.

The cardiovascular population of SSEA-1⁺ progenitors is clonogenic and multipotential.

To characterize the differentiation potential of SSEA-1⁺ progenitors toward several cell lineages, we plated isolated SSEA-1⁺ progenitors as suspensions of single cells on mitotically arrested E14 mouse embryonic fibroblasts (MEFs) in 96-well plates for 5 days, stained them with DAPI, and scanned them using an Arrayscan. Despite being plated by limiting dilution at high or low density (5,000 down to 1 single cell/well), cells generated clones within 5 days. The number of clones per well tightly correlated with the starting number of plated cells (Figure ​(Figure3A).3A). One single cell (monitored by phase contrast microscopy, using the wild-type HUES-24 cell line, or by GFP expression, using the HUES-9 Oct4GFP cell line) was able to generate a clone within 7 days (Figure ​(Figure3,3, A–C). In another set of experiments, a single SSEA-1⁺ cell was observed while carrying out cytokinesis as early as 2 days after plating (Figure ​(Figure3A,3A, above the graph). The limiting dilution experiments were confirmed by adding a single cell into a well of a 96-well plate using a micropipette and monitoring the formation of a colony (data not shown). Altogether, this demonstrates that SSEA-1⁺ cell–derived colonies originate from single cells and not from cell clusters, eliminated by thorough trypsinization and filtering (40 μm) before the passage through the sorting magnetic column.

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Figure 3

BMP2-induced SSEA-1⁺ cardiac progenitors give rise to cardiac, endothelial, and smooth muscle cells.

(A–C) Cell clonogenicity. BMP2-induced SSEA-1⁺–sorted cells were plated at different densities (5,000, 500, 50, 5, and 1 cell/well) in 96-well plates containing MEFs. Five days later, cells were stained with DAPI to be scanned using an Arrayscan. (A) The orange arrow indicates that the colony was generated in a single cell-containing well. Cell colonies were numbered using the Cellomics viewer software. (A and B) A colony generated from a single SSEA-1⁺ cell (arrows) and visualized 2 (B) or 5 (A) days later (above the graph). (C) A single SSEA-1⁺ cell derived from the HUES-9 pOct-4-GFP cell line that was plated in a microwell of 96-well plates and visualized by fluorescence right after plating (top image) or 5 days later (bottom GFP and transmitted light images). (D) Immunofluorescence of BMP2-induced SSEA-1⁺ cells cultured for 5 days on MEFs in the absence of growth factors or challenged by PDGF or VEGF, or cocultured with human cardiac fibroblasts. Top left corner insets show high-magnification images of localization of markers. The inset in the actinin panel shows a high-magnification image of sarcomeres. Experiments were done in triplicate with similar results. (E) BMP2-induced SSEA-1⁺ cells cultured for 5 days on MEFs immunostained with anti-ISL1, anti-MEF2C, or anti-NKX2.5 antibodies were quantified by HCCI using an Arrayscan. The numbers in the bars of the graph indicate the number of scored and validated cells. Original magnification ×20; ×40 (smooth muscle [SM] myosin in D); ×5 (insets in D).

After 1 week in culture on MEFs in the absence of SU5402, BMP2-induced SSEA-1⁺–sorted cell–derived colonies still expressed SSEA-1, together with 4 cardiac-restricted transcription factors, ISL1, MEF2C, NKX2.5, and TBX5. A majority of cells from most colonies expressed these markers (Figure ​(Figure3D),3D), pointing to a rather homogeneous population of cardiac progenitors. ISL1, MEF2C, NKX2.5, and TBX5 were all located into the nucleus. FLK1, poorly expressed in the freshly sorted SSEA-1⁺ cell population (5%–7%) (data not shown), was found later at the membrane of a proportion, but not in all cells, after 1 week of culture on feeder cells in the absence of SU5402. In contrast, HUES-derived SSEA-1^– cells did not express NKX2.5, ISL1, MEF2C, and TBX5 but still expressed OCT4, SOX2, and NANOG as assessed by immunofluorescence (data not shown).

