Recombinant vitronectin is a functionally defined substrate that supports human embryonic stem cell self-renewal via alphavbeta5 integrin.
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 alphaVbeta5 integrins mediated adhesion to vitronectin, alpha5beta1 mediated adhesion to fibronectin, and alpha6beta1 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.
Braam
Stefan R
SR
Leiden University Medical Centre, Postal Zone S-1-P, P.O. Box 9600, 2300 RC Leiden, The Netherlands.
Zeinstra
Laura
L
Litjens
Sandy
S
Ward-van Oostwaard
Dorien
D
van den Brink
Stieneke
S
van Laake
Linda
L
Lebrin
Franck
F
Kats
Peter
P
Hochstenbach
Ron
R
Passier
Robert
R
Sonnenberg
Arnoud
A
Mummery
Christine L
CL
D000818:Animals; D002448:Cell Adhesion; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D003094:Collagen; D003470:Culture Media; D004338:Drug Combinations; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis; D006801:Humans; D007797:Laminin; D051379:Mice; D011509:Proteoglycans; D019030:Receptors, Vitronectin / Q000502:physiology*; D011994:Recombinant Proteins / Q000494:pharmacology; D019096:Vitronectin / Q000494:pharmacology*
D003470:Culture Media; D004338:Drug Combinations; D016326:Extracellular Matrix Proteins; D007797:Laminin; D011509:Proteoglycans; D019030:Receptors, Vitronectin; D011994:Recombinant Proteins; D019096:Vitronectin; C064660:integrin alphaVbeta5; C059485:matrigel; D003094:Collagen