RepSox effectively promotes the induced differentiation of sheep fibroblasts into adipocytes via the inhibition of the TGF‑β1/Smad pathway

Abstract

Previous reports have demonstrated that RepSox can function as a replacement for cMyc and Sox2 in the reprogramming of cells into induced pluripotent stem cells (iPSCs), as well as increasing the levels of bone morphogenetic protein (BMP)-3 and inducing the phosphorylation of Smad1 in mouse embryonic stem cells. In the present study, it was demonstrated that RepSox caused the visible morphological transformation of sheep fibroblasts; however, no significant alterations in cell proliferation, apoptosis or chromosome aberrations were observed. Moreover, RepSox increased the plasticity of long-term cryopreserved sheep fibroblasts, and further promoted differentiation into adipocytes. RepSox treatment led to a notable decrease in the expression of components of the transforming growth factor (TGF)-β signaling pathway, particularly Smad2/3 phosphorylation. RepSox also activated the BMP pathway, promoted the reprogramming of cells from fibroblasts into adipocytes and induced mesenchymal-epithelial transition. It is worth noting that RepSox notably increased the expression of octamer-binding transcription factor 4 and L-Myc, whereas Sox2 and Nanog expression were not detected. The results of high-throughput RNA sequencing revealed that the levels of differentially expressed genes (DEGs) involved in various metabolic processes were markedly upregulated in the RepSox-treated fibroblasts, while the DEGs in the majority of signaling pathways were markedly downregulated. On the whole, the present study demonstrates that RepSox can promote the plasticity of sheep fibroblasts and facilitates the differentiation of adipocytes via increasing BMP expression and inhibiting the activation of the TGF-β signaling pathway.

Therapeutic function of a novel rat induced pluripotent stem cell line in a 6‑OHDA‑induced rat model of Parkinson's disease

“…Therefore, inhibition of GSK3-β by Wnt signaling could promote self-renewal and cell reprogramming by regulating the stability of c-Myc (40,44). Inhibition of TGF-β could induce mesenchymal to epithelial transition (MET) and increase Nanog expression (36). Together with the present findings, it may be concluded that cR could promote the reprogramming process by simultaneously inhibiting GSK3-β and TGF-β signaling.…”

“…At day 2 post-transduction, REFs were re-plated on an irradiated Oricell ® ICR mouse embryonic fibroblast feeder layer (cyagen Biosciences, Inc.). The following day, the culture medium was replaced with dMEM/F-12 supplemented with 10% KnockOut Serum Replacement (Gibco; Thermo Fisher Scientific, Inc.), 0.1 mM β-mercaptoethanol, 1% NEAA, 2 mM L-glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin, which was further supplemented with 3 µM CHIR99021 (Sigma-Aldrich; Merck KGaA), 1 µM RepSox (Selleck Chemicals) and 15 ng/ml fibroblast growth factor 2 (R&D Systems, Inc.) on day 4 (36,37). The GFP + /iPS-like colonies [rat iPScs induced by OcT3/4, Sox2, Klf4, c-Myc, Nanog and Lin28 + cR (RiPScs-6F/cR)] were mechanically picked 20-30 days after viral transduction and re-cultured on feeder layers.…”

“…The cell nuclei were counterstained with DAPI (Thermo Fisher Scientific, Inc.) for 15 min at room temperature. Finally, the coverslips were mounted with ProLong™ Gold Antifade Mountant (Invitrogen; Thermo Fisher Scientific, Inc.) and observed under an inverted fluorescence microscope (Guangzhou Micro-shot Technology co., Ltd.) (1,36).…”

Genomic, transcriptomic and proteomic depiction of iPSC-derived smooth muscle cells as emerging cellular models for arterial diseases

“…Recently, it was reported to induce brown adipogenesis 44 and promote the differentiation of sheep fibroblasts into adipocytes 45 . RepSox induction of contractile markers in SMCs was found to depend on activation of NOTCH signaling pathway 27 .…”

“…RepSox functions as a selective TGFβR-1/ALK5 inhibitor which inhibits ALK5 autophosphorylation 42 and was used to reprogramming of fibroblasts into iPSCs by replacing cMyc and Sox2 43 . Recently, it was reported to induce brown adipogenesis 44 and promote the differentiation of sheep fibroblasts into adipocytes 45 . RepSox induction of contractile markers in SMCs was found to depend on activation of NOTCH signaling pathway 27 .…”