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A developed serum-free medium and an optimized chemical cocktail for direct conversion of human dermal fibroblasts into brown adipocytes.

ABSTRACT: Brown adipocytes coordinate systemic energy metabolism associated with the pathogenesis of obesity and related metabolic diseases including type 2 diabetes. We have previously reported chemical compound-induced brown adipocytes (ciBAs) converted from human dermal fibroblasts without using transgenes. In this study, to reveal a precise molecular mechanism underlying the direct conversion and human adipocyte browning, we developed serum-free brown adipogenic medium (SFBAM) with an optimized chemical cocktail consisting of Rosiglitazone, Forskolin, and BMP7. During the direct conversion, treatment with BMP7 enhanced Ucp1 expression rather than the conversion efficiency in the absence of BMP signalling inhibitors. Moreover, treatment with a TGF-? signalling pathway inhibitor was no longer required in the serum-free medium, likely because the TGF-? pathway was already suppressed. SFBAM and the chemical cocktail efficiently converted human dermal fibroblasts into ciBAs within four weeks. The ciBAs exhibited increased mitochondrial levels, elevated oxygen consumption rate, and a response to ?-adrenergic receptor agonists. Thus the ciBAs converted by the serum-free medium and the chemical cocktail provide a novel model of human brown (beige) adipocytes applicable for basic research, drug screening, and clinical applications.

SUBMITTER: Takeda Y

PROVIDER: S-EPMC7048747 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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A developed serum-free medium and an optimized chemical cocktail for direct conversion of human dermal fibroblasts into brown adipocytes.

Takeda Yukimasa Y Dai Ping P

Scientific reports 20200228 1

Brown adipocytes coordinate systemic energy metabolism associated with the pathogenesis of obesity and related metabolic diseases including type 2 diabetes. We have previously reported chemical compound-induced brown adipocytes (ciBAs) converted from human dermal fibroblasts without using transgenes. In this study, to reveal a precise molecular mechanism underlying the direct conversion and human adipocyte browning, we developed serum-free brown adipogenic medium (SFBAM) with an optimized chemic ...[more]

PMID: 32111895

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Project description:Introduction: The molecular regulation mechanism of fat deposition in bovine and its improvement on beef quality are important research directions in the livestock industry. The research of molecular mechanisms that govern the regulation and differentiation of adipocytes may conduct to understand the mechanism of obesity, lipid disorders, and fat deposition. In the recent decade, small-molecule compounds have been widely used in reprogramming and transdifferentiation fields, which can promote the induction efficiency, replace exogenous genes, or even induce cell fate conversion alone. Furthermore, small-molecule compound induction is expected to be a novel approach to generate new cell types from somatic cells in vitro and in vivo. Methods: In this study, we established rapid chemically induced platform for transdifferentiation of bovine ear fibroblasts into adipocyte-like cells using a small-molecule cocktail (Repsox, VPA, TTNPB). The chemically induced adipocytes (CiADCs) were characterized by lipid staining, qRT-PCR and WB. Bovine natural adipocytes were used as positive control, and the expression of adipocyte-related marker genes in CiADCs were analyzed. Moreover, RNA-Seq explore the mechanism of RVB in the regulation of Bovine adipocyte transdifferentiation. Results: In this study, the chemically induced adipocytes (CiADCs) could be identified as early as day 6. The CiADCs appeared to be circular and rich of lipid droplets. The adipocyte-specific genes of LPL, PPARγ, IGF1, GPD1, C/EBPδ, ADIPOQ, PCK2, FAS, C/EBPβ, PPARGC1A, C/EBPα, and CFD were detected to be significantly upregulated in both CiADCs and natural adipocytes. Western blot analysis also confirmed the increase C/EBPα and PPARγ protein level in induced adipocytes (CiADCs-6d) treated with RVB. In addition, we also found that the signaling pathways (PPAR signaling pathway, PI3K-Akt signaling pathway, p53 signaling pathway, MAPK signaling pathway, and ECM-receptor interaction) regulated by the DEGs played a vital role in adipogenesis. Discussion: In the present study, a combination of small-molecule compounds RVB was used to transdifferentiate bovine ear fibroblasts into the chemically-induced adipocyte cells (CiADCs) that have a large number of lipid droplets. Importantly, the small-molecule cocktail significantly shortened the reprogramming turnaround time. The morphology of CiADCs is close to the "ring type" of natural differentiated adipocytes on sixth day. And, the CiADCs showed similar adipocyte-specific gene expression patterns to natural adipocytes. Furthermore, RVB increased protein expression of PPARγ and C/EBPα in the chemically-induced adipocytes (CiADCs-6d). Our findings reveal that the signaling pathways of C/EBPα and PPARγ play pivotal roles in this transdifferentiation process. In addition, we also found that the signaling pathways (PPAR signaling pathway, PI3K-Akt signaling pathway, p53 signaling pathway, MAPK signaling pathway, and ECM-receptor interaction) regulated by the DEGs played a vital role in adipogenesis. In general, this study provides valuable evidence to deepen our understanding of the molecular mechanism of small molecule cocktails in regulating adipogenesis.

Dataset Information

A developed serum-free medium and an optimized chemical cocktail for direct conversion of human dermal fibroblasts into brown adipocytes.

Publications

A developed serum-free medium and an optimized chemical cocktail for direct conversion of human dermal fibroblasts into brown adipocytes.

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