Induction of Neuronal Differentiation of Human Dermal Mesenchymal Stem Cells by a Defined Arginine-Aspartate-Phenylalanine Peptide Mixture in Vitro

Abstract

The aim of this study is to investigate the effects of a peptide mixture composed of arginine (Arg), aspartic acid (Asp), and phenylalanine (Phe) on the proliferation and neural differentiation of human dermal mesenchymal stem cells (HDMSCs). HDMSCs were isolated from residual skin tissue following circumcision and purified using a combination of long-term trypsin stress and suspension culture. The impact of the peptide mixture on HDMSC proliferation was assessed at various concentrations using a Cell Counting Kit-8 (CCK-8) assay. Based on preliminary screening of proliferative activity, an optimal peptide mixture concentration for promoting proliferation was selected for further HDMSCs culture. During this culture, morphological changes were observed. Differentiation efficiency was evaluated using immunofluorescence staining for the neural markers Nestin and Neurofilament-L (NF-L) at weeks 3 and 6 of culture, respectively. Primary adherent HDMSCs exhibited a long spindle morphology and formed spherical cell clusters in suspension culture. A peptide mixture at concentrations of 5 ng/mL Arg, 20 ng/mL Asp, and 40 ng/mL Phe significantly promoted HDMSC proliferation (p < 0.05). During the induction period, the cells gradually acquired neuronal morphological characteristics, including cell body contraction and process extension. Immunofluorescence results showed that the cells expressed Nestin by week 3, shifting to NF-L positivity by week 6. This indicates that the peptide mixture induced the differentiation of HDMSCs into neural cells. A peptide mixture containing Arg, Asp, and Phe has been shown to effectively induce the differentiation of HDMSCs into neural cells. This provides a novel and safe strategy for the regeneration of neural tissue.