Enhanced pancreatic endocrine differentiation of human pluripotent stem cells following pharmacological inhibition of select kinases

A. Hayek, I. Afrikanova, M. Yebra, M. Simpkinson, Y. Xu, A. Montgomery
UC San Diego, La Jolla, USA

Objective: Stepwise approaches for the derivation of beta-cells from human embryonic stem cells (hESCs) have been described. However, low levels ofendocrine specification limit the final yield of insulin-producing beta-cells. Studies have shown that Src family kinases (SFKs) and focal adhesion kinase (FAK) have a major impact on the differentiation of disparate cell types, yet little is known about how these kinases influence early beta-cell development. In this study we assessed the impact of inhibiting SFK and FAK activity on the endocrine specification of hESCs.

Methods: Selective small compound inhibitors of SFK and FAK activity were assessed for their ability to induce endocrine specification in the context of a differentiation protocol first described by Novocell. Induction of endocrine specification was determined based on the expression of restricted proendocrine transcription factors and on the final yield of insulin-positive cells.

Results: The pyrrolo-pyrimidine SFK inhibitor PP2 was found to effectively promote the endocrine specification of hESC derivatives based on its capacity to induce the expression of the transcription factors NGN3, NEUROD1, NKX2.2 and PAX4 and to significantly increase the final yield of insulin-positive cells. Importantly, PP2 was found to inhibit the downstream activation of FAK and selective inhibition of this kinase was also sufficient to induce early endocrine commitment based on increased expression of NGN3, NEUROD1 and NKX2.2. Additional studies using dominant-negative constructs and isolated human fetal pancreata suggest that c-Src is the SFK primarily responsible for inhibiting early endocrine specification. Finally, we also found that inhibition of SFK/FAK signaling suppresses the proliferation of cultures enriched for pancreatic progenitors and induced the expression of the human beta-cell-associated cyclin-dependent kinase inhibitor p57kip2.

Conclusions: This study has important implications for the derivation of beta-cells for the cell-based therapy of diabetes and sheds new light on signaling events that regulate early endocrine specification.