Mus musculus
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA234369 | ENA |
REPOSITORIES: ENA
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Project description:Recent studies have been successful at utilizing ectopic expression of transcription factors to generate induced cardiomyocytes (iCMs) from fibroblasts, albeit at a low frequency in vitro. This work investigates the influence of small molecules that have been previously reported to improve differentiation to cardiomyocytes as well as reprogramming to iPSCs in conjunction with ectopic expression of the transcription factors Hand2, Nkx2.5, Gata4, Mef2C, and Tbx5 on the conversion to functional iCMs. We utilized a reporter system in which the calcium indicator GCaMP is driven by the cardiac Troponin T promoter to quantify iCM yield. The TGFβ inhibitor, SB431542 (SB), was identified as a small molecule capable of increasing the conversion of both mouse embryonic fibroblasts and adult cardiac fibroblasts to iCMs up to ~5 fold. Further characterization revealed that inhibition of TGFβ by SB early in the reprogramming process led to the greatest increase in conversion of fibroblasts to iCMs in a dose-responsive manner. Global transcriptional analysis at Day 3 post-induction of the transcription factors revealed an increased expression of genes associated with the development of cardiac muscle in the presence of SB compared to the vehicle control. Incorporation of SB in the reprogramming process increases the efficiency of iCM generation, one of the major goals necessary to enable the use of iCMs for discovery-based applications and for the clinic. Mouse embryonic fibroblasts (MEFs) and adult mouse cardiac fibroblasts (CFs) were transfected with an empty vector (0F) or the combination of Hand2, Nkx2.5, Gata4, Mef2C, and Tbx5 (5F). Samples were exposed to the vehicle control (D, DMSO), SB431542 (SB, 0.5 uM MEF, 5 uM CF), or TGFb1 (T, 2 ng/mL) during culture. Transcription factor expression was induced at Day 0 and samples were isolated at Day 3 post-induction.
2014-01-14 | E-GEOD-54022 | biostudies-arrayexpress
Project description:Recent studies have been successful at utilizing ectopic expression of transcription factors to generate induced cardiomyocytes (iCMs) from fibroblasts, albeit at a low frequency in vitro. This work investigates the influence of small molecules that have been previously reported to improve differentiation to cardiomyocytes as well as reprogramming to iPSCs in conjunction with ectopic expression of the transcription factors Hand2, Nkx2.5, Gata4, Mef2C, and Tbx5 on the conversion to functional iCMs. We utilized a reporter system in which the calcium indicator GCaMP is driven by the cardiac Troponin T promoter to quantify iCM yield. The TGFβ inhibitor, SB431542 (SB), was identified as a small molecule capable of increasing the conversion of both mouse embryonic fibroblasts and adult cardiac fibroblasts to iCMs up to ~5 fold. Further characterization revealed that inhibition of TGFβ by SB early in the reprogramming process led to the greatest increase in conversion of fibroblasts to iCMs in a dose-responsive manner. Global transcriptional analysis at Day 3 post-induction of the transcription factors revealed an increased expression of genes associated with the development of cardiac muscle in the presence of SB compared to the vehicle control. Incorporation of SB in the reprogramming process increases the efficiency of iCM generation, one of the major goals necessary to enable the use of iCMs for discovery-based applications and for the clinic.
2014-01-14 | GSE54022 | GEO
Project description:Secretome-mediated signaling from human iPSC-derived cardiomyocytes (TGFβ induced dysfunction) to primary human cardiac fibroblasts was investigated to identify downstream regulators of fibrosis. Quantitative proteomic profiling revealed a dynamic reprogramming of fibroblast global proteome, with dysregulation of proteins implicated in extracellular matrix (ECM) remodelling, cytoskeleton organization, lysosome function, and oxidoreductase- and kinase activity. Protein modification-focused processing analyses of mass spectrometry proteome data further highlight phospho-proteome alterations in pro-fibrotic pathways regulated by various kinases (CK2, CDK1, CDK2, MAPK1, PRKACA, PRKG1). We verified upregulated casein kinase 2 (CK2) substrate levels in secretome-treated fibroblasts, and pharmacological inhibition of CK2 using TBB (4,5,6,7-Tetrabromobenzotriazole) significantly abrogated reactive oxygen species’ levels and activation state (SMA+).
