Conditioned medium from induced pluripotent stem cell-derived mesenchymal stem cells accelerates cutaneous wound healing through enhanced angiogenesis

Abstract

Background: Mesenchymal stem cells (MSCs) can improve cutaneous wound healing via the secretion of growth factors. However, the therapeutic efficacy of MSCs varies depending upon their source. Induced pluripotent stem cells are emerging as a promising source of MSCs with the potential to overcome several limitations of adult MSCs. This study compared the effectiveness of conditioned medium of MSCs derived from induced pluripotent stem cells (iMSC-CdM) with that derived from umbilical cord MSCs (uMSC-CdM) in a mouse cutaneous wound healing model. We also investigated the mechanisms of protection.

Methods: The iMSC-CdM or uMSC-CdM were topically applied to mice cutaneous wound model. The recovery rate, scar formation, inflammation and angiogenesis were measured. We compared angiogenesis cytokine expression between iMSC-CdM and uMSC-CdM and their protective effects on human umbilical vein endothelial cells (HUVECs) under H₂O₂-induced injury. The effects of iMSC-CdM on energy metabolism, mitochondria fragmentation and apoptosis were measured.

Results: Topical application of iMSC-CdM was superior to the uMSC-CdM in accelerating wound closure and enhancing angiogenesis. Expression levels of angiogenetic cytokines were higher in iMSC-CdM than they were in uMSC-CdM. The iMSC-CdM protected HUVECs from H₂O₂ induced injury more effectively than uMSC-CdM did. Administration of iMSC-CdM stimulated HUVEC proliferation, tube formation and energy metabolism via the ERK pathway. Mechanistically, iMSC-CdM inhibited H₂O₂-induced mitochondrial fragmentation and apoptosis of HUVECs.

Conclusion: Collectively, these findings indicate that iMSC-CdM is more effective than uMSC-CdM in treating cutaneous wounds, and in this way, iMSC-CdM may serve as a more constant and sustainable source for cell-free therapeutic approach.

Keywords: Conditioned medium; Induced pluripotent stem cell-derived mesenchymal stem cells; Mitochondria dysfunction; Wound healing.

Fig. 1

Characterization of mesenchymal stem cells derived from umbilical cord mesenchymal stem cells (uMSCs) and mesenchymal stem cells derived from induced pluripotent stem cells (iMSCs). a Surface marker profiling was determined by flow cytometry. b Morphology and trilineage differentiation of uMSCs and iMSCs. Scale bar = 100m. c Schematic diagram for conditioned medium collection

Fig. 2

Conditioned medium of iMSCs effectively accelerated cutaneous wound healing. a Schematic diagram of animal study. Conditioned medium of mesenchymal stem cells derived from umbilical cord mesenchymal stem cells (uMSC-CdM) and conditioned medium of mesenchymal stem cells derived from induced pluripotent stem cells (iMSC-CdM) was applied daily after the surgery. b Schematic diagram depicting delivery method of uMSC-CdM/iMSC-CdM. A sterile cotton pad (blue box) was placed to cover the wound area (red circle) and then uMSC-CdM or iMSC-CdM was topically administered onto the pad. c Representative images of the wounds receiving conditioned medium treatment were taken 1 and 9days after surgery. Scale bar = 1cm. d The recovery rate was measured daily after treatment. *P < .05 iMSC-CdM vs control group; **P < .01 iMSC-CdM vs control group; ***P < .001 iMSC-CdM vs control group; ^#P < .05 uMSC-CdM vs control group; P < .05 iMSC-CdM vs uMSC-CdM group

Fig. 3

Treatment with iMSC-CdM inhibited inflammation and enhanced angiogenesis. a Trichrome staining revealed that the wounds treated with uMSC-CdM and iMSC-CdM had similar granulation tissue at the peri-wound area (arrows indicate keratinocytes; stars indicate granulation tissue). Scale bar = 100m. b Inflammatory cytokine expression was determined by a bead-based immunoassay. c Representative fluorescence images and quantification of CD31 positive blood vessels. Scale bar = 100m. d Expression of pro-angiogenesis factors at the peri-wound area determined by quantitative polymerase chain reaction. e EGF and VEGFA protein expression determined by Elisa assay. ns, non-significance, *P < .05, **P < .01, ***P < .001

Fig. 4

The cytoprotective effects of iMSC-CdM on H₂O₂ treated HUVECs. After H₂O₂ treatment, a apoptosis, b cell reactive oxygen species, c mitochondrial reactive oxygen species, and d mitochondrial permeability transition pore (mPTP) opening were determined by flow cytometry. MFI, mean fluorescent intensity. ns, non-significance, *P < .05, **P < .01, ***P < .001. e Cell proliferation was determined by Cell Counting Kit8 assay. *P < .05 iMSC-CdM vs control group; #P < .05 uMSC-CdM vs control group; P < .05 iMSC-CdM vs uMSC-CdM group. f Representative light photomicrographs and quantitative analysis of HUVEC tube formation assay after uMSC-CdM/iMSC-CdM treatment. Scale bar = 100m. *P < .05, **P < .01. g Protein concentration and expression levels of angiogenic factors in uMSC-CdM and iMSC-CdM. ***P < .001

Fig. 5

iMSC-CdM enhanced energy metabolism and angiogenesis via the ERK pathway. a ATP concentrations were determined in conditioned medium-treated HUVECs with pathway inhibitors as indicated. b Respiratory potential of conditioned medium-treated HUVECs quantified with the Seahorse Metabolic Analyzer. Real-time measurements of oxygen consumption rate (OCR) were obtained basally and then after treatment with oligomycin (ATP synthase inhibitor), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP, electron transport chain accelerator), and rotenone plus antimycin A (electron transport chain inhibitors). c Representative photomicrographs and quantitative analysis of tube formation assay in conditioned medium-treated HUVECs with and without ERK inhibitor U0126. Scale bar = 100m. d Representative immunoblot images and quantitative analysis in conditioned medium-treated HUVECs with and without ERK inhibitor U0126. e Expression of pro-angiogenesis factors in conditioned medium-treated HUVECs with and without ERK inhibitor U0126. ns, non-significance, *P < .05, **P < .01, ***P < .001

Fig. 6

iMSC-CdM treatment suppresses H₂O₂-induced mitochondria fragmentation and apoptosis of HUVECs. a Representative images and quantitative analysis of fragmented mitochondria in H₂O₂-treated HUVECs. Scale bar = 50 um. b Western blotting and quantitative analysis for the expression of MFN1, MFN2, DRP1 and pDRP1 ser616 in H₂O₂-treated HUVECs. c Apoptosis was determined by flow cytometry. ns, non-significance, *P < .05, **P < .01, ***P < .001