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EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS

Recombinant Vitronectin Is a Functionally Defined Substrate That Supports Human Embryonic Stem Cell Self-Renewal via Vβ5 Integrin

^aHubrecht Institute, Developmental Biology and Stem Cell Research, Utrecht, The Netherlands;
^bDepartment of Anatomy and Embryology, Leiden University Medical Centre, Leiden, The Netherlands;
^cHeart and Lung Division and
^dDepartment of Biomedical Genetics, University Medical Centre Utrecht, Utrecht, The Netherlands;
^eDivision of Cell Biology, Netherlands Cancer Institute, Amsterdam, The Netherlands

Key Words. Human embryonic stem cells • Self-renewal • Defined substrate • Integrins • Extracellular matrix

Correspondence: Correspondence: Christine Mummery, Ph.D., Leiden University Medical Centre, Postal Zone S-1-P, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Telephone: ++31-71-526 9307; Fax: ++31-71-526 8289; e-mail: c.l.mummery{at}lumc.nl

Received on April 17, 2008; accepted for publication on June 21, 2008.

First published online in STEM CELLS EXPRESS  July 3, 2008.

Defined growth conditions are essential for many applications of human embryonic stem cells (hESC). Most defined media are presently used in combination with Matrigel, a partially defined extracellular matrix (ECM) extract from mouse sarcoma. Here, we defined ECM requirements of hESC by analyzing integrin expression and ECM production and determined integrin function using blocking antibodies. hESC expressed all major ECM proteins and corresponding integrins. We then systematically replaced Matrigel with defined medium supplements and ECM proteins. Cells attached efficiently to natural human vitronectin, fibronectin, and Matrigel but poorly to laminin + entactin and collagen IV. Integrin-blocking antibodies demonstrated that Vβ5 integrins mediated adhesion to vitronectin, 5β1 mediated adhesion to fibronectin, and 6β1 mediated adhesion to laminin + entactin. Fibronectin in feeder cell-conditioned medium partially supported growth on all natural matrices, but in defined, nonconditioned medium only Matrigel or (natural and recombinant) vitronectin was effective. Recombinant vitronectin was the only defined functional alternative to Matrigel, supporting sustained self-renewal and pluripotency in three independent hESC lines.

Disclosure of potential conflicts of interest is found at the end of this article.