α6β1- and αV-integrins are required for long-term self-renewal of murine embryonic stem cells in the absence of LIF

Abstract

BackgroundThe 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.ResultsHere 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.ConclusionsIn 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-015-0051-y) contains supplementary material, which is available to authorized users.

Jun-Mediated Changes in Cell Adhesion Contribute to Mouse Embryonic Stem Cell Exit from Ground State Pluripotency

“…Indeed, such a hypothesis is supported by the observation that removal of either β1 or αv integrin affects the ability of ESC to exit from the pluripotent state, as demonstrated by their retention of the expression of pluripotency markers. This is consistent with previous data showing a role for αv integrin in human ESC function and a role for αv integrin in maintaining the epistem cell‐like state in mESC cultures . Moreover, α5β1 integrin has a role in hESC function through adhesion to fibronectin .…”

Blood vitronectin is a major activator of LIF and IL-6 in the brain through integrin–FAK and uPAR signaling

“…Regulation of LIF through integrins or FAK was previously unknown. In the absence of LIF, laminin-111 and -511 (laminin-511 is composed of α5, β1 and γ1 chains) promote stem cell renewal in vitro through α6β1 integrin (Cattavarayane et al, 2015). This might involve induction of LIF, since it is widely used to maintain self-renewal.…”

Generation of Cynomolgus Monkey Chimeric Fetuses using Embryonic Stem Cells

“…Looking in more detail at the expression of key signaling pathway components, we found that the LIF-Stat3, PI3K-Akt, Nodal-Smad2/3, and Wnt signaling pathways were all significantly activated in NHSMV cells ( Figure S2D). ECM components can also affect the growth and self-renewal properties of ESCs (Cattavarayane et al, 2015), and we found that a number of ECM molecules, including collagen, integrin, laminin, tenascin, and THBS, were significantly upregulated in NHSMV cells ( Figure S2E). Finally, analysis of PI3K-Akt signaling pathways showed upregulated GT-RTK, ECM-ITGA, Gbg, and Cytokine R and downregulated PP2A (promoting CDK activation and cell-cycle progression) in NHSMV cells ( Figure S2F).…”