The fibroblast feeder cells support

The fibroblast feeder GSK2656157 support hESCs by secreting growth factors and ECM components to the culture medium and also by directly interacting with the hESCs through cell–cell contacts. The factors secreted by fibroblast feeder cells have been studied by analyzing fibroblast CM (Lim and Bodnar, 2002; Prowse et al., 2005; Buhr et al., 2007; Chin et al., 2007; Prowse et al., 2007) and de-cellularized matrices with mass spectrometry (Abraham et al., 2010). Several ECM proteins such as collagens, fibronectin, laminins, nidogen and heparan sulfate proteoglycans have been suggested as key factors provided by the hESC-supportive feeder cells (Lim and Bodnar, 2002; Prowse et al., 2005, 2007; Abraham et al., 2010). Vitronectin (Braam et al., 2008; Prowse et al., 2011), fibronectin (Amit and Itskovitz-Eldor, 2006), laminin (Xu et al., 2001; Rodin et al., 2010) and a combination of collagen IV, vitronectin, fibronectin, and laminin (Ludwig et al., 2006a), have been used as substrata in feeder-independent culture of hESCs, showing that these ECM proteins, in combinations with specific media and growth factors, support attachment and proliferation of hESCs. In addition to ECM proteins, growth factors provided by the feeder cells or in the culture medium are essential for hESC growth. The central role of basic fibroblast growth factor (bFGF) in hESC self-renewal is well established (Dvorak et al., 2005; Levenstein et al., 2006). Basic FGF and transforming growth factor beta (TGFß) family members TGFß, activin A (ActA) and nodal (Vallier et al., 2005; Xiao et al., 2006) co-operate to maintain undifferentiated hESC growth. The gene expression and secretion of these key growth factors have been shown to differ between hESC-supportive and non-supportive feeder cells (Eiselleova et al., 2008; Kueh et al., 2006).
Results
Discussion Feeder cells contribute to maintenance of hESC morphology and pluripotent status in feeder-dependent culture conditions by mechanisms that are poorly known. Likely the feeder cells will be replaced with more defined, feeder-independent hESC culture conditions in the future. Many serum-free media formulations for feeder-independent hESC culture have been published and some, such as mTeSR1 (Stemcell Technologies) (Ludwig et al., 2006b) and STEMPRO hESC SFM (Life Technologies, Invitrogen) are commercially available. These media are routinely used in combination with xeno-derived, undefined matrices like Matrigel or Geltrex but have been shown to support pluripotent stem cell culture also with defined substrates like vitronectin (Braam et al., 2008) and laminin-511 (Rodin et al., 2010). Although the long-term culture of undifferentiated pluripotent stem cells is possible in these feeder-independent conditions, the media still contain undefined and xeno-derived compounds and the success of deriving new pluripotent stem cell lines, both hESC and hiPSC, in completely defined, xeno- and feeder-free conditions is low. Defining the factors provided by fibroblast feeder cells and revealing their complex mechanisms in hESC maintenance provides valuable tools for further development of feeder-independent derivation and culture methods. We studied the differences in ECM and growth factor production between hESC-supportive and non-supportive human fibroblast feeder cells. Many of the genes down-regulated in the non-hESC supportive hDF feeder cells were distinct laminin isoforms and laminin related integrin subunits that have been reported to be associated with undifferentiated hESCs. Laminins are trimeric glycoproteins consisting of one α (1–5), one ß (1–3), and one γ (1–3) chain (Colognato and Yurchenco, 2000), and are the first ECM proteins synthesized by the mouse blastocyst (Miner et al., 2004). A number of studies have shown that in addition to embryogenesis, the specific laminin isoforms regulate cell adhesion, proliferation, migration and differentiation (Colognato and Yurchenco, 2000; Scheele et al., 2007). We have previously shown that undifferentiated hESCs synthesize and deposit laminin-511 (α5ß1γ1) and -111 (α1ß1γ1) and that laminin-511, purified from human cells, can be used as a supportive matrix in feeder-independent hESC cultures (Vuoristo et al., 2009). These results have been confirmed by others for both hESCs (Rodin et al., 2010; Miyazaki et al., 2008; Evseenko et al., 2009) and mESCs (Domogatskaya et al., 2008), indicating that laminin-511 has a central role in the maintenance of hESC survival and pluripotency. Some studies have also reported laminin-332 (α3ß3γ2) to function as hESC substrata (Miyazaki et al., 2008). In the present study, laminin-511 was found to be produced by the most commonly used hESC-supportive feeder types, hFFs and mEFs, but not by the non-supportive hDF feeder cell lines. This indicates that laminin-511 might also act in hESC self-renewal through feeder cells, yet the possible mechanism remains to be studied.