Impact of sustained TGFβ-receptor inhibition on chromatin accessibility and gene expression in cultured endometrial MSC [RNA-Seq]
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE146066 | GEO | 2020/09/23
REPOSITORIES: GEO
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Project description: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 TGFbeta receptor inhibitor, promotes pharmacological expansion of eMSC in culture by blocking differentiation and senescence. We subjected primary eMSC treated with A83-01 to Assay for Transposase Accessible Chromatin with sequencing (ATAC-seq) to map the underlying chromatin changes. A total of 185,084 open DNA loci were mapped accurately in EnSCs. ATAC-seq analysis demonstrated that sustained TGFbeta-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.
2020-09-23 | GSE146065 | GEO
Project description:Mesenchymal stem/stromal cells (MSCs) are multipotent cells with favorable properties for cell therapy and regenerative medicine. Human endometrium harbors a small population of perivascular, clonogenic MSCs (eMSCs) identified by the SUSD2 marker. As for other MSCs, eMSCs require extensive in vitro expansion to generate clinically relevant numbers of cells, resulting in spontaneous differentiation, replicative senescence, and cell death, decreasing therapeutic potency. We previously demonstrated that A83-01, a TGF-b receptor inhibitor maintained eMSCs clonogenicity, promoted proliferation, prevented apoptosis and maintained MSC function in vitro. Here we compare the transcriptome of passaged eMSCs from six women cultured with and without A83-01 for 7 days. We identified 1206 differentially expressed genes using a false discovery rate cut-off at 0.01 and fold change >2. Significant enrichment of genes involved in inflammatory responses, angiogenesis, cell migration and proliferation, and reduced smooth muscle proliferation, collagen fibril and extracellular matrix organization genes were revealed. TGF-b, Wnt and Akt signaling pathways were decreased. Anti-fibrotic and anti-apoptotic genes were induced, and fibroblast proliferation and myofibroblast related genes were down-regulated. We validated the enhanced potency of A83-01-treated eMSCs and found increased MSC potency genes (TWIST1, TWIST2, JAG1, LIFR, and SLIT2) and no pluripotency gene expression. Increased angiogenic capacity was functionally demonstrated in vitro, and our angiogenic and cytokine protein arrays further confirmed the angiogenic, antifibrotic and immunomodulatory phenotype of A83-01-treated eMSCs. Overall, we showed that eMSCs culture expanded with A83-01 have improved potential for cell-therapies and regenerative medicine applications.
2018-12-19 | GSE115137 | GEO
Project description:To investigate the role of ISRIB in human trophoblast stem cell(hTSC) maintenance without activation of WNT & TGFβ. We performed gene expression profiling analysis using data obtained from RNA-seq of hTSCs cultured in the medium contained 0.5 µM ISRIB and 10 µM Y27652, after removing CHIR99021, A83-01, SB431542.
2023-12-31 | GSE221409 | GEO
Project description:Fibroblasts can be reprogrammed into cardiomyocyte-like cells by overexpressing transcription factors, GATA4, Hand2, Mef2C and Tbx5 (GHMT). A83-01, an inhibitor of ALK4, ALK5 and ALK7 and two microRNA, miR-1 and miR-133 increase the efficiency of cardiac reprogramming. RNA_Seq was performed to anyalyze effects of these factors on gene expression. Total RNAs were prepared from mouse embryonic fibroblasts (MEFs); Reprogramming fibroblasts including MEFs transduced with retroviruses encoding GHMT, MEFs transduced with with retroviruses encoding GHMT plus miR-1 and miR-133 (GHMT2m), MEFs transduced with with retroviruses encoding GHMT2m treated with A83-01, at day 7 after viral transduction; and neonatal mouse cardiomyocytes (NMCMs).
