A purified population of multipotent cardiovascular progenitors derived from primate pluripotent stem cells engrafts in postmyocardial infarcted nonhuman primates

Abstract

Cell therapy holds promise for tissue regeneration, including in individuals with advanced heart failure. However, treatment of heart disease with bone marrow cells and skeletal muscle progenitors has had only marginal positive benefits in clinical trials, perhaps because adult stem cells have limited plasticity. The identification, among human pluripotent stem cells, of early cardiovascular cell progenitors required for the development of the first cardiac lineage would shed light on human cardiogenesis and might pave the way for cell therapy for cardiac degenerative diseases. Here, we report the isolation of an early population of cardiovascular progenitors, characterized by expression of OCT4, stage-specific embryonic antigen 1 (SSEA-1), and mesoderm posterior 1 (MESP1), derived from human pluripotent stem cells treated with the cardiogenic morphogen BMP2. This progenitor population was multipotential and able to generate cardiomyocytes as well as smooth muscle and endothelial cells. When transplanted into the infarcted myocardium of immunosuppressed nonhuman primates, an SSEA-1+ progenitor population derived from Rhesus embryonic stem cells differentiated into ventricular myocytes and reconstituted 20% of the scar tissue. Notably, primates transplanted with an unpurified population of cardiac-committed cells, which included SSEA-1- cells, developed teratomas in the scar tissue, whereas those transplanted with purified SSEA-1+ cells did not. We therefore believe that the SSEA-1+ progenitors that we have described here have the potential to be used in cardiac regenerative medicine.

Figure 1. Gene and protein profiles of SSEA-1⁺ cardiac progenitors.

ESCs were treated or not (CTRL) for 4 days with BMP2 (10 ng/ml) and (A) monitored by flow cytometry using a FITC-conjugated anti–SSEA-1 antibody or (B and C) separated using the anti–SSEA-1 Miltenyi kit and the MACS columns. SSEA-1^– and SSEA-1⁺ cDNAs were run in real-time PCR. Data are from 8 experiments, performed on the HUES-24 cell line, and reproduced in different HUESC lines (H9, HUES-9, HUES-24, HUES-26, I3, and I6). The inset in C illustrates SSEA-1⁺ cells immunostained with anti-Tbx6 and anti-Mesp1/2 antibody 12–24 hours after sorting. (D and E) ChIP assay using (D) anti-H3triMeK4 or anti-triMeK27 antibodies or (E) the anti-PolII or serine-phosphorylated PolII (Ser5-PPolII, left) antibodies. Q-PCR was used to amplify the chromatin-bound DNA, using primers specific for OCT4, TBX6, ISL1, MEF2C, NKX2.5, ACTC1, and PAX4 promoters (sequence within the 700 bp in 3′ of ATG; Table 2). Data (n = 5) show fold enrichment of methylated histones on promoters in the SSEA-1⁺ versus SSEA-1^– population. The gels illustrate the specificity of PCR products (input, SSEA-1⁺ DNA samples from anti-triMeH3K4 IP, anti-rabbit IgG, or no antibody). OCT4IA, OCT-4 isoform A. (E) The left side shows PCR gel of DNA products after real-time PCR and indicates a complete loss of serine-phosphorylated PolII on both the NANOG and SOX2 promoters. ChiP experiments have been mostly performed using the HUES-24 cell line and were validated in 2 other experiments using the I6 cell line. *P ≤ 0.05; ^#P ≤ 0.01.

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.

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).

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).

Figure 5. SSEA-1⁺ cells safely engraft in infarcted primate myocardium.

(A) BMP2-induced gene expression in ORMES cells. (B) GFP-expressing cells following engraftment of actin promoter EGFP ORMES cells in the scar area of the myocardium. (C) Teratoma formed after cell engraftment (the left and right images show low and high magnification, respectively). (D) Flow cytometry analysis of FITC-conjugated SSEA-1⁺ ORMES cells. (E) Q-PCR of mesodermal, cardiac genes (or pluripotency genes, inset) expressed in SSEA-1⁺ cells sorted after 4 days BMP2 treatment. Data were normalized to GAPDH and to the SSEA-1^– population right after sorting. (F) Kinetic of gene expression in SSEA-1⁺ cells expressed as fold changes versus SSEA-1^– cells normalized to undifferentiated ORMES-2, following 4 days BMP2 treatment after sorting and following 7 days culture on MEFs. (G) H&E staining of the myocardial section from a postinfarction primate having received the medium (left image, sham) or the cells together (right image) with an image illustrating the low background of the green GFP fluorescence channel in the non–cell-grafted myocardium (middle panel). (H) Upper panels show, from left to right, GFP-expressing cells 2 months after engraftment of actin promoter EGFP and SSEA-1⁺ ORMES cells, ×40 original magnification of a myocardial section; costaining of actin promoter EGFP and SSEA-1⁺ ORMES cells, with anti-CD31 and anti-SMA antibodies; costaining of actin promoter EGFP and SSEA-1⁺ ORMES cells, with anti-GFP, anti-actinin, and anti-MLC2v antibodies. The inset shows costaining of GFP and MLCK in a myocardial section grafted with SSEA-1⁺ ORMES.

Figure 6. Directed specification of pluripotent stem cells toward a cardiac fate.

BMP2-induced Oct-4⁺, SSEA-1⁺ cells give rise to endo/mesendodermal cells secreting cardiogenic factors, further directing the cell fate toward a cardiac phenotype when plated on MEFs releasing FGF2. Addition of PDGF or VEGF directs the fate of cells toward a smooth muscle and endothelial phenotype, respectively. SMMyo, smooth muscle myosin.