Synergistic effects of micropatterned substrates and transforming growth factor-β1 on differentiation of human mesenchymal stem cells into vascular smooth muscle cells through modulation of Krϋppel-like factor 4

Abstract

The functionality and structural integrity of the cardiovascular system are critically dependent on vascular smooth muscle cells (VSMCs). Human mesenchymal stem cells (hMSCs) have significant potential for differentiating into VSMCs, making them a valuable resource in regenerative medicine and the development of vascular grafts. This study explored the synergistic effects of micropatterned substrates and TGF-β1 on the differentiation of hMSCs into VSMCs. HMSCs were cultured on both micropatterned and flat substrates for a duration of 6 days, with some groups receiving TGF-β1 treatment, after which cell morphology and the expression of specific smooth muscle markers were evaluated through Western blotting, immunofluorescence staining, and RT-qPCR. Results indicated that hMSCs on micropatterned substrates treated with TGF-β1 exhibited significantly elevated protein levels of smooth muscle myosin heavy chain (MYH11) compared with hMSCs on flat substrates without TGF-β1 (p < 0.001). Additionally, MYH11 expression was markedly enhanced in samples cultured on micropatterned substrates with TGF-β1. Furthermore, hMSCs treated with TGF-β1 on flat substrates exhibited increased cadherin-11 mRNA expression compared with both micropatterned and flat substrates lacking TGF-β1 (p < 0.05). Interestingly, KLF4 protein levels were significantly higher in hMSCs on flat substrates without TGF-β1 compared to those cultured on micropatterned substrates with TGF-β1 treatment (p < 0.001). In conclusion, this study demonstrated that the combination of micropatterned substrates and TGF-β1 treatment preferentially enhances MYH11 expression, indicative of advanced smooth muscle cell organization, along with modulating KLF4 levels and upregulating cadherin-11 expression in hMSCs. These findings provide critical insights into the differentiation pathways of MSCs into VSMCs and may inform the design of improved vascular grafts that better replicate the properties of native blood vessels.

Keywords: Human mesenchymal stem cells; Krϋppel-like factor 4; Micropatterned substrates; Myosin heavy chain (MYH11); Vascular smooth muscle cells.

Figure 1.

Phase-contrast microscopy images of MSCs. (A₁) MSCs cultured on flat PDMS substrates for 6 d display a typical cell morphology. (A₂) MSCs on micropatterned PDMS substrates show an elongated cell shape induced by the micropatterns.

Figure 2.

Synergistic effects of micropatterned substrates and TGF-β1 on MYH11 expression. (A) The expression of MYH11 protein in MSCs cultured on micropatterned versus flat substrates in normal DMEM medium, with or without TGF-β1, was assessed after 6 d. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as an internal control in all samples. The expression of MYH11 was normalized to that of GAPDH. The data are presented as mean ± SEM of band intensity normalized to MSCs on flat substrates (n = 3). (B) Immunofluorescence staining of MYH11 and DAPI in MSCs: (B₁) MSCs cultured on flat substrates without TGF-β1 show the lowest MYH11 expression. (B₂) MSCs on micropatterned substrates with TGF-β1 exhibit the highest MYH11 protein expression as detected by immunoblotting. DAPI staining indicates cell nuclei. (C) The expression levels of MYH11 mRNA were evaluated using RT-qPCR. The results are expressed as mean ± SEM, normalized to flat substrates (n = 3). Statistical significance was indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Key abbreviations include F (flat substrate), MP (micropatterned substrate), N (normal medium), and D6 (day 6). MYH11 refers to the smooth muscle myosin heavy chain.

Figure 3.

Micropattern and TGF-β1 modulate KLF4 expression in MSCs. (A) Immunoblotting analysis of KLF4 in mesenchymal stem cells (MSCs) cultured on micropatterned and flat substrates in normal DMEM, administered with or without TGF-β1 treatment for 6 d. KLF4 protein expression was normalized to GAPDH levels. The data are shown as mean ± SEM of band intensity, with values normalized to MSCs on flat substrates (n = 3). (B) Immunofluorescence staining of KLF4 and DAPI in MSCs: (B₁) MSCs cultured on flat substrates without TGF-β1 exhibit the highest KLF4 protein expression. (B₂) MSCs on micropatterned substrates with TGF-β1 show the lowest KLF4 expression as detected by immunoblotting. DAPI staining indicates cell nuclei. (C) Relative gene expression analysis of KLF4: KLF4 mRNA levels were quantified via RT-qPCR, normalized against flat substrate controls. Results are presented as mean ± SEM (n = 3), with statistical significance indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Abbreviations used include F (flat substrate), MP (micropatterned substrate), N (normal medium), and D6 (day 6). KLF4 refers to Krϋppel-like factor 4.

Figure 4.

Elongation and TGF-β1 regulate cadherin-11 gene. Relative gene level expression analyses of cadherin-11. The expression of cadherin-11 mRNA was examined by RT-qPCR. The graphic data is mean ± SEM of the value normalized to flat substrates (n = 3). **p < 0.01. The terms F, MP, N, and D6 represent a flat substrate, a micropatterned substrate, a normal medium, and day 6, respectively.