In vitro induction of differentiation of human adipose-derived mesenchymal stem cells into melanocytes: an experimental study

Abstract

Objective To establish a method for inducing human adipose-derived mesenchymal stem cells (hADSCs) to differentiate into melanocytes in vitro.Methods hADSCs were isolated from human subcutaneous fat tissue,and subjected to phenotypic analysis by flow cytometry.The differentiation potential of the hADSCs was evaluated by induction of osteogenic and adipogenic differentiation.Then,the fifth passage hADSCs were induced to differentiate into melanocytes by conditioned medium containing stem cell factor (SCF),endothelin 3,basic fibroblast growth factor (bFGF) for ten weeks.Those hADSCs receiving no induction treatment served as the normal control.Subsequently,real-time fluorescence-based quantitative PCR (RT-PCR) was performed to determine the mRNA expressions of melanin-associated genes including microophthalmia-associated transcription factor (MITF),ckit receptor tyrosine kinase (KIT),dopachrome tautomerase (DCT),tyrosinase,tyrosinase-related protein-1 (TYRP1),and sex determining region Y-box 10 (SOX10).Statistical analysis was done by one-factor analysis of variance and the least significant difference (LSD) test.Further more,the hADSC-derived melanocytes were identified by immunocytochemistry and immunofluorescence assay.Results As flow cytometry revealed,the isolated hADSCs showed high expressions of CD29,CD44,CD73,CD90,CD105 and CD166 with the positivity rates being more than 95％,but low or absent expressions of CD31,CD34,CD45 and human leukocyte antigen (HLA)-DR.Additionally,the osteogenic and adipogenic differentiation of hADSCs was successfully induced.The relative mRNA expression levels of MITF,KIT,DCT,tyrosinase,TYRP1 and SOX10 were 0.325 ± 0.012,0.042 ± 0.006,0.046 ± 0.013,0.036 ± 0.005,0.041 ± 0.003,and 0.225 ± 0.014 respectively in hADSCs induced for 10 weeks,which were significantly upregulated compared with the normal control hADSCs (all P ＜ 0.05).Moreover,the induced hADSCs showed positive immunocytochemical staining for MITF and HMB45,as well as positive cytoimmunofluorescence staining for TYRP1 and S100.Conclusion The hADSCs have been successfully induced to differentiate into cells with the biological characteristics of melanocytes. Key words: Human adipose-derived mesenchymal stem cells; Cell differentiation; Melanocytes