Both natural and synthetic approaches have

Both natural and synthetic approaches have been used to generate fully defined hPSC culture substrates. ECM proteins such as vitronectin and laminins 511 and 521 have been shown to maintain hESC pluripotency and self-renewal (Braam et al., 2008; Domogatskaya et al., 2012; Miyazaki et al., 2012; Rodin et al., 2010, 2014a, 2014b; Yurchenco, 2011). However, not all ECM proteins are suitable for hPSC maintenance because collagens and fibronectin are not able to support an undifferentiated hPSC population (Azarin and Palecek, 2010; Braam et al., 2008; Evseenko et al., 2009; Miyazaki et al., 2012; Rodin et al., 2010; Villa-Diaz et al., 2013). Other fully defined hPSC culture environments have been produced by modification of substrates with recombinant E-cadherin (marketed as StemAdhere) (Nagaoka et al., 2010) or engineered peptide coatings (marketed as Synthemax) (Melkoumian et al., 2010).
Results
Discussion In this study, we first identified laminin as a prevalent signature component of the ECM produced endogenously by undifferentiated H9 hESCs and 19-9-11 iPSCs cultured on three different defined substrates and subsequently examined the role of endogenously produced α-5 laminin in regulating the self-renewal and pluripotency marker expression of these stem cells. We targeted the α-5 chain specifically because the majority of integrin binding activity in the laminin molecule is thought to be conferred by the C-terminal globular domains encoded on the α strand (Beck et al., 1990; Deutzmann et al., 1990; Ido et al., 2006; Li et al., 2002; Miner and Yurchenco, 2004). Furthermore, hPSCs predominantly express the α6β1 integrin, which interacts strongly with these laminin globular domains (Evseenko et al., 2009; Li et al., 2002; Meng et al., 2010; Miyazaki et al., 2012; Rodin et al., 2010). Using two different techniques to disrupt α-5 laminin production in both hESC and iPSC lines, we found that hPSC expression of the pluripotency marker Nanog was not strongly dependent on α-5 laminin production but that hPSC self-renewal diminished as a result of apoptosis in the absence of this specific ECM component. Importantly, this apoptosis could be reduced by supplying the knockout EHT 1864 Supplier with an exogenous source of α-5 laminin, such as culturing them on a laminin-521 substrate (Rodin et al., 2014b). To link this apoptotic response to an ECM-dependent mechanism, we tested heterozygous and knockout LAMA5 lines against several small-molecule inhibitors and identified blebbistatin and the ROCK inhibitor Y27632 as being able to reduce the apoptotic response of the knockout lines cultured under defined conditions. Blebbistatin and Y27632 are known to act in pluripotent stem cells by preventing membrane blebbing, a phenomenon that was observed during culture of α-5 laminin knockout cells under defined conditions. These agents prevent blebbing via inhibition of contractility (Chen et al., 2010, 2014; Ohgushi et al., 2010; Ohgushi and Sasai, 2011; Watanabe et al., 2007), which may reduce anoikis, a type of apoptotic cell death that occurs because of a lack of cell-cell and cell-ECM contacts (Chen et al., 2010; Kovács et al., 2004; Ohgushi et al., 2010; Ohgushi and Sasai, 2011; Watanabe et al., 2007). Although contractility inhibition did reduce apoptosis in α-5 laminin-deficient cells, the restoration of hPSC self-renewal was only partial, which does suggest the involvement of additional signaling mechanisms in the hPSC response to α-5 laminin. The specificity of the hPSC apoptotic response to a lack of proper ECM contacts highlights the importance of substrate interactions in regulating hPSC survival. Work by our own group suggests that differentiating pluripotent stem cells produce an ECM that is synergistic with the differentiation cues given to the cells (Laperle et al., 2015). Meanwhile, recent work by Meng et al. (2012) suggests that matrices, media, and the soluble growth factors to which hPSCs are exposed during culture have a synergistic effect on the growth and attachment of hPSCs under defined culture conditions. For instance, in mouse mesangial and early endoderm progenitor cells, transforming growth factor β (TGF-β) signaling has been shown to drive laminin production and increase the production of other ECM components (Jiang et al., 2005; Sugiyama et al., 2013; Tonary and Carnegie, 1996; Wells and Discher, 2008). It is possible that the α-5 laminin self-renewal signal, combined with other soluble components, drives hPSCs to produce a matrix that synergizes with these self-renewal signals. The hPSCs subsequently produce a matrix rich in α-5 laminin and receive supportive self-renewal signals from that matrix in an autocrine and paracrine signaling loop (Figure 6). Although this endogenous ECM may be necessary for efficient self-renewal, it is unlikely to be sufficient to fully support this process on its own.