Abstract
Mesenchymal stem/stromal cells (MSCs) are self-renewing multipotent cells with regenerative, secretory and immunomodulatory capabilities that are beneficial for the treatment of various diseases. To avoid the issues that come with using tissue-derived MSCs in therapy, MSCs may be generated by the differentiation of human embryonic stems cells (hESCs) in culture. However, the changes that occur during the differentiation process have not been comprehensively characterized. Here, we combined transcriptome, proteome and phosphoproteome profiling to perform an in-depth, multi-omics study of the hESCs-to-MSCs differentiation process. Based on RNA-to-protein correlation, we determined a set of high confidence genes that are important to differentiation. Among the earliest and strongest induced proteins with extensive differential phosphorylation was AHNAK, which we hypothesized to be a defining factor in MSC biology. We observed two distinct expression waves of developmental HOX genes and an AGO2-to-AGO3 switch in gene silencing. Exploring the kinetic of noncoding ORFs during differentiation, we mapped new functions to well annotated long noncoding RNAs (CARMN, MALAT, NEAT1, LINC00152) as well as new candidates which we identified to be important to the differentiation process. Phosphoproteome analysis revealed ESC and MSC-specific phosphorylation motifs with PAK2 and RAF1 as top predicted upstream kinases in MSCs. Our data represent a rich systems-level resource on ESC-to-MSC differentiation that will be useful for the study of stem cell biology.
Highlights
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To avoid the issues that come with using tissue-derived Mesenchymal stem/stromal cells (MSCs) in therapy, MSCs may be generated by the differentiation of human embryonic stems cells in culture
MSCs may be generated from ESCs or induced pluripotent stem cells by protocols ranging from co-culture with OP9 cells, selection of mesenchymal-like cells after undirected differentiation, to directed differentiation using small molecular compounds (4 –7)
Summary
Multi-omics study we characterized the differentiation of human embryonic stem cells into mesenchymal stem cells using transcriptomics, quantitative MS-based proteomics and phosphoproteomics. Based on RNA-to-protein correlation, we determined a set of high confidence genes that are important to differentiation with AHNAK hypothesized to be a defining factor in MSC biology. Mesenchymal stem/stromal cells (MSCs) are self-renewing multipotent cells with regenerative, secretory and immunomodulatory capabilities They have great potential in cell-based therapy and have proven beneficial for the treatment of various diseases [1]. Using an integrative multi-omics approach combining transcriptome, proteome and phosphoproteome profiling of MSC differentiation from hESCs we here present a comprehensive view of MSC development yielding insights into molecular mechanisms, reveal players in MSC biology and include a phosphoproteomic angle, which lays a foundation to decipher the evolution of molecular events during differentiation. We extracted affected biological functions, signaling pathways, and differentiation potential in developing MSCs and identified important transcription factors, kinases and phosphatases, as well as noncoding transcripts associated with the process
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