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Impact of sustained TGFβ-receptor inhibition on chromatin accessibility and gene expression in cultured endometrial MSC [ATAC-Seq]

ABSTRACT: Impact of sustained TGFβ-receptor inhibition on chromatin accessibility and gene expression in cultured endometrial MSC [ATAC-Seq]

PROVIDER: PRJNA609218 | ENA |

REPOSITORIES: ENA

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Project description:In the current study, we hypothesized that if bone-marrow derived MSC contribute to endometrial regeneration and are progenitors to hESF, their treatment with agents known to regulate hESF differentiation could promote their differentiation down the stromal fibroblast lineage. To this end, we treated bone marrow-derived MSC with estradiol (E2) and progesterone (P4), BMP2, and activators of the PKA pathway and investigated specific markers of hESF differentiation (decidualization). Furthermore, we investigated the transcriptome of these cells in response to cAMP and compared this to the transcriptome of hESF decidualized in response to activation of the PKA pathway. The data support that MSC can be differentiated down the hESF pathway, as evidenced by changes in cell shape and common expression of decidual markers and other genes important in hESF differentiation and function. Bone marrow-derived MSC were expanded in DMEM (high glucose) medium containing 10% fetal bovine serum, 1% penicillin and streptomycin. At 80% confluency MSC were trypsinized and plated at 5x103 cells/cm2 in 6cm plates. Nearly confluent cells were serumstarved overnight and thereafter treated for 0, 3, 7, 14 and 21 days in low-serum medium (2% FBS) with the following treatments: (a) 10nM E2/1mM P4; (b) 0.5mM or 1mM 8-Br-cAMP (cAMP); (c) 50nM or 100nM BMP-2; (d) 0.5mM and 1mM 8-Br-cAMP + BMP-2; (d) 0.5mM and 1mM 8-BrcAMP + BMP-2 + E2/P4; (e) 50nM or 100nM human FSH, with vehicle controls at each time point. Each experiment was conducted in duplicate and repeated at least 3 times. Media were changed every third day. Endpoints were prolactin (PRL) and IGF binding protein-1 (IGFBP1) mRNA, assessed by quantitative real-time RT-PCR, and protein in conditioned media, assessed by ELISA (see below), after 3, 7, 14 and 21 days of incubation. Immunostaining for vimentin, cytokeratin 7, and Oct-4 was performed before and after MSC treatments to assess the stromal lineage of MSC and their differentiation status. MSC expressed vimentin and did not express cytokeratin or Oct-4, independent of culture conditions or duration of culture or treatment. The morphological appearance of MSC under different treatment conditions was photographed using a Leica CTR 6500 microscope. Microarray analysis was performed with MSC, n=3, at t=0 (untreated, uncultured control), 14day vehicle control, and 14day treated with 8-Br-cAMP. Only high quality samples (RIN=9.1-10) were used for microarray analysis. Hybridization was performed with Affymetrix Human Gene 1.0 ST arrays.

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Impact of sustained TGFβ-receptor inhibition on chromatin accessibility and gene expression in cultured endometrial MSC [ATAC-Seq]

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