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. 2022 May;22(9):e2100265.
doi: 10.1002/pmic.202100265. Epub 2022 Feb 9.

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

Bogdan Budnik  1 , Juerg Straubhaar  2 , John Neveu  3 , Dmitry Shvartsman  3   4
Affiliations

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

Bogdan Budnik et al. Proteomics. 2022 May.

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, following differentiation protocol perturbations at all stages to identify proteoform profiles. Our modeling recognizes outliers on a phenomic landscape of endocrine differentiation, and we describe new biological pathways involved. We have validated our proteomic data by analyzing independent single-cell RNAseq datasets for in-vitro pancreatic islet production and corroborated our findings for several proteins suggestive as targets for future research. The 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 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 community's use to improve endocrine differentiation protocols and developmental studies.

Keywords: NKX6-1, pancreas; beta cell (β-cell), cell signaling, chromogranin, insulin, integrin, laminin; mass spectrometry (MS), multiomics, nano LC-MS/MS; pancreatic islet, phosphoproteomics, phosphorylation, protein dynamic, proteomics, signaling networks, single-cell RNA sequencing, stem cells, systems biology, Type 1 diabetes, Type 2 diabetes.

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