Skip to main content
Biointerphases

Journal for Biophysical Chemistry

Download PDF

Impact of vitronectin concentration and surface properties on the stable propagation of human embryonic stem cells

Biointerphases volume 5, pages FA132–FA142 (2010)Cite this article

Abstract

The standard method for culturing human embryonic stem cells (hESC) uses supporting feeder layers of cells or an undefined substrate, MatrigelTM, which is a basement membrane extracted from murine sarcoma. For stem cell therapeutic applications, a superior alternative would be a defined, artificial surface that is based on immobilized human plasma vitronectin (VN), which is an adhesion-mediating protein. Therefore, VN adsorbed to diverse polymer surfaces was explored for the continuous propagation of hESC. Cells propagated on VN-coated tissue culture polystyrene (TCPS) are karyotypically normal after >10 passages of continuous culture, and are able to differentiate into embryoid bodies containing all three germ layers. Expansion rates and pluripotent marker expression verified that a minimal VN surface density threshold is required on TCPS. Further exploration of adsorbed VN was conducted on polymer substrates with different properties, ranging from hydrophilic to hydrophobic and including cationic and anionic polyelectrolyte coatings. Despite differing surface properties, these substrates adsorbed VN above the required surface density threshold and were capable of supporting hESC expansion for >10 passages. Correlating wettability of the VN-coated surfaces with the response of cultured hESC, higher cell expansion rates and OCT-4 expression levels were found for VN-coated TCPS, which exhibits a water contact angle close to 65°. Importantly, this simple, defined surface matches the performance of the benchmark Matrigel, which is a hydrogel with highly complex composition.

Reference

  1. J. A. Thomson, Science 282, 1145 (1998).

    Article  CAS  Google Scholar 

  2. M. Amit, M. K. Carpenter, M. S. Inokuma, C. P. Chiu, C. P. Harris, M. A. Waknitz, J. Itskovitz-Eldor, and J. A. Thomson, Dev. Biol. 227, 271 (2000).

    Article  CAS  Google Scholar 

  3. C. H. Xu, M. S. Inokuma, J. Denham, K. Golds, P. Kundu, J. D. Gold, and M. K. Carpenter, Nat. Biotechnol. 19, 971 (2001).

    Article  CAS  Google Scholar 

  4. O. Genbacev et al., Fertil. Steril. 83, 1517 (2005).

    Article  Google Scholar 

  5. A. B. H. Choo, J. Padmanabhan, A. C. P. Chin, and S. K. W. Oh, Biotechnol. Bioeng. 88, 321 (2004).

    Article  CAS  Google Scholar 

  6. A. Choo, J. Padmanabhan, A. Chin, W. J. Fong, and S. K. W. Oh, J. Biotechnol. 122, 130 (2006).

    Article  CAS  Google Scholar 

  7. H. Hakala, K. Rajala, M. Ojala, S. Panula, S. Areva, M. Kellomäki, R. Suuronen, and H. Skottman, Tissue Eng. A 15, 1775 (2009).

    Article  CAS  Google Scholar 

  8. R. Derda, L. Li, B. Orner, R. Lewis, J. Thomson, and L. Kiessling, ACS Chem. Biol. 2, 347 (2007).

    Article  CAS  Google Scholar 

  9. Z. Melkoumian et al., Nat. Biotechnol. 28, 606 (2010).

    Article  CAS  Google Scholar 

  10. P. Kolhar, V. R. Kotamraju, S. T. Hikita, D. O. Clegg, and E. Ruoslahti, J. Biotechnol. 146, 143 (2010).

    Article  CAS  Google Scholar 

  11. L. G. Villa-Diaz, H. Nandivada, J. Ding, N. C. Nogueira-de-Souza, P. H. Krebsbach, K. S. O'Shea, J. Lahann, and G. D. Smith, Nat. Biotechnol. 28, 581 (2010).

    Article  CAS  Google Scholar 

  12. Y. Mei et al., Nature Mater. 9, 768 (2010).

    Article  CAS  Google Scholar 

  13. T. E. Ludwig et al., Nat. Biotechnol. 24, 185 (2006).

    Article  CAS  Google Scholar 

  14. M. Amit and J. Itskovitz-Eldor, Methods Mol. Biol. 331, 105 (2006).

    Google Scholar 

  15. T. Miyazaki et al., Biochem. Biophys. Res. Commun. 375, 27 (2008).

    Article  CAS  Google Scholar 

  16. S. R. Braam et al., Stem Cells 26, 2257 (2008).

    Article  CAS  Google Scholar 

  17. S. Rodin, A. Domogatskaya, S. Strom, E. M. Hansson, K. R. Chien, J. Inzunza, O. Hovatta, and K. Tryggvason, Nat. Biotechnol. 28, 611 (2010).

