The following are the supplementary data related to

The following are the supplementary data related to this article.
Acknowledgments This work was supported by Marie-Curie IEF FellowshipPIEF-GA-2009-254644 (J.F.) and the Netherlands Institute for Regenerative Medicine (NIRM) (E.W., M. vd. B. and T.N.).
Introduction Human ES (hES) cells are pluripotent stem cells derived from the inner cell mass of sulfanilamide stage human embryos produced by in vitro fertilization (Thomson et al., 1998). Like mouse ES (mES) cells, hES cells are pluripotent and can form embryoid bodies in vitro and teratomas in vivo upon injection into immunodeficient mice. Although hES cells are of the same blastocyst origin as mES cells, they depend on distinct receptor tyrosine kinase signaling pathways for maintenance in culture. For example, hES cells require bFGF and TGFβ/Activin signals to maintain the undifferentiated state. In contrast, factors essential for mES cell self-renewal, such as LIF and BMPs, either have no effect on hES cells or induce their differentiation, respectively (Yu and Thomson, 2008). In hES cells, bFGF signals through the FGF receptor tyrosine kinase to activate Erk signaling which inhibits differentiation and the PI3K-Akt pathway to promote survival (Dvorak et al., 2005; Li et al., 2007). In addition, the TGFβ/Nodal/Activin signaling axis inhibits neuronal differentiation, and works synergistically with bFGF to maintain hES cell pluripotency (Vallier et al., 2005). Despite these differences in growth factor requirements between mES and hES cells, the core transcription factors governing pluripotency are similar, with both mES and hES cells expressing the master pluripotency factors, Oct4, Nanog and Sox2 (Boyer et al., 2005). While the growth factor conditions, receptor kinase signaling, and transcription factor networks governing hES cell fate have been examined in detail, the intracellular signaling pathways downstream of receptor tyrosine kinases have not been fully explored. The Src family of non-receptor tyrosine kinases is coupled to many growth factor receptors (including the FGFR) to regulate cell adhesion, proliferation, growth and survival (Parsons and Parsons, 2004; Boggon and Eck, 2004). There are eleven Src-related kinases in the human genome (Manning et al., 2002), eight of which have been studied extensively in mammalian cells (Blk, Fgr, Fyn, Lck, Lyn, Hck, c-Src and c-Yes) plus three phylogenetically related kinases (Srm, Frk and Brk). In adult mice, c-Src, Fyn and c-Yes are ubiquitously expressed, while Lck, Lyn, Hck, Blk and Fgr are more restricted in their expression patterns, primarily to hematopoietic cells (Lowell and Soriano, 1996). Surprisingly, at least seven members of the Src kinase family are expressed in mES cells, and individual family members appear to play distinct roles in regulating their developmental fate (Meyn et al., 2005). For example, the expression of Hck is rapidly silenced as mES cells differentiate to embryoid bodies (EBs), suggesting a role in self-renewal or the suppression of differentiation. In contrast, c-Src is expressed and active in both pluripotent mES cells and EBs derived from them, and its activity alone is sufficient to induce differentiation of mES cells to primitive ectoderm and endoderm (Meyn et al., 2005; Meyn and Smithgall, 2009). Other studies have shown that both Hck and c-Yes are important for mES cell self-renewal downstream of LIF (Ernst et al., 1994; Anneren et al., 2004; Tamm et al., 2011), and that c-Yes may have a similar role in human ES cells (Anneren et al., 2004). In this study, we performed a comprehensive analysis of Src-family kinase (SFK) expression and signaling during hES cell self-renewal and differentiation. The hES cell lines H1, H7 and H9 all express comparable mRNA levels of the Src-family members c-Src, c-Yes, Fyn, Lck and Lyn under culture conditions for self-renewal. Remarkably, Lck expression decreased dramatically as hES cells differentiated; Lck transcript and protein levels were lost more rapidly than those of the well-known pluripotency marker Oct4 during EB formation. The observed changes in Lck expression and activity are unique to hES cells as compared to mES cells, and suggest a possible role in the maintenance of the undifferentiated state. In contrast, c-Src, c-Yes and Fyn message levels did not change during differentiation, similar to previous observations in mES cells. We also found differences in SFK activity as a function of hES cell differentiation. c-Src, Fyn and Lyn were active in renewing hES cells and differentiated EBs, while Lck and c-Yes were active only in self-renewing hES cells. To investigate the requirement for SFK activity in differentiation, hES cell cultures were treated with small molecule SFK inhibitors previously tested in mES cells (Meyn and Smithgall, 2009; Meyn et al., 2005). Remarkably, hES cells treated with the potent pan-SFK inhibitor A-419259 retained colony morphology and marker expression characteristic of pluripotency even under culture conditions for differentiation. Immunoblots confirmed that this compound blocked all endogenous SFK activity, including that of Lck and c-Src. These observations demonstrate that the activities of several Src-family members are required for hES cells to initiate the differentiation program, and that SFK signals for differentiation may be dominant to those for self-renewal.