Mechanical Stretching Promotes Skin Tissue Regeneration via Enhancing Mesenchymal Stem Cell Homing and Transdifferentiation

Abstract

Skin tissue expansion is a clinical procedure for skin regeneration to reconstruct cutaneous defects that can be accompanied by severe complications. The transplantation of mesenchymal stem cells (MSCs) has been proven effective in promoting skin expansion and helping to ameliorate complications; however, systematic understanding of its mechanism remains unclear. MSCs from luciferase-Tg Lewis rats were intravenously transplanted into a rat tissue expansion model to identify homing and transdifferentiation. To clarify underlying mechanisms, a systematic approach was used to identify the differentially expressed genes between mechanically stretched human MSCs and controls. The biological significance of these changes was analyzed through bioinformatic methods. We further investigated genes and pathways of interest to disclose their potential role in mechanical stretching-induced skin regeneration. Cross sections of skin samples from the expanded group showed significantly more luciferase(+) and stromal cell-derived factor 1α (SDF-1α)(+), luciferase(+)keratin 14(+), and luciferase(+)CD31(+) cells than the control group, indicating MSC transdifferentiation into epidermal basal cells and endothelial cells after SDF-1α-mediated homing. Microarray analysis suggested upregulation of genes related to hypoxia, vascularization, and cell proliferation in the stretched human MSCs. Further investigation showed that the homing of MSCs was blocked by short interfering RNA targeted against matrix metalloproteinase 2, and that mechanical stretching-induced vascular endothelial growth factor A upregulation was related to the Janus kinase/signal transducer and activator of transcription (Jak-STAT) and Wnt signaling pathways. This study determines that mechanical stretching might promote skin regeneration by upregulating MSC expression of genes related to hypoxia, vascularization, and cell proliferation; enhancing transplanted MSC homing to the expanded skin; and transdifferentiation into epidermal basal cells and endothelial cells.

Significance: Skin tissue expansion is a clinical procedure for skin regeneration to cover cutaneous defects that can be accompanied by severe complications. The transplantation of mesenchymal stem cells (MSCs) has been proven effective in promoting skin expansion and ameliorating complications. This study, which sought to provide a systematic understanding of the mechanism, determined that mechanical stretching could upregulate MSC expression of genes related to hypoxia, vascularization, and cell proliferation; enhance transplanted MSC homing to the expanded skin tissue; and promote their transdifferentiation into epidermal basal cells and endothelial cells.

Keywords: Mechanical stretching; Mesenchymal stem cell; Skin regeneration; Stem cell therapy; Tissue expansion.

Figure 1.

Transplanted mesenchymal stem cells (MSCs) transdifferentiated into KRT14⁺ cells and CD31⁺ cells after SDF-1α-induced homing in the expanded skin. Luciferase was stained to locate the transplanted MSCs. (A): Skin from the expanded group showed significantly more SDF-1α and luciferase colocalization than the control group, especially the outer root sheath of the hair follicles (arrow), verifying the essential role of SDF-1α in MSC homing under mechanical stretching. (B): More KRT14⁺/luciferase⁺ cells were observed in the skin from the expanded group than the control group, especially in the outer root sheath of the hair follicles (arrow). (C): More CD31⁺/luciferase⁺ cells were observed in blood vessels (arrow) of the skin from the expanded group than the control group. ∗∗∗, p < .001; scale bars = 100 μm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; KRT, keratin; SDF, stromal cell-derived factor.

Figure 2.

Upregulation of SDF-1α might be induced by HIF-1α in the expanded skin. (A): Immunohistochemistry staining showed enhanced HIF-1α expression after skin tissue expansion. (B): Western blot results also showed higher HIF-1α in the expanded skin. (C): We used small interfering RNA to knock down HIF-1α expression in the expanded skin. After 21-day expansion, the expanded skin samples were subjected to Western blot. The results showed that SDF-1α expression was significantly lower when HIF-1α was knocked down in the expanded skin. ∗∗∗, p < .001; scale bars = 100 μm. Abbreviations: HIF, hypoxia-inducible factor; SDF, stromal cell-derived factor.