    Article  CAS  Google Scholar 

  18. T. J. Rowland, L. M. Miller, A. J. Blaschke, E. L. Doss, A. J. Bonham, S. T. Hikita, L. V. Johnson, and D. O. Clegg, Stem Cells. Dev. 19, 1231 (2010).

    Article  CAS  Google Scholar 

  19. I. Schvartz, D. Seger, and S. Shaltiel, Int. J. Biochem. Cell Biol. 31, 539 (1999).

    Article  CAS  Google Scholar 

  20. L. Y. W. Yap et al., Tissue Eng. C: Methods (in press, doi:10.1089/ten.tec.2010.0328).

  21. J. Racine, E. Luong-Van, Y. Sadikin, R. K. C. Kang, Y. S. Chu, V. Racine, J. P. Thiery, and W. R. Birch, J. Adhes. Sci. Technol. 24, 975 (2010).

    Article  CAS  Google Scholar 

  22. S. K. W. Oh, A. K. Chen, A. B. H. Choo, and I. Reading, “Quantitative 2D Imaging of Human Embryonic Stem Cells, in Emerging Technology Platforms for Stem Cells, edited by U. Lakshmipathy, J. D. Chestnut, and B. Thyagarajan (Wiley, Hoboken, NJ, 2009), pp. 283–290.

    Chapter  Google Scholar 

  23. H. L. Tan, W. J. Fong, E. H. Lee, M. Yap, and A. Choo, Stem Cells 27, 1792 (2009).

    Article  CAS  Google Scholar 

  24. A. C. Chin, W. J. Fong, L. T. Goh, R. Philp, S. K. Oh, and A. B. Choo, J. Biotechnol. 130, 320 (2007).

    Article  CAS  Google Scholar 

  25. See supplementary material for the primers used in qRT-PCR.

  26. E. A. Vogler, J. Biomater. Sci., Polym. Ed. 10, 1015 (1999).

    Article  CAS  Google Scholar 

  27. C. C. Barrias, M. C. L. Martins, G. Almeida-Porada, M. A. Barbosa, and P. L. Granja, Biomaterials 30, 307 (2009).

    Article  CAS  Google Scholar 

  28. J. Y. Lim, X. M. Liu, E. A. Vogler, and H. J. Donahue, J. Biomed. Mater. Res. 68A, 504 (2004).

    Article  CAS  Google Scholar 

  29. E. Occhiello, M. Morra, P. Cinquina, and F. Garbassi, Polymer 33, 3007 (1992).

    Article  CAS  Google Scholar 

  30. S. Guruvenket, G. M. Rao, M. Komath, and A. M. Raichur, Appl. Surf. Sci. 236, 278 (2004).

    Article  CAS  Google Scholar 

  31. L. Feng, Y. L. Song, J. Zhai, B. Q. Liu, J. Xu, L. Jiang, and D. B. Zhu, Angew. Chem., Int. Ed. 42, 800 (2003).

    Article  CAS  Google Scholar 

  32. M. R. Doran, J. E. Frith, A. B. J. Prowse, J. Fitzpatrick, E. J. Wolvetang, T. P. Munro, P. P. Gray, and J. J. Cooper-White, Biomaterials 31, 5137 (2010).

    Article  CAS  Google Scholar 

  33. N. T. Kohen, L. E. Little, and K. E. Healy, BioInterphases 4, 69 (2009).

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Materials Research and Engineering, Agency for Science, Technology and Research (A*STAR), 117602, Singapore

    Jian Li

  2. Stem Cell Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 138668, Singapore

    Jo'an Bardy, Allen Chen & Steve K. W. Oh

  3. Stem Cells and Tissue Repair Group, Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR), 138648, Singapore, Singapore

    Lynn Y. W. Yap, Victor Nurcombe & Simon M. Cool

  4. Graduate School for Integrative Sciences and Engineering, National University of Singapore, 117597, Singapore

    Lynn Y. W. Yap

  5. Department of Orthopedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, 119074, Singapore

    Victor Nurcombe & Simon M. Cool

  6. Institute of Materials Research and Engineering, Agency for Science, Technology and Research (A*STAR), 117602, Singapore

    William R. Birch

Authors
  1. Jian Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jo'an Bardy
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lynn Y. W. Yap
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Allen Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Victor Nurcombe
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Simon M. Cool
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Steve K. W. Oh
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. William R. Birch
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Steve K. W. Oh.

Additional information

This paper is part of an In Focus section on Biointerphase Science in Singapore, sponsored by Bruker Optik Southeast Asia, IMRE, the Provost's Office and School of Materials Science and Engineering of Nanyang Technological University, and Analytical Technologies Pte. Ltd.

These authors contributed equally to the work described in this article.

Authors to whom correspondence should be addressed.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Li, J., Bardy, J., Yap, L.Y.W. et al. Impact of vitronectin concentration and surface properties on the stable propagation of human embryonic stem cells. Biointerphases 5, FA132–FA142 (2010). https://doi.org/10.1116/1.3525804

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1116/1.3525804