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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.

Summary Maturity-onset diabetes of the young 1 (MODY1) is a monogenic diabetes condition caused by heterozygous HNF4A mutations. We investigate how HNF4A haploinsufficiency from a MODY1/HNF4A mutation influences the development of foregut-derived liver and pancreatic cells through differentiation of human induced pluripotent stem cells from a MODY1 family down the foregut lineage. In MODY1-derived hepatopancreatic progenitors, which expressed reduced HNF4A levels and mislocalized HNF4A, foregut genes were downregulated, whereas hindgut-specifying HOX genes were upregulated. MODY1-derived hepatocyte-like cells were found to exhibit altered morphology. Hepatic and β cell gene signatures were also perturbed in MODY1-derived hepatocyte-like and β-like cells, respectively. As mutant HNF4A (p.Ile271fs) did not undergo complete nonsense-mediated decay or exert dominant negativity, HNF4A-mediated loss of function is likely due to impaired transcriptional activation of target genes. Our results suggest that in MODY1, liver and pancreas development is perturbed early on, contributing to altered hepatic proteins and β cell defects in patients., Graphical Abstract, Highlights • HNF4A is downregulated and predominantly mislocalized in the cytoplasm in MODY1 • Foregut markers, pancreatic and hepatic genes, were downregulated in MODY1-HPPs • A reciprocal upregulation of hindgut HOX genes was observed in MODY1-HPPs • Mutant HNF4A resulted in loss of transcriptional activation of target genes, Molecular Mechanism of Gene Regulation; Diabetology; Stem Cells Research; Developmental Biology

Cdc20 and Cdh1 activate the anaphase-promoting complex/cyclosome, a master cell cycle regulator. Although cell cycle modifications occur during differentiation of stem cells, a role for the anaphase-promoting complex/cyclosome on stem cell fate has not been established in embryonic or adult human tissues. We found that differentiated human primary keratinocytes from the skin express extremely low levels of Cdc20 compared with human primary keratinocyte stem cells (holoclones). In agreement with this, staining of human skin biopsies showed that Cdc20 is expressed in occasional cells from the basal and epibasal layers of the epidermis and is absent from the differentiated layers. Conversely, Cdh1 is preferentially expressed in differentiated cells. Interestingly, partial silencing of Cdc20 enhanced differentiation, indicating that loss of Cdc20 might be a cause rather than a consequence of terminal differentiation. By contrast, Cdh1 silencing induced the opposite cellular phenotype, which was characterized by an increase in stemness, cellular proliferation, and loss of differentiation markers. These data pinpoint the anaphase-promoting complex/cyclosome as a key regulator of adult stem cell fate. They also demonstrate the critical and opposing roles of Cdc20 and Cdh1 in controlling the balance between human primary keratinocyte proliferation and differentiation, and therefore in regulating skin homeostasis. ASTAR (Agency for Sci., Tech. and Research, S’pore) Published version