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TGF-beta couples pluripotency and epithelial character via ZNF398

ABSTRACT: Human pluripotent stem cells (hPSCs) have the capacity to give rise to all differentiated cells of the adult. TGF-beta is used routinely for expansion of conventional hPSCs as flat epithelial colonies expressing the transcription factors POU5F1/OCT4, NANOG, SOX2. Here we report a global analysis of the transcriptional program controlled by TGF-beta followed by an unbiased gain-of-function screening in multiple hPSC lines to identify factors mediating TGF-beta activity. We identify a quartet of transcriptional regulators promoting hPSC self-renewal including ZNF398, a human-specific mediator of pluripotency and epithelial character in hPSCs. Mechanistically, ZNF398 binds active promoters and enhancers together with SMAD3 and the histone acetyltransferase EP300, enabling transcription of TGF-beta targets. In the context of somatic cell reprogramming, inhibition of ZNF398 abolishes activation of pluripotency and epithelial genes and iPSC colony formation. Our findings have clear implications for the generation of bona fide hPSCs for regenerative medicine.

ORGANISM(S): Homo sapiens

PROVIDER: GSE133630 | GEO | 2020/04/06

REPOSITORIES: GEO

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Project description:Human pancreatic stellate cells (HPSCs) are an essential stromal component and are the mediators of pancreatic ductal adenocarcinoma (PDAC) progression. Small extracellular vesicles (sEVs) are membrane-enclosed nanoparticles released from stellate cells adjacent to the PDAC. sEVs are believed to play a key role in cell-cell communications and may play a critical role in disease progression. The role of membrane proteins of HPSC sEVs in the PDAC tumor microenvironment is unclear and to date, there has not been a quantitative proteomic comparison of sEVs from normal pancreatic stellate cells (HPaStec) and from PDAC-associated stellate cells (HPSCs). We hypothesized there would be differences in sEVs secretion and membrane protein expression between the 2 conditions that might contribute to PDAC biology. To test these hypotheses, we isolated sEVs using ultracentrifugation followed by characterization by electron microscopy and Nanoparticle Tracking Analysis. HPSCs secreted more sEVs compared to HPaStec, and these sEVs were enriched with exosomal markers, which was confirmed by Western blotting and flow cytometry. HPSC-sEVs also restore the activation of normal stellate cells. Next, we showed that intact membrane-associated proteins may be essential for sufficient uptake of stellate cell sEVs by both normal epithelial and cancer cells. Importantly, we demonstrated that stellate cells in general modulate the cellular proliferations of pancreatic cancer cells although stellate cell sEVs did not change the proliferation of cancer cells. We then compared sEV proteins isolated from HPSCs and HPaStecs cells using liquid chromatography–tandem mass spectrometry. Most of the 1,481 protein groups identified were shared with the exosome database, ExoCarta (http://exocarta.org/; curated by the Mathivanan Lab). Eighty-seven protein groups were differentially expressed (selected by 2-fold difference and adjusted P value ≤ 0.05) between HPSC and HPaStec sEVs. HPSC sEVs contained dramatically more CSE1L, a poor prognostic marker for pancreatic cancer. In conclusion, our findings using mass spectrometry–based proteomics may direct further studies to understand the biology and role of protein composition of HPSC sEVs in PDAC progression or to develop novel strategies based on delivery of exosome cargo to PDAC tumor cells.

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TGF-beta couples pluripotency and epithelial character via ZNF398

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