The Effects Of Neurotrophic Factors On Differentiation Of Neural Stem Cells And Marrow Stromal Cells From Rat

Objective To investigate the effects of neurotrophic factors on differentiation of neural stem cells and marrow stromal cells from rat and to understand the possibilities of the applications of NSCs and MSCs in clinical therapy for central nerve system injury.Methods (1) NSCs were isolated from postnatal day 2~3 rat cerebral cortex using serum-free neurospheres method, and NSCs in different culture intervals were identified by BrdU incorporation, with selective marker nestin, and the abilities of differentiation into neurons (MAP2 or β-tubulinⅢ positive), astrocytes (GFAP positive) and oligodendrocytes (CNPase positive). The percentages of neurospheres expressing nestin in different culture intervals were measured by flow cytometry (FCM) and the ultrastructure of these neurospheres was examined by electron microscopy. (2) Neuronal induction on neural stem cells was examined using BDNF, GDNF, PDGF and forskolin, and immunofluoresence staining of neural stem cells after induction was carried out. The variation of TrkB receptor in cultured NSCs over time was detected by RT-PCR and Western blot. (3) The survival, migration, and differentiation of transplanted cells from cultured 1 month old NSCs in normal and chronic ischemic adult rat cortex or hippocampus were observed by double staining of BrdU and P-tubulinⅢ in brain slices. (4) MSCs were isolated from adult rat and identified by cell surface markers CD11b, CD90, CD44, CD70 and CD45. MSCs were treated with P-mercaptoethanol, BDNF, GDNF, NGF, PDGF and forskolin, and identified their differentiation by immunofluoresence staining. The survival, migration, and differentiation of transplanted MSCs in normal adult rat cerebral cortex were observed.Results (1) The NSCs isolated from postnatal rat cortex can be grown in vitro inneurospheres. Cultured neurospheres were immunopositive to BrdU and nestin. The neurospheres differentiated into neurons, astrocytes, and oligodendrocytes after induction. The percentages of nestin positive cells in 7 days, 1 month and 3 months old neurospheres were 42.2 ± 7.6, 41.7 ± 7.3 ft 33.8 ± 12.9 respectivily. Some differentiated neural cells, apoptotic and necrotic cells were observed in the ultrastructure of neurospheres. (2) The percentage of MAP2 positive cells in one month old NSCs increased about 6 times compared to the control after neurotrophic factors induction (p<0.001), and BDNF was the most effective among them. About 90% MAP2 positive cells were also GABA positive. Only a few MAP2 positive cells were seen in 3 month old NSCs by combination of BDNF and forskolin. The results of RT-PCR and western blot showed that the expression of mRNA for the TrkB receptor in 3 months old NSCs decreased 3 times compared to that of 1 month old NSCs. (3) Some NSCs migrated away from injection site 4 weeks after cultured 1 month old NSCs transplanted into adult rat cortex and hippocampus. There were some anti-BrdU/anti-P-tubulinlll double positive cells (NSCs differentiated into neurons). BDNF enhanced migration of NSC, but there were no differences in differentiation into neurons when transplanted NSCs into adult rat brain with or without BDNF. (4) Cultured MSCs were exposured to P-mercaptoethanol, BDNF, GDNF, NGF, PDGF and forskolin, there were no obvious effects on differentiation into neurons and glia. MSCs migrated after transplantation into normal adult rat cortex for 4 weeks, but there were no anti-BrdU/anti-P-tubulinlll or/and anti-BrdU/anti-GFAP double positive cells (MSCs differentiated into neurons or glia) could be seen in brain slices.Conclusions NSCs isolated from postnatal rat cortex retained equilibrium of proliferation and differentiation under our experiment condition, and maintained relative stable NSCs population. Neurotrophic factors, including BDNF, GDNF, PDGF and forskolin, could induce neuronal differentiation of cultured 1 month old NSCs, and BDNF was the most effective among these neurotrophic factors. However there were very week effect on differentiation of 3 months old NSCs when combination of BDNF and forskolin, which might relate to decrease of theexpression level of TrkB mRNA. Transplanted NSCs could survive, migrate, and differentiate into neurons after implanted 1 month old NSCs into normal and chronic ischemic adult rat cerebral cortex and/or hippocampus for 4 weeks. The range of migration was related to the implanted area with or without injury. BDNF promoted migration of transplanted NSCs. In our experiment conditions, cultured MSCs didn't differentiate into neurons and glia after induction with P-mercaptoethanol, BDNF, GDNF, NGF, PDGF and forskolin. Although MSCs survived and migrated after transplantation into normal adult rat cerebral cortex for 4 weeks, they could not differentiate into neurons and glia.