Regeneration of Full-Thickness Skin Defects by Differentiated Adipose-Derived Stem Cells into Fibroblast-like Cells by Fibroblast-Conditioned Medium

Abstract

Fibroblasts are ubiquitous cells in the human body and they are absolutely necessary for wounds such as injured skin to heal. Their indispensable role in would healing is the reason why we aim to differentiate hADSCs into fibroblasts. Recently articles relative to human adipose-derived stem cells (hADSCs) derived conditioned medium (hADSCs-CM) have reported on the phenomenon and mechanism of stimulating both collagen synthesis as well as migration of dermal fibroblasts. Similarly, Human fibroblast-derived Conditioned medium (F-CM) was reported to contain a variety of factors known to be important in the growth of skin. However, it is still unknown how F-CM stimulates hADSCs in order to secrete Type I collagen. Our data showed that protein level of type I pro-collagen secreted by hADSCs in F-CM increased significantly compared to hADSCs in normal medium treated for 72 hours. In addition, we performed a sircoll collagen assay and found that the amount of collagen in F-CM treated hADSCs (72hr) was markedly raised in both the normal medium treated hADSCs (72hr) and the F-CM (24hr). We aim to validate 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 induces hADSCs into fibroblast cells, we performed FACS analysis and verified that both F-CM treated hADSCs and HS 27 contained the similar expression patterns for CD 13, CD 54, CD 105, whereas, normal medium treated hADSCs is significant different. RT-PCR (nanog, oct4A, and sox2 as un-differentiation markers, and vimentin, HSP47, and desmin as matured fibroblast markers), moreover, supported the characterization that hADSCs in F-CM highly differentiated into fibroblast cells. We also observed the TGF-beta/Smad signaling pathway in order to discover the mechanism of type I pro-collagen expression in hADSCs by F-CM and smad 2/3 protein up-regulation in F-CM treated hADSCs. In vivo, furthermore, we injected cells into balb/c nude mouse skin with a 10 mm punch wound, and obtained a significantly positive wound healing effect in the full-thickness excision wounded mice in F-CM treated hADSCs group rather than in the hADSCs or PBS group. In conclusion, we differentiated F-CM treated hADSCs into fibroblasts and tested the efficiency of the cells into a skin injured model.