Addition of PDGF (10 ng/ml) or VEGF (50 ng/ml) to BMP2-induced SSEA-1⁺ cells cultured on MEFs strongly triggered expression of smooth muscle actin and myosin as well as of CD31, respectively, while only 12% of freshly sorted BMP2-induced SSEA-1⁺ cells (Supplemental Figure 2A) were CD31⁺ (Supplemental Figure 2B). Plated on collagen 1–coated dishes and treated with VEGF (50 ng/ml for 10 days), 80%–90% of SSEA-1⁺ cells acquired the morphology of endothelial cells and expressed high levels of CD31, CD34, and FLK1 mRNAs as well as the protein CD31 for 30% of them (Supplemental Figure 2C) but not cardiac troponin T (data not shown), as investigated by immunofluorescence.

When plated on human fibroblasts in the presence of 5% fetal calf serum or in a mix of human cardiac fibroblasts and cardiomyocytes, 10% and 60%–80% of cells, respectively, expressed actinin arranged in sarcomeric units within a week (Figure ​(Figure3D3D and Figure ​Figure4A).4A). A similar effect was observed in 70% of cells when cells were treated with the conditioned medium of a coculture of human cardiac fibroblasts and cardiomyocytes.

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Figure 4

BMP2-induced SSEA-1⁺ primate cardiac progenitors differentiate in mature cardiomyocytes.

Immunostaining of actin promoter EGFP SSEA-1⁺ ORMES cells cultured on (A and B) both cardiac fibroblasts and myocytes. (A) Anti-GFP and anti-actinin, with merged image on the right. (B) Anti-GFP immunostaining and DAPI staining of nuclei of SSEA-1⁺ cells; z-stack of images was acquired in a laser-scanning confocal microscope, with the green and blue channels together with differential interference contrast images. The merged image of a 200-nm focal plane is shown. (C) Merged image of anti-actinin (red), anti-GFP (green), and Cx43 (white) immunostaining. (D) Actin promoter EGFP SSEA-1⁺ ORMES cells cultured on cardiomyocytes only and (E) fibroblasts only (GFP, green; actinin, red; CX43 or phosphorylated P-CX43, white). (F) Actin promoter EGFP SSEA-1⁺ ORMES cells cultured on both cardiac fibroblasts and myocytes immunostained with the anti-GFP (green), anti-actinin (red), and anti-MLC2v (blue) antibodies. These images are representative of 3 coculture experiments. Original magnification, ×100 (inset). (G) Actin promoter EGFP SSEA-1⁺ ORMES cells were cultured for 2 weeks on matrigel in the presence of filtered conditioned medium of a mix of fibroblasts and cardiomyocytes and actin stained with phalloidin. Original magnification, ×63 (inset). (H) Actin promoter EGFP SSEA-1⁺ ORMES cells cultured on both cardiac fibroblasts and myocytes immunostained with the anti–β-MHC (red) and anti-GFP (green). Merged image is shown on the right. The sarcomeric staining of GFP is due to its binding to myosin (60).

Interestingly, SSEA-1⁺ cells in culture on MEFs for 4 days expressed some genes specific for the secondary heart lineage. This was further enhanced by FGF8 (5 × 10^–8 M) (36) treatment of cells for an additional 4 days. FGF8, indeed, definitively turned on expression of TBX1, retinaldehyde dehydrogenase 2 (RALDH2) (37, 38), HES1 (39), and FOXH1 (40) (Supplemental Figure 3A). The FGF8 effect was also observed when genes were monitored at the level of single colonies (data not shown).

To better quantify the cardiogenic fate of BMP2-induced SSEA-1⁺ cardiovascular progenitors cultured for 1 week on MEFs, immunostained cells cultured in 96-well plates were scored by high-content cell imaging (HCCI), using an Arrayscan, in 2 experiments. This technological approach allowed us both to quantify and to visualize cells positive for each cardiac marker. The BMP2-induced SSEA-1⁺ population expanded as cardiovascular progenitors within colonies (Figure ​(Figure3E,3E, right) expressed NKX2.5 and MEF2C (63% and 91%, respectively) as well as ISL1 (89%). The cells lost expression of OCT4 and SOX2 (data not shown). This quantitative analysis by HCCI was confirmed by flow cytometry (data not shown).

