Using the potent Src family kinase inhibitor A we also

Using the potent Src-family kinase inhibitor, A-419259, we also explored the role of SFK activity in hES cell fate. Treatment of hES caspase inhibitor with this inhibitor blocked all endogenous Src-family kinase activity, including that of c-Src and Lck. As a consequence, hES cells treated with the inhibitor grew as domed, morphologically undifferentiated colonies even when cultured in differentiation medium. Similar results were obtained with two additional Src-family kinase inhibitors from other chemical classes: the pyrazolopyrimidine, PP2, and the anilino-quinazoline, SKI-1 (data not shown) (Meyn et al., 2005). Furthermore, hES cells continued to express the well-established cell-surface marker for self-renewal, Tra-1-60, when cultured with A-419259 under differentiation conditions. Finally, addition of A-419259 to AggreWell cultures of hES cells completely blocked the formation of EBs (data not shown), a result similar to that observed previously with mES cells (Meyn et al., 2005). Taken together, these observations strongly suggest that SFK activity is required for hES cell differentiation to occur. A-419259 inhibits all members of the Src-kinase family to a similar extent, including kinases linked to opposing functions in ES cell self-renewal (e.g., Lck or c-Yes) vs. differentiation (c-Src). However, the net effect of global SFK inhibition is suppression of hES cell differentiation, a phenotype very similar to that reported previously in mES cells (Zhang et al., 2014; Meyn and Smithgall, 2009; Meyn et al., 2005). This observation supports the idea that a hierarchy of SFK signaling exists in ES cells, in which kinases that influence self-renewal are dominant over those that initiate differentiation. When both classes of kinases are blocked, then the ES cells become trapped in the undifferentiated state. A complete understanding of the complex and opposing roles of individual Src-family members in hES cell differentiation will require future studies of individual kinase functions. One approach is to use RNA interference to selectively block the expression of individual kinases. For example, RNAi knockdown of c-Yes caused ES cells to lose self-renewal marker expression and undergo differentiation (Anneren et al., 2004). However, knockdown approaches may suffer from the same limitations as knockout mice, in that functional compensation through upregulation of redundant kinases may occur during the selection process required to establish the knockdown cell population. In addition, knockdown and knockout approaches, in contrast to pharmacological inhibition, eliminate the kinase protein entirely thus removing possible adaptor or other functions that are independent of protein kinase activity. Another approach is the use of selective kinase inhibitors. However, because of the highly conserved nature of Src-family kinase domains, truly selective ATP-competitive inhibitors for individual SFKs are not currently available. An alternative is to use a chemical genetics approach, in which small molecule kinase inhibitors are combined with genetic mutations to achieve selective inhibition or resistance for a specific kinase. Previous work showed that the expression of an inhibitor-resistant mutant of c-Src rescued mES cells from the A-419259-induced differentiation block, providing important evidence that c-Src activity is essential for mES cell differentiation (Meyn and Smithgall, 2009). Future studies will explore the development of isoform-selective inhibitors and activators of individual Src-family members. Such compounds may represent valuable chemical probes for the role of individual Src-family kinases in both mouse and human ES cell differentiation and may ultimately have translational value in the control of hES cell fate.
Acknowledgments This work was supported in part by National Institutes of Health grants GM077629 to T.E.S. and HD047675 to G.S.
Introduction Despite our ability to genetically engineer animals to mimic human physiology under normal conditions and in disease, animal models often fail to fully recapitulate human processes. For this reason chimeric animals engrafted with human cells and/or tissues play a very important role in biomedical research, including stem cell biology. Xenografting of human cells began with tumor transplants and first became possible when immune deficient strains of mice became available (Rygaard and Povlsen, 1969). Currently, many different kinds of primary cells can be permanently engrafted in mice, among them hematopoietic stem cells, thymic epithelium, neurons and hepatocytes. Hematopoietic chimeras generated by human stem cell transplants have been particularly important as they can be used to study human immunological and inflammatory processes in vivo (Greiner et al., 1998; Pearson et al., 2008; Shultz et al., 2007). When blood stem cell transplantation is combined with engraftment of thymic epithelium, significant aspects of the cellular immune response can be recapitulated.