Figure 3.

Functional categorization of significantly differentially expressed genes in mechanically stretched hMSCs. Differentially expressed genes from hMSCs cultured in the presence and absence of mechanical stretching were classified into the following categories: biological process, molecular function, and cellular components. Abbreviation: hMSC, human mesenchymal stem cell.

Figure 4.

MMP2 is a crucial factor for mesenchymal stem cells (MSCs) homing. (A): Western blot analysis showed higher MMP2 expression in the expanded skin after MSC transplantation. (B): MSCs were transfected with siRNAs targeting MMP2, and the expression levels of the target mRNAs were analyzed by reverse transcription-polymerase chain reaction. (C): si-MMP2-transfected MSCs and si-control-transfected MSCs were transplanted into the rat skin expansion model. The expanded skin area was collected after 21 days. Immunohistochemistry staining showed that expanded skin transplanted with si-MMP2-transfected MSCs contained significantly fewer luciferase-positive cells than control. (D): Western blot analysis showed that expanded skin transplanted with si-MMP2-transfected MSCs had significantly lower MMP2 and SDF-1α expression levels than control. (E,F): Costaining of luciferase/KRT14 and luciferase/CD31 were performed to determine the fate of siRNA-treated MSCs after transplantation into the rat expansion model. The results showed that skin transplanted with si-MMP2 MSCs had significantly fewer luciferase⁺/KRT14⁺ and luciferase⁺/CD31⁺ cells than that with si-control MSCs. ∗∗, p < .01; ∗∗∗, p < .001; scale bars = 100 μm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; KRT, keratin; MMP, matrix metalloproteinase; mRNA, messenger RNA; SDF, stromal cell-derived factor; si, small interfering.

Figure 5.

The Jak-STAT and Wnt signaling pathways are involved in mechanical stretching-induced VEGFA upregulation. (A): Human mesenchymal stem cells (hMSCs) under mechanical stretching were treated with the Jak inhibitor AG490 and the Wnt-pathway inhibitor ICG-001. Reverse transcription-polymerase chain reaction results showed that hMSCs under mechanical stretching treated with AG490 or ICG-001 expressed significantly lower VEGFA mRNA levels than those treated with DMSO. (B): Rat skin samples from the expanded and control groups were analyzed by Western blot to evaluate the expression of VEGFA, p-STAT3 (marker of the Jak-STAT signaling pathway), and p-GSK3β (marker of the Wnt signaling pathway); the results showed that expression of VEGFA and markers of the Jak-STAT and Wnt signaling pathways were upregulated in the expanded skin compared with unexpanded skin after MSC transplantation. ∗∗, p < .01; ∗∗∗, p < .001. Abbreviations: DMSO, dimethyl sulfoxide; Jak-STAT, Janus kinase/signal transducer and activator of transcription; mRNA, messenger RNA; NS, no strain; S, strain; VEGFA, vascular endothelial growth factor A.

Figure 6.

Schematic diagram of the homing and transdifferentiation of transplanted MSCs. Mechanical stretching might enhance MSC homing to the outer root sheath of hair follicles and blood vessels through feedback between MMP2 and the SDF-1α/C-X-C chemokine receptor 4 axis. MSCs transdifferentiate into KRT14⁺ cells in the microenvironment of the hair follicle stem cell niche, and into CD31⁺ cells in blood vessels. MSCs promote vascularization in the expanded skin through upregulation of VEGFA (via the Jak-STAT and Wnt signaling pathways) and other proangiogenic factors. Abbreviations: Jak-STAT, Janus kinase/signal transducer and activator of transcription; KRT, keratin; MET, mesenchymal-epithelial transition; MMP, matrix metalloproteinase; MSC, mesenchymal stem cell; SDF, stromal cell-derived factor; VEGFA, vascular endothelial growth factor A.