To assess the transcriptomic homogeneity of the clones, individual colonies grown for 7 days on MEFs were randomly picked up through a micropipette and screened for gene expression by RT-Q-PCR. These experiments revealed that each colony expressed, to a similar and high extent, NKX2.5, MEF2C, ACTC1, and MYOCD and, to a lesser degree, FLK1. ISL1 and TBX20 were downregulated in 50% of colonies; NES and PAX3 were expressed in 50% of clones, actually those expressing ISL1 and TBX20. None of the colonies expressed SOX17, an endodermal marker (Supplemental Figure 3B). The multipotentiality of a single clone of SSEA-1⁺ cells was further demonstrated by cutting single colonies grown on MEFs for 5 days into several cell clusters. Cells were then separately replated on collagen 1–coated dishes and treated with PDGF or VEGF or plated on human fibroblasts. In the later case, cardiomyocytes (Supplemental Figure 4A) were observed 2–3 weeks later, while CD31-positive cells (Supplemental Figure 4B) and smooth muscle actin-positive cells (Supplemental Figure 4C) were present in growth factor treated cells after 1 week (Supplemental Figure 4).

In the perspective of a potential use of SSEA-1⁺ progenitors in customized regenerative medicine, we used iPS cells. BMP2-induced SSEA-1⁺ cells could also be derived from iPS cells (Supplemental Figure 5A). A similar pattern of expression of cardiac proteins (i.e., ISL1, MEF2C, and NKX2.5) was found in BMP2-triggered SSEA-1⁺ cell–derived and sorted cells cultured for 1 week on MEFs, as assessed by immunofluorescence. Furthermore, a minor population of BMP2-induced SSEA-1⁺ iPS cells (4%) stained positive for CD31 (Supplemental Figure 5B).

Cardiovascular BMP2-induced SSEA-1⁺ progenitors differentiate into mature cardiomyocytes ex vivo.

To test the cardiogenicity and the functionality of BMP2-induced SSEA-1⁺ progenitors prior to animal graft experiments, we used an ex vivo experimental set up. In order to track the fate of cells in culture, Rhesus ESCs (ORMES-2 cell line) were genetically engineered to express GFP under the transcriptional control of the human cardiac α-actin promoter. BMP2-induced SSEA-1⁺ Rhesus cardiac progenitors were then cocultured for 4 weeks with human atrial myocytes, cardiac fibroblasts, or a mix of cardiomyocytes and fibroblasts (Figure ​(Figure4).4). After 4 weeks, microscopic examination revealed the presence of GFP-positive cells (Figure ​(Figure4)4) in the 3 cultures, thereby confirming that human cardiac/fibroblastic cells provide the paracrine environment that triggers the cardiomyogenic differentiation of BMP2-induced SSEA-1⁺ progenitors. The extent of differentiation of BMP2-induced SSEA-1⁺ cardiac progenitors was prominent and reached 70% ± 10% (in 3 experiments) for cells cocultured on the mix of cardiac fibroblasts and cardiomyocytes or conditioned medium from the coculture (Figure ​(Figure4G).4G). More interestingly, under this experimental situation, a high proportion of cells (60%–80%), as monitored by concomitant GFP and actinin expression, differentiated in mature cardiomyocytes expressing organized and sarcomeric aligned structures (Figure ​(Figure4,4, A and B). To investigate a potential cell fusion phenomenon, we acquired z-stacks of images (every 200 nm) of BMP2-induced SSEA-1⁺ cardiac progenitors cultured on a mix of cardiac fibroblasts and cardiomyocytes and stained with an anti-GFP antibody and DAPI to visualize the nucleus. Examination of images showed that each GFP cell featured a single nucleus in the same focal plane (200 nm) as the one where GFP gave the brightest fluorescence (Figure ​(Figure4B),4B), excluding a fusion with cells sitting below.

