Synthetic peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte differentiation of human embryonic stem cells

Abstract

Human embryonic stem cells (hESCs) have two properties of interest for the development of cell therapies: self-renewal and the potential to differentiate into all major lineages of somatic cells in the human body. Widespread clinical application of hESC-derived cells will require culture methods that are low-cost, robust, scalable and use chemically defined raw materials. Here we describe synthetic peptide-acrylate surfaces (PAS) that support self-renewal of hESCs in chemically defined, xeno-free medium. H1 and H7 hESCs were successfully maintained on PAS for over ten passages. Cell morphology and phenotypic marker expression were similar for cells cultured on PAS or Matrigel. Cells on PAS retained normal karyotype and pluripotency and were able to differentiate to functional cardiomyocytes on PAS. Finally, PAS were scaled up to large culture-vessel formats. Synthetic, xeno-free, scalable surfaces that support the self-renewal and differentiation of hESCs will be useful for both research purposes and development of cell therapies.

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Figure 1: Development of the PAS surface.
Figure 2: PAS supports long-term culture and pluripotency of H7 hESCs in chemically defined medium X-VIVO 10 + GF.
Figure 3: Direct differentiation of H7 hESCs into cardiomyocytes on PAS and scalability of PAS production.

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Acknowledgements

We would like to thank our sponsors from Corning Incorporated, J. Mooney and M. McFarland, and from Geron Corporation, J. Lebkowski and A.H. Davies. From Corning Incorporated we thank A. Frutos for critical reading and helpful comments on the manuscript, M. Lewis for his chemistry expertise, T. Garvey for his assistance in PAS fabrication, P. Gagnon and P. Szlosek for substrate plate supply and T. Heck for his confocal imaging help. From Geron Corporation we thank K. Delavan-Boorsma and S. Edell for their support of in vivo studies.

Author information

Z.M. conceived, designed, performed and analyzed PAS validation with H7 cells. J.L.W. conceived, designed, performed and analyzed PAS validation with H1 cells, wrote supplementary materials. P.D.-S. and J.Y. designed, performed and analyzed cardiomyocyte differentiation on PAS. J.Y. designed, performed and analyzed EB differentiation. L.Q. assisted J.Y. in performing and analyzing cardiomyocytes and EB differentiation. C.A.P. performed teratoma formation studies. D.M.W., A.G.F. and Y.Z. conceived, designed, developed and fabricated PAS. D.M.W. identified peptides for the study, characterized surface peptide uniformity, contributed to design of peptide conjugation scheme. A.G.F. developed and optimized peptide conjugation and characterized surface peptide density. C.S. assisted Y.Z. in acrylate coating development and fabrication. A.M., J.N. and P.W. developed coating conditions for PAS in T75 format, fabricated PAS-T75. Z.M. and R.B. wrote the manuscript. J.P.B., S.P. and R.B. provided direction and guidance for the various areas of the project.

Correspondence to Zara Melkoumian or Ralph Brandenberger.

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Competing interests

Z.M., J.L.W., P.D.-S., J.P.B., S.P., D.M.W., A.G.F., Y.Z., C.S., A.M., J.N. and P.W. are employees of Corning Incorporated. J.Y., L.Q., C.A.P. and R.B. are employees of Geron Corporation.

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Supplementary Tables 1,2 and Supplementary Figs. 1–6 (PDF 1273 kb)

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Melkoumian, Z., Weber, J., Weber, D. et al. Synthetic peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte differentiation of human embryonic stem cells. Nat Biotechnol 28, 606–610 (2010) doi:10.1038/nbt.1629

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