2025-02-11 | PXD038854 | Pride
Project description:Direct conversion of fibroblasts to induced cardiomyocytes (iCMs) has great potential for regenerative medicine. Recent publications have reported significant progress, but the evaluation of reprogramming has relied upon non-functional measures such as flow cytometry for cardiomyocyte markers or GFP expression driven by a cardiomyocyte-specific promoter. The issue is one of practicality: the most stringent measures - electrophysiology to detect cell excitation and the presence of spontaneously contracting myocytes - are not readily quantifiable in the large numbers of cells screened in reprogramming experiments. However, excitation and contraction are linked by a third functional characteristic of cardiomyocytes: the rhythmic oscillation of intracellular calcium levels. We set out to optimize direct conversion of fibroblasts to iCMs with a quantifiable calcium reporter to rapidly assess functional transdifferentiation. We constructed a reporter system in which the calcium indicator GCaMP is driven by the cardiomyocyte-specific Troponin T promoter. Using calcium activity as our primary outcome measure, we compared several published combinations of transcription factors along with novel combinations in mouse embryonic fibroblasts. The most effective combination consisted of Hand2, Nkx2.5, Gata4, Mef2c, and Tbx5 (HNGMT). This combination is >50-fold more efficient than GMT alone and produces iCMs with cardiomyocyte marker expression, robust calcium oscillation, and spontaneous beating that persists for weeks following inactivation of reprogramming factors. HNGMT is also significantly more effective than previously published factor combinations for the transdifferentiation of adult mouse cardiac fibroblasts to iCMs. Quantification of calcium function is a convenient and effective means for the identification and evaluation of cardiomyocytes generated by direct reprogramming. Using this stringent outcome measure, we conclude that HNGMT produces iCMs more efficiently than previously published methods. Mouse embryonic fibroblasts were treated with different combinations of transcription factors to drive transdifferentiation to induced cardiomyocytes (iCMs). Putative iCMs were enriched by zeocin selection. Zeocin resistance was conferred to iCMs via the TroponinT-GCaMP5-Zeo lentiviral reporter.
2013-04-22 | E-GEOD-45274 | biostudies-arrayexpress
Project description:Psychological stress results in increased susceptibility to infection yet the mechanisms responsible for this major healthcare burden are incompletely understood. In mice, stress induced by restraint increases infection by Staphylococcus aureus while adrenalectomy or chemical inhibition of adrenergic signaling blocks this response. Single-cell transcriptomics and lipidomic profiling of the skin after stress reveal fibroblasts undergoing adipogenesis are the major cell type responding to stress. Fibroblasts are critical to the increase in infection as adrenaline inhibits fibroblast Camp expression and bacterial killing, and stress did not increase infection in mice that lack Camp in fibroblasts. The suppression of the antimicrobial function of Camp in fibroblasts occurs due to activation of TGFβ signaling and is critical to the capacity of stress to increase susceptibility to infection since treatment of mice with a neutralizing TGFβ antibody or a TGFβ receptor inhibitor restores expression of Camp and alleviates increased susceptibility to infection. These data show that susceptibility to infection after psychological stress is due to a brain-skin axis that induces TGFβ and subsequently inhibits host defense by immune acting fibroblasts in the dermis. This identifies TGFβ as an unexpected target that can ameliorate increased bacterial infections associated with stress.
2025-05-29 | GSE270713 | GEO
Project description:Psychological stress results in increased susceptibility to infection yet the mechanisms responsible for this major healthcare burden are incompletely understood. In mice, stress induced by restraint increases infection by Staphylococcus aureus while adrenalectomy or chemical inhibition of adrenergic signaling blocks this response. Single-cell transcriptomics and lipidomic profiling of the skin after stress reveal fibroblasts undergoing adipogenesis are the major cell type responding to stress. Fibroblasts are critical to the increase in infection as adrenaline inhibits fibroblast Camp expression and bacterial killing, and stress did not increase infection in mice that lack Camp in fibroblasts. The suppression of the antimicrobial function of Camp in fibroblasts occurs due to activation of TGFβ signaling and is critical to the capacity of stress to increase susceptibility to infection since treatment of mice with a neutralizing TGFβ antibody or a TGFβ receptor inhibitor restores expression of Camp and alleviates increased susceptibility to infection. These data show that susceptibility to infection after psychological stress is due to a brain-skin axis that induces TGFβ and subsequently inhibits host defense by immune acting fibroblasts in the dermis. This identifies TGFβ as an unexpected target that can ameliorate increased bacterial infections associated with stress.
2025-05-29 | GSE270712 | GEO
Project description:Direct Conversion of Fibroblasts into Oligodendrocyte Progenitor Cells
| PRJNA226272 | ENA
Project description:Combined inhibition of TGFβ and PDGF signaling attenuates radiation-induced pulmonary fibrosis
| PRJNA272714 | ENA
Project description:Forced expression of pro-neural transcription factors was shown to mediate direct neuronal conversion of human fibroblasts. Since neurons are postmitotic, the conversion efficiency represents an important parameter. Here we present a minimalist approach combining two factor neuronal programming with small molecule-based inhibition of GSK3ß and SMAD signaling, which gives rise to functional neuron-like cells (iNs) of various neurotransmitter phenotypes with an overall yield of up to >200% and a final neuronal purity of up to >80%. Timcourse of reprogramming of fibroblasts towards an neuronal phenotype in two independent fibroblast lines
2012-04-07 | E-GEOD-36246 | biostudies-arrayexpress
Project description:Direct conversion of mouse fibroblasts into cholangiocyte progenitor cells
| PRJNA382180 | ENA