No cells were labelled with an anti-SMA antibody. The remaining non- or poorly actinin-positive cells still acquired a cardiac fate, as they expressed GFP under the transcriptional control of the cardiac α-actin promoter but failed to set a contractile apparatus. When cultured on myocytes alone, cells remains as fetal (sarcomere size, 1.12 ± 0.02; n = 137 sarcomeres, 12 cells) (Figure ​(Figure4C).4C). On fibroblasts, they started to form more mature sarcomeres (1.80 ± 0.03; n = 70 sarcomeres, 4 cells) (Figure ​(Figure4D),4D), while on both myocytes and fibroblasts (Figure ​(Figure4,4, A and B), sarcomeres reached an adult size (2.01 ± 0.03 n = 127, 10 cells). Furthermore, aligned ORMES-derived cardiomyocytes were the only ones to express the ventricular isoform connexin 43 (CX43) (Figure ​(Figure4C)4C) at their membrane, the ventricular myosin light chain for 80% of them (Figure ​(Figure4F)4F) and the adult β-MHC isoform (Figure ​(Figure4H)4H) for 50% of them, an index of acquisition of an adult ventricular phenotype. When cultured alone on matrigel in the only presence of conditioned medium of a mix of fibroblasts and cardiomyocytes, 70% of BMP2-induced SSEA-1⁺ cardiac progenitors adopted a morphology of embryonic cardiomyocytes and developed within 2 weeks a sarcomeric actin network and became hypertrophied, indicating the presence in the conditioned medium of both differentiating and hypertrophic agents (Figure ​(Figure4G;4G; n = 6). While quiescent, as no pacemaker cell was present in the dish, when electrically paced, these cells featured a Ca²⁺ transient (data not shown). CX43 was phosphorylated in the cells in contrast to cytosolic microtubule-associated unphosphorylated CX43, which was expressed in nonmature ORMES-derived cardiomyocytes observed when the latter were cultured on atrial myocytes alone (Figure ​(Figure4D)4D) or fibroblasts alone (Figure ​(Figure4E).4E). These data further document that cell transplantation should be performed in the border zone of infarcted myocardium or in patchy scars composed of both fibrosis and reversibly damaged cardiac fibers.

Real-time Q-PCR by SYBR Green detection.

RNA was extracted from SSEA-1⁺ or SSEA-1^– cells or single clones using Zymo research kits. One μg of RNA or the total RT reaction (single clone) was reverse-transcribed using the SuperScript II Reverse Transcriptase (Invitrogen) and oligo(16)dT. Q-PCR was performed using a LightCycler LC 1.5 or 480 (Roche Diagnostics). Amplification and specificity of amplicons was carried out as previously described (19). Data were analyzed according to the method described by Pfaffl et al. (59).

Cell sorting.

Trypsinized cells were incubated for 30 minutes with anti–SSEA-1 antibody–coated Miltenyi beads in D-PBS supplemented with 0.5% (wt/vol) BSA and 2 mM EDTA and transferred to a L50 Miltenyi cartridge set on the magnet. Cells were washed 3 times with D-PBS/BSA/EDTA and eluted from the column removed from the magnet using 3 ml of D-PBS/BSA/EDTA (19). FACS, performed 48 hours later when beads detached from cells, revealed a purity of 95% of SSEA-1⁺ cells.

Flow cytometry analysis.

BMP2-treated cells were trypsinized, washed twice with PBS, and filtered through a 70-μm mesh filter before flow cytometry analysis using FACSCalibur (Becton Dickinson) and CELL QUEST software. Gates were set taking both undifferentiated HUESCs and SSEA-1^– cells, stained with both the primary and secondary antibody as negative controls.

Antibodies.

The following antibodies were used for flow cytometry or immunofluorescence: anti-NKX2.5 (mab2444) was from Abcam; anti-TBX6, anti-MESP1/2, and anti-MEF2C were from Aviva System Biology; anti-TBX5 and anti-MYOD were from Abcam; anti-SMA (SMA clone 1A4) and smooth muscle myosin (SMM) were from Dako; anti-ISL1 was from the Developmental Hybridoma Bank of Iowa University, Iowa City, Iowa, USA; the FITC-conjugated anti–SSEA-1, anti-CD31, anti-CD34, and anti-CD45 were from Becton Dickinson. The anti-MLC2v raised against the specific human myosin light chain residues 45–59 was from Biocytex, and the anti-MLCK (clone K36) was from Sigma-Aldrich. Specificity of antibodies was controlled using the respective isotype of the Igs in FACS and using undifferentiated HUESCs for immunostaining.

Cell imaging.

Slides were observed with confocal microscopy (Zeiss LSM 510 META) and low-power laser, allowing us to check for the right spectrum of the fluorochrome that were used (Alexa or GFP), further confirmed by using the META sensitive detector of the LSM 510 scanning microscope. A unique feature of the LSM 510 META scan head is its ability to acquire l stacks in 10-nm increments over a broad spectral range. GFP was detected within a 520–540 nm window. Some myocardial sections have been also treated to decrease autofluorescence (using NaBH4 treatment of sections). GFP was then labelled by an anti-GFP antibody (Covance) and a secondary Alexa Fluor 488– or Texas red–conjugated antibody.

