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A defined glycosaminoglycan-binding substratum for human pluripotent stem cells

Nature Methods volume 7, pages 989–994 (2010)Cite this article

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Abstract

To exploit the full potential of human pluripotent stem cells for regenerative medicine, developmental biology and drug discovery, defined culture conditions are needed. Media of known composition that maintain human embryonic stem (hES) cells have been developed, but finding chemically defined, robust substrata has proven difficult. We used an array of self-assembled monolayers to identify peptide surfaces that sustain pluripotent stem cell self-renewal. The effective substrates displayed heparin-binding peptides, which can interact with cell-surface glycosaminoglycans and could be used with a defined medium to culture hES cells for more than 3 months. The resulting cells maintained a normal karyotype and had high levels of pluripotency markers. The peptides supported growth of eight pluripotent cell lines on a variety of scaffolds. Our results indicate that synthetic substrates that recognize cell-surface glycans can facilitate the long-term culture of pluripotent stem cells.

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Figure 1: Strategy for the identification of peptide-substituted surfaces for hES cell adhesion and survival.
Figure 2: Surfaces displaying heparin-binding peptides support hES cell adhesion and self-renewal.
Figure 3: Synthetic surfaces support the long-term culture of pluripotent stem cells.
Figure 4: Pluripotent stem cells grown on synthetic surfaces maintain their ability to differentiate.
Figure 5: Streptavidin-coated surfaces presenting heparin-binding peptides support robust adhesion and self-renewal.

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References

  1. Thomson, J.A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).

    Article  CAS  Google Scholar 

  2. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

    Article  CAS  Google Scholar 

  3. Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).

    Article  CAS  Google Scholar 

  4. Ludwig, T.E. et al. Derivation of human embryonic stem cells in defined conditions. Nat. Biotechnol. 24, 185–187 (2006).

    Article  CAS  Google Scholar 

  5. Yao, S. et al. Long-term self-renewal and directed differentiation of human embryonic stem cells in chemically defined conditions. Proc. Natl. Acad. Sci. USA 103, 6907–6912 (2006).

    Article  CAS  Google Scholar 

  6. Wang, L. et al. Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling. Blood 110, 4111–4119 (2007).

    Article  CAS  Google Scholar 

  7. Xu, C. et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat. Biotechnol. 19, 971–974 (2001).

    Article  CAS  Google Scholar 

  8. Martin, M.J., Muotri, A., Gage, F. & Varki, A. Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat. Med. 11, 228–232 (2005).

    Article  CAS  Google Scholar 

  9. Amit, M., Shariki, C., Margulets, V. & Itskovitz-Eldor, J. Feeder layer- and serum-free culture of human embryonic stem cells. Biol. Reprod. 70, 837–845 (2004).

    Article  CAS  Google Scholar 

  10. Braam, S.R. et al. Recombinant vitronectin is a functionally defined substrate that supports human embryonic stem cell self renewal via alphavbeta5 integrin. Stem Cells 26, 2257–2265 (2008).

    Article  CAS  Google Scholar 

  11. Vuoristo, S. et al. Laminin isoforms in human embryonic stem cells: synthesis, receptor usage and growth support. J. Cell. Mol. Med. 13, 2622–2633 (2009).

    Article  Google Scholar 

  12. Derda, R. et al. Defined substrates for human embryonic stem cell growth identified from surface arrays. ACS Chem. Biol. 2, 347–355 (2007).

    Article  CAS  Google Scholar 

  13. Gerecht, S. et al. Hyaluronic acid hydrogel for controlled self-renewal and differentiation of human embryonic stem cells. Proc. Natl. Acad. Sci. USA 104, 11298–11303 (2007).

    Article  CAS  Google Scholar 

  14. Li, Y.J., Chung, E.H., Rodriguez, R.T., Firpo, M.T. & Healy, K.E. Hydrogels as artificial matrices for human embryonic stem cell self-renewal. J. Biomed. Mater. Res. A 79, 1–5 (2006).

    Article  Google Scholar 

  15. Harb, N., Archer, T.K. & Sato, N. The Rho-Rock-Myosin signaling axis determines cell-cell integrity of self-renewing pluripotent stem cells. PLoS ONE 3, e3001 (2008).

    Article  Google Scholar 

  16. Melkoumian, Z. 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).

    Article  CAS  Google Scholar 

  17. Villa-Diaz, L.G. et al. Synthetic polymer coatings for long-term growth of human embryonic stem cells. Nat. Biotechnol. 28, 581–583 (2010).

    Article  CAS  Google Scholar 

  18. Derda, R. et al. High-throughput discovery of synthetic surfaces that support proliferation of pluripotent cells. J. Am. Chem. Soc. 132, 1289–1295 (2010).

    Article  CAS  Google Scholar 

  19. Meng, Y. et al. Characterization of integrin engagement during defined human embryonic stem cell culture. FASEB J. 24, 1056–1065 (2010).

    Article  CAS  Google Scholar 

  20. Kolhar, P., Kotamraju, V.R., Hikita, S.T., Clegg, D.O. & Ruoslahti, E. Synthetic surfaces for human embryonic stem cell culture. J. Biotechnol. 146, 143–146 (2010).

    Article  CAS  Google Scholar 

  21. Orner, B.P., Derda, R., Lewis, R.L., Thomson, J.A. & Kiessling, L.L. Arrays for the combinatorial exploration of cell adhesion. J. Am. Chem. Soc. 126, 10808–10809 (2004).

