BACKGROUND: The growth properties and self-renewal capacity of embryonic stem 
(ES) cells are regulated by their immediate microenvironment such as the 
extracellular matrix (ECM). Integrins, a central family of cellular ECM 
receptors, have been implicated in these processes but their specific role in ES 
cell self-renewal remains unclear.
RESULTS: Here we have studied the effects of different ECM substrates and 
integrins in mouse ES cells in the absence of Leukemia Inhibitory Factor (LIF) 
using short-term assays as well as long-term cultures. Removal of LIF from ES 
cell culture medium induced morphological differentiation of ES cells into 
polarized epistem cell-like cells. These cells maintained epithelial morphology 
and expression of key stemness markers for at least 10 passages in the absence 
of LIF when cultured on laminin, fibronectin or collagen IV substrates. The 
specific functional roles of α6-, αV- and β1-integrin subunits were dissected 
using stable lentivirus-mediated RNAi methodology. β1-integrins were required 
for ES cell survival in long-term cultures and for the maintenance of stem cell 
marker expression. Inhibition of α6-integrin expression compromised self-renewal 
on collagen while αV-integrins were required for robust ES cell adhesion on 
laminin. Analysis of the stemness marker expression revealed subtle differences 
between α6- and αV-depleted ES cells but the expression of both was required for 
optimal self-renewal in long-term ES cell cultures.
CONCLUSIONS: In the absence of LIF, long-term ES cell cultures adapt an epistem 
cell-like epithelial phenotype and retain the expression of multiple stem cell 
markers. Long-term maintenance of such self-renewing cultures depends on the 
expression of β1-, α6- and αV-integrins.