For high-content imaging, after immunostaining, 96-well plates were scanned using the Arrayscan (Cellomics ThemoFisher), using the Cell Health Profiling Bioapplication. The fluorescence threshold was determined from the background obtained in undifferentiated HUESCs and set as a fixed threshold value to scan the wells of SSEA-1⁺ cells. Thirty fields per well and 18 wells per cardiac marker were scanned as described (19). SSEA-1^– HUESCs, stained with the same antibodies as those used for SSEA-1⁺ cells, were used to set low- and high-intensity thresholds independently for each channel (green, red, blue) and exposure parameters (time of exposure, 300–600 ms). We also set a background correction. Parameters were saved, and all wells were scanned with the same setting. The visualization of images and analysis were then performed using the vHCS software.

Sorted SSEA-1⁺ cells were plated by limiting dilution into 96-well plates (from 1,000 down to a single cell per well), stained 7 days later with DAPI, and scanned at a magnification of ×10, using the Health Cell Profiling application from Cellomics. The size and fragmentation of objects were set to identify only clusters of cells (cell colonies). The number of colonies was then scored using the software vHCS (Cellomics). To ensure that wells contained a single cell, we also used the HUES-9 pOct-4-GFP cell line and monitored the presence of the cell by epifluorescence.

Culture of human fibroblasts and human atrial myocytes.

Cells were isolated from the right atrial appendage, collected from 3-month-old patients during surgery required to relieve congenital diseases, and cultured as previously described (60). Approval of human tissue collection, care, and use was given by the ethics committee of Hospital Kremlin Bicêtre (AP-HP), Kremlin Bicêtre, France (Committee, protocol 04-26). Parents of patients provided informed consent prior to their participation in the present study.

Generation of α-actin promoter GFP ORMES-2 cell line.

The 10⁶ ORMES-2 cells, cultured as described above, were nucleofected with 3 μg of vector pActin-GFP, purified using a Pharmingen kit and linearized. Cells were then cultured on neomycin-resistant MEFs for 2 days and G418 (100 μg/ml) was added for the next 10 days. Colonies were cut with a needle and picked out of the dish to be further cultured and amplified. Clones were genotyped using GFP primers and further expanded.

Monitoring of gene expression by real-time Q-PCR.

To monitor gene expression in separate single clones, single cell-derived colonies grown for 7 days on MEFs were picked up randomly using a needle and used for RNA extraction (Zymo research kit). RT-Q-PCR was performed using the LightCycler 1.5 as described previously (46) and analyzed according to Pfaffl et al. (59).

ChIP.

The ChIP assay was performed according to the Fast ChIP protocol (61). Briefly, cells were crosslinked for 15 minutes with 1% formaldehyde, and the reaction was stopped for 5 minutes by 125 mM glycine. The cell pellet was then washed with cold PBS, and the cells were lysed with the ChIP buffer (61). One hundred μg chromatin and 3 μg antibody were used in each assay. The antibodies used were the anti-PolII (Santa Cruz Biotechnology Inc.), anti-phospho serine 5 PolII (Covance), the anti-H3triMeK4, and anti-H3triMeK27 (UBI or Abcam), and they were added overnight at 4°C. The Dynabead (protein A–conjugated or anti-IgM–conjugated phosphoserine 5 PolII; Invitrogen) was used to bind the immunocomplex H3-DNA or PolII-DNA. No antibody and nonrelevant rabbit IgG were used as negative controls.

The beads were extensively washed using the ChIP buffer. The protein-DNA interaction was reverse cross-linked by adding chelex to the beads and by boiling the samples for 10 minutes. DNA was then purified on microcartridges. Q-PCR was used to amplify the DNA using primers specific for promoters (sequence within the 700 bp in 3′ of ATG; Table ​Table2).2). Absolute enrichment was calculated assuming that at most 1% of nucleosomes were immunoprecipitated (62). Genomic region was thus considered enriched if 10 ng IP samples showed a greater enrichment when compared with 0.1 ng of input DNA. Quantification of the immunoprecipitated DNA was normalized against the starting input material.

Table 2

PCR primer sequences

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miRNA profiling.