2015-07-28 | E-GEOD-71405 | biostudies-arrayexpress
Project description:Background: Embryonic trancription factor TBX18 re-expression has been demonstrated to be capable of converting ventricular myocytes to pacemakers cells. The goals of this study are to investigate the effects of TBX18 on myocytes and non-myocytes at day 3, day 6 and day 14, as well as tracking the key node of reprogramming process and mechanism. Methods: Neonatal rat ventricular myocytes (NRVC) were treated by adeno-TBX18 or GFP for 3 days to investigate the early effects of TBX18 on myocytes and non-myocytes. Except for these 2 treatments, Adeno-TBX18 with one specific Tgfβ inhibitor, A83-01 (0.2uM) was treated for NRVC for 14 days for explore the effects of Tgfβ inhibition on fibroblast activation and myocyte reprogramming process. Attached NRVC monolayers were digested with 0.25% trypsin (Cat# LS003707, Corning, NY) for 5 min at 37 °C, and filtered through a 100-μm cell strainer (Fischer Scientific, Waltham, MA) to generate a single-cell suspension. Single cells were resuspended to a concentration of 700-2,000 cells per microliter. Gel beads-in-emulsion (GEMs) were generated with Chromium X (10X Genomics, Pleasanton, CA) and RNA libraries were prepared using the Chromium Next GEM Single Cell 3' GEM kit, v3.1 (10X Genomics Inc., Pleasanton, CA) with a targeted cell recovery of 4,000-10,000 cells per reaction. Quality control of cDNA and scRNA-seq libraries were performed using the Bioanalyzer High Sensitivity kit (Agilent Technologies Inc., CA). Libraries were sequenced using Novaseq 6000 (Illumina Inc., CA) with a minimum depth of 50,000–65,000 read pairs per cell. Result: Upon QA/QC filtering, 13,437 cells met the quality thresholds for subsequent analysis. We focused on three cell types: cardiomyocytes (CMs, Tnni3-positive), total FBs (Pdgfrb and Tcf21-positive) with 2 subpopulations, including myoFBs (Pdgfrb, Tcf21, Acta2-high) and quiescent FBs (Pdgfrb, Tcf21, Acta2-low). Pathway analysis further revealed that upregulated DEGs by TBX18 are involved in fibroblast activation and ECM remodeling. Among those, Tgfβ signaling, which is one of the major molecular mediators of fibrosis in the heart, figured prominently in TBX18-transduced CMs and FBs. This was evidenced by upregulated expression of Tgfβ ligands and profibrotic genes such as Tgfb1, Tgfb2, and Acta2 in TBX18-NRVCs. Accordingly, higher proportion of activated myofibroblasts (39.3% vs. 25.4%) and lower proportion of quiescent FBs were observed in TBX18-NRVCs compared to control (Fig. 2F). To test the causative role of Tgfβ signaling in triggering fibrosis upon TBX18 re-expression, monolayers of NRVCs were treated with a small molecule inhibitor of type I Tgfβ receptors, A83-01. Treatment with A83-01 significantly decreased the population of myoFBs from 68.0% in TBX18-NRVC to 49.5% of total cell population at d14. Heat map analysis of all genes indicated that TBX18-CMs show upregulation of fibroblast-related genes, which is mitigated with A83-01 treatment. The upregulated DEGs in TBX18-FBs pointed to ECM remodeling as well as inflammatory response involving Tgfβ and other cytokines. Again, treatment with A83-01 abrogated the rise of these pathways. Profibrotic genes such as Tgfb2, Tgfb3, Acta2, and inflammatory genes were upregulated in FBs transduced with Ad-TBX18, which was mitigated by A83-01 treatment. The upregulated gene clusters in TBX18-FBs were also upregulated in TBX18-CMs, particularly, interferon signaling, interleukin-4 regulation of apoptosis, which may have led to the loss of TBX18-CMs over time. The inflammatory and profibrotic pathways were suppressed by treatment with A83-01 and may explain the higher population of TBX18-CMs with A83-01 at d14.We also quantified the number of de novo iPMs based on: Tnni3high, Hcn4+, Nkx2-5low, Gja1low through our scRNA-seq analysis and demonstrated the proportions of Hcn4+ iPMs was similar in TBX18-CMs regardless of Tgfβ inhibition but was higher compared to control. Conclusion: Tgfβ signaling inhibition with a83-01 prevents against myofibroblast activation, cardiomyocytes apoptosis and inflammatory response induced by TBX18, and short term treatment of A8301 does not strongly interfere the reprogramming process and efficiency.
