Abstract
Tissue-engineered vascular grafts with long-term patency are greatly needed in the clinical settings, and smooth muscle cells (SMCs) are a critical graft component. Human mesenchymal stem cells (MSCs) are used for generating SMCs, and understanding the underlying regulatory mechanisms of the MSC-to-SMC differentiation process could improve SMC generation in the clinic. Here, we found that in response to stimulation of transforming growth factor-β1 (TGFβ1), human umbilical cord–derived MSCs abundantly express the SMC markers α-smooth muscle actin (αSMA), smooth muscle protein 22 (SM22), calponin, and smooth muscle myosin heavy chain (SMMHC) at both gene and protein levels. Functionally, MSC-derived SMCs displayed contracting capacity in vitro and supported vascular structure formation in the Matrigel plug assay in vivo. More importantly, SMCs differentiated from human MSCs could migrate into decellularized mouse aorta and give rise to the smooth muscle layer of vascular grafts, indicating the potential of utilizing human MSC-derived SMCs to generate vascular grafts. Of note, microRNA (miR) array analysis and TaqMan microRNA assays identified miR-503 and miR-222-5p as potential regulators of MSC differentiation into SMCs at early time points. Mechanistically, miR-503 promoted SMC differentiation by directly targeting SMAD7, a suppressor of SMAD-related, TGFβ1-mediated signaling pathways. Moreover, miR-503 expression was SMAD4-dependent. SMAD4 was enriched at the miR-503 promoter. Furthermore, miR-222-5p inhibited SMC differentiation by targeting and down-regulating ROCK2 and αSMA. In conclusion, MSC differentiation into SMCs is regulated by miR-503 and miR-222-5p and yields functional SMCs for use in vascular grafts.
Highlights
Tissue-engineered vascular grafts with long-term patency are greatly needed in the clinical settings, and smooth muscle cells (SMCs) are a critical graft component
Functional SMCs can be derived from human mesenchymal stem cells (MSCs) upon transforming growth factor-1 (TGF1) stimulation
Treatment of MSCs with 5 ng/ml TGF1 in ␣MEM with 1% serum induced the optimal differentiation toward SMC lineages
Summary
To establish the optimal conditions to induce SMC differentiation from human MSCs, the concentration of serum and TGF1 were first optimized (data not shown). Transfection of miR-503 mimics in MSCs in medium with 1% FBS promoted SMC differentiation with increased expression of SMC markers, including calponin, SM22, ␣SMA, and SMMHC at the mRNA level after 3 days as shown by Q-PCR (Fig. 3D). Results showed that, compared with the empty plasmid, the plasmid containing the WT ROCK 3Ј-UTR demonstrated lower relative luciferase activity when co-transfected with miR-222-5p mimics, and this effect was not observed when the plasmids were co-transfected with miRNA mimic negative control (Fig. 8C). Mutation of both target sites, but not a single target site, rescued the inhibited relative luciferase activity (Fig. 8C). Data are presented as the mean Ϯ S.D. from three independent experiments. *, p Ͻ 0.05, and ***, p Ͻ 0.001. mim ctrl, miRNA mimic negative control; mim 503, miR-503 mimic; ctrl, plasmid without SMAD7 3Ј-UTR; wt, plasmid bearing WT SMAD7 3Ј-UTR
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