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SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells

Abstract

To identify cell-surface markers specific to human cardiomyocytes, we screened cardiovascular cell populations derived from human embryonic stem cells (hESCs) against a panel of 370 known CD antibodies. This screen identified the signal-regulatory protein alpha (SIRPA) as a marker expressed specifically on cardiomyocytes derived from hESCs and human induced pluripotent stem cells (hiPSCs), and PECAM, THY1, PDGFRB and ITGA1 as markers of the nonmyocyte population. Cell sorting with an antibody against SIRPA allowed for the enrichment of cardiac precursors and cardiomyocytes from hESC/hiPSC differentiationcultures, yielding populations of up to 98% cardiac troponin T-positive cells. When plated in culture, SIRPA-positive cells were contracting and could be maintained over extended periods of time. These findings provide a simple method for isolating populations of cardiomyocytes from human pluripotent stem cell cultures, and thereby establish a readily adaptable technology for generating large numbers of enriched cardiomyocytes for therapeutic applications.

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Figure 1: Specification of the cardiovascular lineage from hESCs.
Figure 2: Expression of the cell-surface receptor SIRPA during hESC differentiation.
Figure 3: Enrichment of cardiomyocytes from hESC-derived cultures by cell sorting based on SIRPA expression.
Figure 4: Enrichment of cardiomyocytes from hiPSC-derived cultures by cell sorting based on SIRPA expression.
Figure 5: Expression of SIRPA on human fetal cardiomyocytes and in adult human heart.
Figure 6: Using SIRPA to predict cardiac differentiation efficiency.
Figure 7: Enrichment of cardiomyocytes through negative selection.

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Acknowledgements

We would like to thank members of the Keller laboratory for discussion and critical reading of the manuscript, G. Daley (Harvard Medical School, Boston) for providing the MSC-iPS1 and 38-2 cell line, B. Neel for providing the Sirpa-mutant mice and for discussion and suggestions, L. Ailles, J. Paterson and E. Hyatt from the antibody core facility, R. Hamilton for assistance in obtaining fetal tissue samples and the Sick Kids/UHN Flow Cytometry Facility for their assistance with cell sorting. This work was supported by funding from VistaGen Therapeutics (San Francisco) and by grants from Canadian Institute of Health Research (CIHR, MOP-84524) and Ontario's Ministry of Research and Innovation (MRI, GL2-CBD3P3) to G.K. N.C.D. was supported by a Swiss National Foundation postdoctoral fellowship. A.G. and P.S. were supported by the Heart and Stroke Foundation of Ontario (T6281 and NS6636) and CIHR (MOP-106538). A.G.E., E.G.S. and D.A.E. are supported by grants from the Australian Stem Cell Centre, the National Health and Medical Research Council (NHMRC, Australia) and the National Heart Foundation (Australia). E.G.S. and A.G.E. are Senior Research Fellows of the NHMRC (Australia).

Author information

Authors and Affiliations

Authors

Contributions

N.C.D. and G.K. designed the study and wrote the paper. N.C.D., A.M.C., P.S. and A.G. designed and performed experiments and analyzed the data. D.A.E., A.G.E. and E.G.S. generated and provided the NKX2-5–GFP hESC line.

Corresponding author

Correspondence to Gordon Keller.

Ethics declarations

Competing interests

G.K. is a paid consultant for, and N.B. and G.K. own stock in, VistaGen Therapeutics, which funded this study in part. N.C.B., A.M.C. and G.K. are employees of University Health Network, which has filed a patent application based on this study.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–3 and Supplementary Figs. 1–12 (PDF 1843 kb)

Supplementary Movie 1

Embryoid bodies at day 20 derived from NKX2-5-GFP HES3 cells (MOV 1202 kb)

Supplementary Movie 2

Unsorted cardiac monolayer culture from HES2-derived EB's (MOV 20809 kb)

Supplementary Movie 3

Cardiac monolayer cultures from SIRPA+ sorted populations at day 20 (MOV 27410 kb)

Supplementary Movie 4

Cardiac monolayer cultures from SIRPA- sorted populations at day 20 (MOV 8154 kb)

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Dubois, N., Craft, A., Sharma, P. et al. SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells. Nat Biotechnol 29, 1011–1018 (2011). https://doi.org/10.1038/nbt.2005

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