2024-09-27 | GSE189815 | GEO
Project description:Primary human hepatocytes (PHHs) were cultured with or without a cocktail of four small molecules, Y-27632, PD0325901, A83-01 and CHIR99021 (termed YPAC), and their transcriptomes after a long-trm culture were compared.
2019-09-01 | GSE135011 | GEO
Project description:Trophoblast differentiation from human ESC has been achieved by exposing the cells to BMP4 with or without supplementation of ALK4/5/7 inhibitor (A83-01) and FGF2 signaling inhibitor (PD173074) (BAP). Here the two differentiation conditions, BMP4 and BAP were applied to two sets of human PSC lines, H1 ESC and iPSC that latter was generated by DOX-inducible lentiviral (V) transductions of umbilical cord mesenchymal cells. The V-iPSC showed residual transgene expressions from the viral vectors in DOX-free culture condition. When the both ESC and iPSC lines were differentiated simultaneously, similar time dependent morphological changes were observed but BMP4 treated V-iPSC showed a minor yet consistent lag in the differentiation progression compared to BMP4 treated hESC. Although both differentiated ESC and V-iPSC showed dominant trophoblast phenotypes, the BMP4 treated V-iPSC also expressed gene markers consistent with the presence of mesoendoderm. The BAP condition provided more efficient differentiation than BMP4 alone, and the BAP-differentiated iPSC and ESC never expressed mesoendoderm markers. Five samples, one control of undifferentiated V-iPSC (FGF2) and four differentiated samples included two H1 ESC (treated with BMP4 or BMP4+A83-01) and similarly treated two V-iPSC were analyzed. Trophoblast differentiation was conducted with BMP4 (10 ng/ml) with or without supplementation of ALK4/5/7 inhibitor (A83-01; 1 μM) to H1 ESC and V-iPSC, respective reagents were added to FGF2-free MEF-CM from the second day culture of following passages for up to six additional days.
2015-08-31 | E-GEOD-64272 | biostudies-arrayexpress
Project description:Fibroblasts can be reprogrammed into cardiomyocyte-like cells by overexpressing transcription factors, GATA4, Hand2, Mef2C and Tbx5 (GHMT). A83-01, an inhibitor of ALK4, ALK5 and ALK7 and two microRNA, miR-1 and miR-133 increase the efficiency of cardiac reprogramming. RNA_Seq was performed to anyalyze effects of these factors on gene expression.
2015-07-28 | GSE71405 | GEO
Project description:Currently, much effort is directed to the development of new cell sources for clinical therapy using cell fate conversion approaches by small molecules. Direct lineage reprogramming to a progenitor state has been reported in terminally differentiated rodent hepatocytes, yet remains a challenge in human hepatocytes. Human hepatocytes were isolated from healthy and diseased donor livers and reprogrammed into progenitor cells by two small molecules, A83-01 and CHIR99021 (AC), in the presence of EGF and HGF. The stemness properties of human chemically derived hepatic progenitors (hCdHs) were tested by standard in vitro and in vivo assays and transcriptome profiling. We developed a robust culture system for generating hCdHs with therapeutic potential. The use of HGF proved to be an essential determinant of fate conversion process. Based on functional evidence, activation of HGF/MET signal transduction system collaborated with A83-01 and CHIR99021 to allow a rapid expansion of progenitor cells through activation of ERK pathway. hCdHs expressed hepatic progenitor marker genes and proteins, and could self-renew for at least 10 passages while retaining normal karyotype and potential to differentiate into functional hepatocytes and biliary epithelial cells in vitro. RNASeq gene expression profiling confirmed transcriptional reprogramming of hCdHs toward a progenitor state and suppression of mature hepatocyte transcripts. Upon intrasplenic transplantation into immunocompromised mice with acute liver injury, hCdHs effectively repopulated damaged parenchyma. Our study is a first report of successful reprogramming of human hepatocytes to a population of proliferating bipotent cells with regenerative potential. hCdHs may provide a nove tool that permits expansion and genetic manipulation of patient-specific progenitors to study regeneration and repair of diseased liver.
2019-05-08 | GSE114643 | GEO
Project description:This SuperSeries is composed of the SubSeries listed below.
2020-09-23 | GSE146067 | GEO