The differentiation of human pluripotent stem cells into pancreatic cells involves cellular proliferation and apoptosis during cell fate transitions. However, their implications for establishing cellular identity are unclear. Here, we profiled the expression of BCL-2 family of proteins during pancreatic specification and observed an upregulation of BCL-xL, downregulation of BAK and corresponding downregulation of cleaved CASP3 representative of apoptosis. Experimental inhibition of BCL-xL reciprocally increased apoptosis and resulted in a decreased gene expression of pancreatic markers despite a compensatory increase in anti-apoptotic protein BCL-2. RNA-Seq analyses then revealed a downregulation of multiple metabolic genes upon inhibition of BCL-xL. Follow-up bioenergetics assays revealed broad downregulation of both glycolysis and oxidative phosphorylation when BCL-xL was inhibited. Early perturbation of BCL-xL during pancreatic specification also had subsequent detrimental effects on the formation of INS + pancreatic beta-like cells. In conclusion, the more differentiated pancreatic progenitors are dependent on anti-apoptotic BCL-xL for survival, whereas the less differentiated pancreatic progenitors that survived after WEHI-539 treatment would exhibit a more immature phenotype. Therefore, modulation of the expression level of BCL-xL can potentially increase the survival and robustness of pancreatic progenitors that ultimately define human pancreatic beta cell mass and function.

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A comprehensive characterization of the molecular processes controlling cell fate decisions is essential to derive stable progenitors and terminally differentiated cells that are functional from human pluripotent stem cells (hPSCs). Here, we report the use of quantitative proteomics to describe early proteome adaptations during hPSC differentiation toward pancreatic progenitors. We report that the use of unbiased quantitative proteomics allows the simultaneous profiling of numerous proteins at multiple time points, and is a valuable tool to guide the discovery of signaling events and molecular signatures underlying cellular differentiation. We also monitored the activity level of pathways whose roles are pivotal in the early pancreas differentiation, including the Hippo signaling pathway. The quantitative proteomics data set provides insights into the dynamics of the global proteome during the transition of hPSCs from a pluripotent state toward pancreatic differentiation.