[Effect of Schwann cells on differentiation of rat bone marrow mesenchymal stem cells at different ages].

OBJECTIVE: Bone marrow mesenchymal stem cells (BMSCs) are multipotent and thus are able to differentiate into a number of different cell types under certain culture condition. However, the effect of age on the differentiation remains unknown. To explore the effect of the microenvironment formed by Schwann cells (SCs) on BMSCs differentiation into neurons and oligodendrocytes in rats at different ages in vitro.

METHODS: SCs were extracted and purified from the distal sciatic nerves of neonatal Wistar rats. BMSCs were isolated from bone marrow of Wistar rats (aged 1 month, 6 months, and 12 months, respectively) and cultured in vitro. The cells were identified by immunofluorescent staining. The BMSCs at passage 2 were labeled by PKH26 and cocultured with SCs at passage 3 in equal proportions in two layer Petri dish. According to the BMSCs from the rats at different ages, experiment was divided into 3 groups: SCs were cocultured with 1-month-old rat BMSCs (group A), 6-month-old rat BMSCs (group B), and 12-month-old rat BMSCs (group C), respectively. The morphological changes of cocultured BMSCs were observed by inverted phase contrast microscope, the expressions of neuron-specific enolase (NSE) and myelin basic protein (MBP) in the cocultured BMSCs were tested by immunofluorescent staining, and the expression of neuregulin 1 (NRG1) was detected by ELISA method.

RESULTS: SCs and BMSCs were isolated and cultured successfully. The identification of SCs showed positive expression of S-100 and BMSCs showed positive expressions of CD29, CD44, and CD90. At 7 days after coculture, the BMSCs in group A began retraction, and became round or tapered with the processes and had a nerve cells or oligodendrocytes-like morphology, but most BMSCs in groups B and C showed no obvious morphological changes under inverted phase contrast microscope. Immunofluorescent staining showed that the positive expression rates of NSE in groups A, B, and C were 22.39% +/- 2.86%, 12.89% +/- 1.78%, and 2.69% +/- 0.80%, respectively, and the positive expression rates of MBP in groups A, B, and C were 16.13% +/- 2.39%, 6.33% +/- 1.40%, and 0.92% +/- 0.17%, respectively. There were significant differences in terms of NSE and MBP positive expression rates among 3 groups (P < 0.05). ELISA analysis showed that NRG1 in the supernatant of group A was increased after coculture in a time-dependent manner. At 6, 9, and 12 days of coculture, NRG1 content was higher in group A than in groups B and C, and in group B than in group C, showing significant differences (P < 0.05).

CONCLUSION: The microenvironment formed by SCs can promote BMSCs differentiation into neurons and oligodendrocytes, but the differentiation capability of BMSCs decreases with aging, and the variety of growth factors secreted by SCs is likely important factors that induce the differentiation of BMSCs into neurons and oligodendrocytes.