Total RNA was extracted from cells using the mirVANA kit (Ambion). Five μg of RNA for each condition were subjected to dye/swap labelling and were hybridized on printed chips of 2,000 miRs, originating from 44 species; each spot was done in quadruplet. Raw data were analyzed to obtain a log2 intensity value for each spot. Log2 intensity was taken for each spot for both sorted SSEA-1⁺ cells and control conditions (sorted SSEA-1^– cells or HUESCs) and a log2 ratio was obtained. Data were clusterized using Cluster view as previously described (63). The heat map was generated using Tree View software. Data were validated by real-time PCR. Ten ng of total RNA extracted with mirVana kit were used in a RT reaction, using stem-loop microRNA specific primers (Applied Biosystems). Subsequently, the diluted RT product was used in a Q-PCR reaction with TaqMan miR-specific probes (Applied Biosystems). Normalization was done using small RNA RNU48 as a reference.

Myocardial infarction model.

Induction anesthesia of primates was performed with propofol (3 mg/kg) and sufentanyl (0.5 mg/kg), and maintenance anesthesia was ensured with the same drugs, albeit, at a lower dose (1 mg/kg and 0.25 mg/kg, respectively). Intravenous atracurium besilate (1 mg/kg) was added whenever required, and all animals received heparin (50 IU/kg) during the procedure. A myocardial infarction was created in Macaca Mulatta Rhesus monkeys (average weight, 8–10 kg) by a 90-minute balloon inflation in the left circumflex coronary artery, followed by reperfusion and angiographic confirmation of the patency of the revascularized infarct vessel. The occurrence of myocardial necrosis was consistently documented by a postprocedural rise in creatine phosphokinase values. Two weeks later, primates underwent a left thoracotomy and received unsorted or freshly sorted SSEA-1⁺ ESC-derived progenitors or just cell medium over the scar and at its borders. The monkeys were treated with either FK-506, started 5 days before cell transplantation at the dose of 1 mg/kg/d, or cyclosporine, given intramuscularly for 3 days and then by oral gavage at doses adjusted to target serum drug concentrations in the range of 100 ng/ml. Treatments were continued until sacrifice. Primates were humanely euthanized at 2 and 3 months after transplantation. The area of infarction and cell grafting was identified by sutures that had been placed circumferentially at the time of cell injections. This area was cut into 2 halves, of which 1 was used for assessment for teratoma. The second half was divided into 4 blocks, which were snap frozen in isopentane cooled in liquid nitrogen and subsequently processed as cryosections for identification and characterization of GFP⁺-grafted cells. In addition, a whole-body autopsy was performed in each animal and used for the search for extracardiac tumors. The experiments with primates were authorized by the Institutional Review Board on Ethics for primate experiments, IMASSA.

Cell transplantation in immunosuppressed mice.

The 10⁵ BMP2-induced SSEA-1⁺ cells were injected in the myocardium (n = 8), after opening the chest, or subcutaneously in the neck (n = 10) of immunodeficient Rag^–/–, γ chain^–/–, C5^–/–, MHC^–/– mice humanized mice. Undifferentiated cells and SSEA-1^– cells were also injected subcutaneously in the neck (n = 5 in each group). Four months later, the animals were euthanized and each organ was collected, sliced, and examined for the presence of tumor.

Guillaume Blin is a fellow from the Ministère de la Recherche et de la Technologie. David Nury and Sonia Stefanovic were funded by the grants “Programme Blanc stem cell signature” and “Specistem” of the National Research Agency (to M. Pucéat). This study was funded by the National Research Agency (grants Programmes Blanc stem cell signature and Maladies rares to M. Pucéat), the Genopole Evry, the French Association against Myopathies (AFM grant no. 13968 to C. Rücker-Martin), the LeDucq Foundation (CAPTAA) (Paris, to P. Menasché and M. Pucéat), the Foundation Coeur et Artères, and the Fonds d’Amorçage des Biothérapies (AP-HP, to P. Menasché). Tui Neri is a fellow from the LeDucq Foundation (MITRAL, to M. Pucéat). We thank Laurent Hamon for experimental assistance; S. Garcia (Pasteur Institute) for the teratomas formation in mice; P. Pradeau, D. Lici, and the team of the IMASSA for the care of primates; and Daniel Stockholm (Cell Imaging Facility, Genethon, Evry) for help in cell imaging. We acknowledge Lionel Bonnevie (Hôpital d’Instruction des Armées Bégin) for carrying out the myocardial infarction surgery in primates. We thank Virginie Magnone (Nice, CNRS) for analysis of microarrays.