A New Two Step Induction Protocol for Neural Differentiation of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells

In this study, we examined a new two step induction protocol for improving the differentiation of human umbilical cord blood-derived mesenchymal stem cells into neural progenitor cells. Human umbilical cord blood-derived mesenchymal stem cells were first cultured in Dulbecco’s modified eagle medium supplemented with 10% fetal bovine serum in a humidified incubator equilibration with 5% CO2 at 37oC. To stimulate neural-differentiation of human umbilical cord blood-derived mesenchymal stem cells, Dulbecco’s modified eagle medium was removed and replaced with pre-induction media including retinoic acid, basic fibroblast growth factor, epidermal growth factor, and basal medium for 2 days. Then, we used nerve growth factor, 3-isobutylmethyl-xanthine, ascorbic acid, and basal medium for 6 days. Real-time PCR was performed to analyze the expression of neural specific genes such as glial fibrillary acidic protein, microtubule-associated protein 2, major basic protein, and Nestin. Real-time-PCR showed that the expression of glial fibrillary acidic protein, major basic protein, and microtubule-associated protein 2 genes, after two step induction, significantly increased compared to common induction protocol. In addition, our study showed that retinoic acid might play a main role in neural-differentiation and fate of mesenchymal stem cells compared to other neural inducers. The combination of chemicals and growth factors in a two step induction protocol may improve the efficiency of differentiation of human umbilical cord blood-derived mesenchymal stem cells into neural progenitor cells and provide a new method for easy and fast application of human umbilical cord blood-derived mesenchymal stem cells in regenerative medicine. Keywords: Umbilical cord blood, mesenchymal stem cell, neural progenitor cells, neural-differentiation.