    Article  CAS  Google Scholar 

  22. Whitesides, G.M., Ostuni, E., Takayama, S., Jiang, X. & Ingber, D.E. Soft lithography in biology and biochemistry. Annu. Rev. Biomed. Eng. 3, 335–373 (2001).

    Article  CAS  Google Scholar 

  23. Mrksich, M. & Whitesides, G.M. Using self-assembled monolayers to understand the interactions of man-made surfaces with proteins and cells. Annu. Rev. Bioph. Biom. 25, 55–78 (1996).

    Article  CAS  Google Scholar 

  24. Ruoslahti, E. & Pierschbacher, M.D. New perspectives in cell adhesion: RGD and integrins. Science 238, 491–497 (1987).

    Article  CAS  Google Scholar 

  25. Lutolf, M.P., Gilbert, P.M. & Blau, H.M. Designing materials to direct stem-cell fate. Nature 462, 433–441 (2009).

    Article  CAS  Google Scholar 

  26. Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686 (2007).

    Article  CAS  Google Scholar 

  27. Vogel, B.E. et al. A novel integrin specificity exemplified by binding of the alpha v beta 5 integrin to the basic domain of the HIV Tat protein and vitronectin. J. Cell Biol. 121, 461–468 (1993).

    Article  CAS  Google Scholar 

  28. Bishop, J.R., Schuksz, M. & Esko, J.D. Heparan sulphate proteoglycans fine-tune mammalian physiology. Nature 446, 1030–1037 (2007).

    Article  CAS  Google Scholar 

  29. Ying, Q.L. et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519–523 (2008).

    Article  CAS  Google Scholar 

  30. Engler, A.J., Sen, S., Sweeney, H.L. & Discher, D.E. Matrix elasticity directs stem cell lineage specification. Cell 126, 677–689 (2006).

    Article  CAS  Google Scholar 

  31. Ludwig, T.E. et al. Feeder-independent culture of human embryonic stem cells. Nat. Methods 3, 637–646 (2006).

    Article  CAS  Google Scholar 

  32. Prokhorova, T. et al. Teratoma formation by human embryonic stem cells is site-dependent and enhanced by the presence of Matrigel. Stem Cells Dev. 18, 47–54 (2008).

    Article  Google Scholar 

  33. Laflamme, M.A. et al. Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nat. Biotechnol. 25, 1015–1024 (2007).

    Article  CAS  Google Scholar 

  34. D'Amour, K.A. et al. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat. Biotechnol. 23, 1534–1541 (2005).

    Article  CAS  Google Scholar 

  35. Chambers, S.M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27, 275–280 (2009).

    Article  CAS  Google Scholar 

  36. Luk, Y.Y., Kato, M. & Mrksich, M. Self-assembled monolayers of alkanethiolates presenting mannitol groups are inert to protein adsorption and cell attachment. Langmuir 16, 9604–9608 (2000).

    Article  CAS  Google Scholar 

  37. Klein, E.A., Yung, Y., Castagnino, P., Kothapalli, D. & Assoian, R.K. Cell adhesion, cellular tension, and cell cycle control. Methods Enzymol. 426, 155–175 (2007).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank R. Derda, B.P. Orner and J.A. Thomson for helpful discussions, J.R. Torrealba for analysis and interpretation of the teratoma data and G.L. Case for help with the automated peptide synthesis. This research was supported by the US National Institutes of Health (R01 grants AI055258 and GM49975) and the University of Wisconsin Materials Research Science and Engineering Center (DMR-0520527). We thank the W.M. Keck Foundation for supporting the Center for Chemical Genomics and the WiCell Research Institute and members of the University of Wisconsin Paul P. Carbone Comprehensive Cancer Center Flow Cytometry Facility (5P30 CA014520-3S) for technical assistance.

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Authors and Affiliations

  1. Cell and Molecular Biology Program, University of Wisconsin–Madison, Madison, Wisconsin, USA

    Joseph R Klim & Laura L Kiessling

  2. Department of Chemistry, University of Wisconsin–Madison, Madison, Wisconsin, USA

    Lingyin Li & Laura L Kiessling

  3. Department of Biochemistry, University of Wisconsin–Madison, Madison, Wisconsin, USA

    Paul J Wrighton & Laura L Kiessling

  4. WiCell Research Institute, Madison, Wisconsin, USA

    Marian S Piekarczyk

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  1. Joseph R Klim
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Contributions

J.R.K., L.L., P.J.W. and L.L.K. conceived the experiments and interpreted the results. J.R.K. performed the in vitro experiments. L.L. synthesized and purified the molecules used to fabricate the surfaces. J.R.K. and M.S.P. conducted the teratoma assay, and P.J.W. conducted the directed differentiation assays. J.R.K. and L.L.K. wrote the manuscript.

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Correspondence to Laura L Kiessling.

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Competing interests

L.L.K. is an author on a patent on self-assembled monolayers for stem cell culture (US patent 2007/0207543). L.L.K., J.R.K. and L.L. are authors on a pending patent that describes surfaces for the long-term culture of pluripotent cells (US patent application 20100087004).

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Supplementary Figures 1–11 and Supplementary Tables 1–2 (PDF 37252 kb)

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Klim, J., Li, L., Wrighton, P. et al. A defined glycosaminoglycan-binding substratum for human pluripotent stem cells. Nat Methods 7, 989–994 (2010). https://doi.org/10.1038/nmeth.1532

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