Abstract
Adipose-derived stem cell (ADSC) is an alternative and less invasive source of mesenchymal stem cells which can be used to develop biological treatment strategies for tissue regeneration, and their therapeutic applications hinge on an understanding of their physiological characteristics. N6-Methyladenosine (m6A) is the most common chemical modification of mRNAs and has recently been revealed to play important roles in cell lineage differentiation and development. However, the role of m6A modification in the vascular smooth muscle cell (VSMC) differentiation of ADSCs remains unclear. Herein, we investigated the expression of N6-adenosine methyltransferases (Mettl3) and demethylases (Fto and Alkbh5) and found that Mettl3 was upregulated in ADSCs undergoing vascular smooth muscle differentiation induction. Moreover, silence of Mettle3 reduced the expression level of VSMC-specific markers, including α-SMA, SM22α, calponin, and SM-MHC. Meanwhile, Mettl3 knockdown also decreased the expression of paracrine factors, including VEGF, HGF, TGF-β, GM-CSF, bFGF, and SDF-1. In addition, our results suggested that hypoxia stress promotes the ADSC differentiate into VMSCs and regulates the secretion of VEGF, HGF, TGF-β, GM-CSF, bFGF, and SDF-1 by mediating Mettl3 gene expression. These observations might contribute to novel progress in understanding the role of epitranscriptomic regulation in the VSMC differentiation of ADSCs and provide a promising perspective for new therapeutic strategies for tissue regeneration.
Highlights
N6-Methyladenosine (m6A), a methylation modification at the N6 position of adenosine in RNAs, is the most abundant modification in eukaryotic mRNAs [1, 2]
The results showed a positive expression of CD13, CD90, and CD44, but negative for CD34, and CD45, indicating that the Adipose-derived stem cell (ADSC) were of high purity and excluding the contamination of endothelial and hematopoietic (Figures 1(a) and 1(b))
The ADSCs were able to be differentiated into osteoblast-like cells and adipose-like cells when cultured in a differentiating induction medium, as determined by Alizarin Red staining and Oil Red O staining, respectively (Figures 1(h) and 1(i))
Summary
N6-Methyladenosine (m6A), a methylation modification at the N6 position of adenosine in RNAs, is the most abundant modification in eukaryotic mRNAs [1, 2]. Mesenchymal stem cells (MSCs) are a heterogeneous population of nonhematopoietic adult stem cells that present in a verity of tissues, such as bone marrow, adipose, and muscle [16, 17]. They have the property of self-renewal and multilineage differentiation capacity and have been widely utilized in stem cell transplantation, gene therapy, tissue engineering, and immunotherapy [18,19,20]. Human adiposederived stem cells (ADSCs) are a type of MSCs isolated from adipose tissue, which demonstrate the multidirectional differentiation potential and have multiple advantages of abundant storage in vivo, easy acquisition, and expansion [18,19,20], suggesting that a broader source of stem cells is available for
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