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bioRxiv · 10.1101/2020.05.01.073346

Impact of sustained TGFβ receptor inhibition on chromatin accessibility and gene expression in cultured human endometrial MSC

Lucciola, R.·Vrljicak, P.·Filby, C.·Darzi, S.·Gurung, S.·Muter, J.·Ott, S.·Brosens, J.·Gargett, C. E.
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Abstract

Endometrial mesenchymal stem cells (eMSC) drive the extraordinary regenerative capacity of the human endometrium. Clinical application of eMSC for therapeutic purposes is hampered by spontaneous differentiation and cellular senescence upon large-scale expansion in vitro. A83-01, a selective transforming growth factor-{beta} receptor (TGF{beta}-R) inhibitor, promotes pharmacological expansion of eMSC in culture by blocking differentiation and senescence, but the underlying mechanisms are incompletely understood. In this study, we combined RNA-seq and ATAC-seq to study the impact of sustained TGF{beta}-R inhibition on gene expression and chromatin architecture of eMSC. Treatment of primary eMSC with A83-01 for 5 weeks resulted in differential expression of 1,463 genes. Gene ontology analysis showed enrichment of genes implicated in cell growth whereas extracellular matrix genes and genes involved in cell fate commitment were downregulated. ATAC-seq analysis demonstrated that sustained TGF{beta}-R inhibition results in opening and closure of 3,555 and 2,412 chromatin loci, respectively. Motif analysis revealed marked enrichment of retinoic acid receptor (RAR) binding sites, which was paralleled by the induction of RARB, encoding retinoic acid receptor beta (RAR{beta}). Selective RAR{beta} inhibition attenuated proliferation and clonogenicity of A83-01 treated eMSC. Taken together, our study provides new insights into the gene networks and genome-wide chromatin changes that underpin maintenance of an undifferentiated phenotype of eMSC in prolonged culture. Significance statementCycling human endometrium is a rich source of adult stem/progenitor cells that could be exploited for clinical purposes. Small molecules, such as A83-01, that modulate cell identity may open new avenues to maintain the functional properties of eMSC upon expansion in culture. By integrating complementary genome-wide profiling techniques, we mapped the dynamic changes in chromatin landscape and gene expression in response to prolonged A83-01 treatment of eMSC. Our findings provide new insights into the mechanisms of action of TGF{beta}-R inhibition that may lead to the development of more targeted pharmacological approaches for MSC expansion.

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Lucciola, R., Vrljicak, P., Filby, C., Darzi, S., Gurung, S., Muter, J., Ott, S., Brosens, J., Gargett, C. E.. 2020-05-03. Impact of sustained TGFβ receptor inhibition on chromatin accessibility and gene expression in cultured human endometrial MSC. https://doi.org/10.1101/2020.05.01.073346

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