Relationship Between Rac1 And The Proliferation And Differentiation Of Neural Precursor Cells

Objective To investigate the relationship between Rac1 and the proliferation and differentiation of neural precursor cells, broadening our understanding of the underlying molecular mechanism and thus theoretically lending support to the potential use of neural precursor cells in nerve injury repair.Methods Three models were used: (1) Mixed primary glial cultures were prepared from the cerebral cortex of newborn SD rats. Cells were cultured in DMEM (H) plus 10% FBS, and subsequently in serum-free medium (SF) after confluence. Immunofluorescence staining was used to characterize the mixed cultures. Rac1 expression was analyzed by immunofluorescence and western blot. Rac1/Cdc42 activity was analyzed by Pull-down assay. (2) An in intro wound model: Mixed glial cultures were switched to serum-free medium after confluence and a lesion area was made by scratching. O-2A precursor cells moved into the lesion area and the characterization and the expression of Rac1 were then analyzed by immunofluorescence. (3) O-2A cell cultures: Cells were obtained from the cerebral cortex of newborn SD rats and isolated by shaking the cultures on an orbital shaker for 12 h at 37°C. The serum-free medium containing bFGF and PDGF was used to expand O-2A precursor cells. Expression of A2B5 (marker for O-2A precursor cells) or GFAP (marker for differentiated type-2 astrocyte) was identified by immunofluorescence staining. Rac1/Cdc42 expression and Rac1/Cdc42 activity were analyzed by western blot and Pull-down assay.Results (1) Cerebral cortex mixed glial culture system was established. After cultured for 6 day, glial cells were of the most abundant cell type in the primary mixed neuronal-glial cultures of newborn rat cortex and formed a cell monolayer. Neural precursor cells grew on top of the monolayer. The mixed cell cultures were positive for GFAP andβ-Ⅲ-Tubulin by immunofluorescence staining. After confluence of astrocytes, change of medium to SF resulted in a considerable proliferation of the neural precursor cells for up to 4 d and the differentiation of these cells afterwards, which was accompanied by varied expression level of Rac1 and Cdc42 detected by immunofluorescence and western blot. The expression of Rac1 and Cdc42 protein increased in the process of the neural precursor cell proliferation, but decreased when these cells began to differentiate. Rac1 and Cdc42 activity also increased in the process of the neural precursor cell proliferation and differentiation. (2) A lesion model of newborn rat cerebral cortex mixed glial culture was established. O-2A precursor cells moved into the lesion area and then differentiated type 2 astrocyte. Rac1 expression was always positive in this process by immunofluorescence staining. (3) O-2A precursor cells were successfully expanded in vitro and showed A2B5 expression with oval-shaped, bipolar morphology. In the presence of defined serum-free medium, these cells gave rise to oligodendrocytes and in the presence of fetal bovine serum, the type 2 astrocytes. The expression of Rac1 and Cdc42 protein decreased, while Rac1 and Cdc42 activity were upregulated during the differentiation of O-2A precursor cells.Conclusion Rac1 is closely related to the proliferation and differentiation of neural precursor cells. Rac1 is activated during the differentiation of neural precursor cells.