In-depth analysis of proteomic and genomic fluctuations during the time course of human embryonic stem cells directed differentiation into beta cells.

Abstract

Pluripotent stem cells (PSC) endocrine differentiation at a large scale allows sampling of transcriptome and proteome with phosphoproteome (proteoform) at specific time points. We describe the dynamic time course of changes in cells undergoing directed beta-cell differentiation and show target proteins or previously unknown phosphorylation of critical proteins in pancreas development, NKX6-1, and Chromogranin A (CHGA). We describe fluctuations in the correlation between gene expression, protein abundance, and phosphorylation, which follow differentiation protocol perturbations of cell fates at all stages to identify proteoform profiles. Our computational modeling recognizes outliers on a phenomic landscape of endocrine differentiation, and we outline several new biological pathways involved. We have validated our proteomic data by analyzing two independent single-cell RNA sequencing datasets for in-vitro pancreatic islet productions using the same cell starting material and differentiation protocol and corroborating our findings for several proteins suggest as targets for future research. Moreover, our single-cell analysis combined with proteoform data places new protein targets within the specific time point and at the specific pancreatic lineage of differentiating stem cells. We also suggest that non-correlating proteins abundances or new phosphorylation motifs of NKX6.1 and CHGA point to new signaling pathways that may play an essential role in beta-cell development. We present our findings for the research communitys use to improve endocrine differentiation protocols and developmental studies.