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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 αVβ5 integrins mediated adhesion to vitronectin, α5β1 mediated adhesion to fibronectin, and α6β1 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.

Recombinant Vitronectin Is a Functionally Defined Substrate That Supports Human Embryonic Stem Cell Self‐Renewal via αVβ5 Integrin