Regeneration of full-thickness skin defects by differentiated adipose-derived stem cells into fibroblast-like cells by fibroblast-conditioned medium

Abstract

Background: Fibroblasts are ubiquitous cells in the human body and are absolutely necessary for wound healing such as for injured skin. This role of fibroblasts was the reason why we aimed to differentiate human adipose-derived stem cells (hADSCs) into fibroblasts and to test their wound healing potency. Recent reports on hADSC-derived conditioned medium have indicated stimulation of collagen synthesis as well as migration of dermal fibroblasts in wound sites with these cells. Similarly, human fibroblast-derived conditioned medium (F-CM) was reported to contain a variety of factors known to be important for growth of skin. However, it remains unknown whether and how F-CM can stimulate hADSCs to secrete type I collagen.

Methods: In this study, we obtained F-CM from the culture of human skin fibroblast HS27 cells in DMEM media. For an in-vivo wound healing assay using cell transplantation, balb/c nude mice with full-thickness skin wound were used.

Results: Our data showed that levels of type I pro-collagen secreted by hADSCs cultured in F-CM increased significantly compared with hADSCs kept in normal medium for 72 h. In addition, from a Sircol collagen assay, the amount of collagen in F-CM-treated hADSC conditioned media (72 h) was markedly higher than both the normal medium-treated hADSC conditioned media (72 h) and the F-CM (24 h). We aimed to confirm that hADSCs in F-CM would differentiate into fibroblast cells in order to stimulate wound healing in a skin defect model. To investigate whether F-CM induced hADSCs into fibroblast-like cells, we performed FACS analysis and verified that both F-CM-treated hADSCs and HS27 cells contained similar expression patterns for CD13, CD54, and CD105, whereas normal medium-treated hADSCs were significantly different. mRNA level analysis for Nanog, Oct4A, and Sox2 as undifferentiation markers and vimentin, HSP47, and desmin as matured fibroblast markers supported the characterization that hADSCs in F-CM were highly differentiated into fibroblast-like cells. To discover the mechanism of type I pro-collagen expression in hADSCs in F-CM, we observed that phospho-smad 2/3 levels were increased in the TGF-β/Smad signaling pathway. For in-vivo analysis, we injected various cell types into balb/c nude mouse skin carrying a 10-mm punch wound, and observed a significantly positive wound healing effect in this full-thickness excision model with F-CM-treated hADSCs rather than with untreated hADSCs or the PBS injected group.

Conclusions: We differentiated F-CM-treated hADSCs into fibroblast-like cells and demonstrated their efficiency in wound healing in a skin wound model.

Keywords: Cell transplantation; Human adipose-derived stem cell-derived conditioned medium; Type I collagen; Wound healing.

Figures

Fig. 1

a In-vivo study design. b In-vitro study design. F-CM fibroblast-derived conditioned medium, hADSC human adipose-derived stem cell, PBS phosphate-buffered saline

Fig. 2

Comparison of pro-collagen type I protein levels for 1% FBS + DMEM only (24 h), 1% FBS + DMEM hADSCs (72 h), fibroblast CM only (24 h), and F-CM hADSCs (72 h): according to first or second density of DMEM and F-CM medium through western blot analysis; and in baseline concentration and double concentration DMEM and F-CM medium through western blot analysis. CM derived conditioned medium, hADSC human adipose-derived stem cell

Fig. 3

a Band intensities were significantly increased for type I pro-collagen protein expression shown at higher concentration of F-CM-treated hADSCs, when compared with 1% FBS + DMEM only, 1% FBS + DMEM ADSCs, or F-CM only after incubation for 72 h. b Amount of collagen (μg/ml) measured for each of the four groups in baseline concentration and double concentration normal and F-CM medium through Sircol assay. *P < 0.05, **P < 0.01. CM derived conditioned medium, hADSC human adipose-derived stem cell

Fig. 4

FACS analysis for characterization of differentiation of untreated hADSCs (72 h), HS27 cells (72 h), and F-CM-treated hADSCs (72 h) using cell surface makers: a CD14, CD19, CD45, CD34, and CD105; b CD13, CD54, and CD73. F-CM fibroblast-derived conditioned medium, hADSC human adipose-derived stem cell

Fig. 5

a, b mRNA levels of Nanog, Oct4a, and Sox2 as undifferentiated markers along with vimentin, HSP47, and desmin as mature fibroblast differentiation markers with RT-PCR in untreated hADSCs (72 h), HS27 cells (72 h), and F-CM-treated hADSCs (72 h). a semi-quantitative RT-PCR image for RNA levels of Nanog, Oct4a, and Sox2, vimentin, HSP47, and desmin. b the graph for relative comparision of each band thickness image in semi-quantitative RT-PCR image (a). *P < 0.05, **P < 0.01. F-CM fibroblast-derived conditioned medium, hADSC human adipose-derived stem cell

Fig. 6

a Immunohistochemistry and b human vimentin localization in balb/c nude mice skin after injection of F-CM-treated hADSCs, untreated hADSCs, and PBS. Alexa-546 as secondary antibody and Hoechst 33342 as counterstain. Arrows indicate positive cells for immunofluorescence. Scale bars represent 100 μm. *P < 0.05. CTL control, F-CM fibroblast-derived conditioned medium, hADSC human adipose-derived stem cell, PBS phosphate-buffered saline

Fig. 7

a Protein level comparison between normal medium (48 h) and F-CM (48 h) with anti-TGF-β, anti-FGF2, and anti-VEGF blotting. b For cell lysates, protein level comparison of untreated hADSCs (72 h), HS27 (72 h), and F-CM-treated hADSCs (72 h) through western blot analysis. CM conditioned medium, hADSC human adipose-derived stem cell

Fig. 8

a Immunocytochemistry and b pro-collagen type I localization for untreated hADSCs (72 h), HS27 cells (72 h), and F-CM-treated hADSCs (72 h). Arrows indicate positive cells for immunofluorescence. Scale bars represent 100 μm. *P < 0.05. F-CM fibroblast-derived conditioned medium, hADSC human adipose-derived stem cell

Fig. 9

a Image for wound healing measurement and b wound healing rate percentages following injection of F-CM-treated hADSCs, untreated hADSCs, and PBS according to day post injection (0, 3, 7, and 10 days after cell implantation in balb/c nude mice skin). **P < 0.01. F-CM fibroblast-derived conditioned medium, hADSC human adipose-derived stem cell, PBS phosphate-buffered saline

Fig. 10

Histological evaluation of wound samples from F-CM-treated hADSC, untreated hADSC, and PBS injected animals at 1 week (1 W) and 2 weeks (2 W) after cell implantation in balb/c nude mice skin using H&E staining. Magnification: 40×, 100×. F-CM fibroblast-derived conditioned medium, hADSC human adipose-derived stem cell, PBS phosphate-buffered saline, G Granulation tissue, W Wounded area

Fig. 11

a Histological evaluation and b F-CM-treated hADSC, untreated hADSC, and PBS injected animals at 1 week (1 W) and 2 weeks (2 W) after cell implantation in balb/c nude mice skin with anti-collagen type I staining. Magnification: 40×, 100×. *P < 0.05, **P < 0.01. CTL control, F-CM fibroblast-derived conditioned medium, hADSC human adipose-derived stem cell, PBS phosphate-buffered saline