The Studies On Isolation And Differentiation Of Neural Stem Cells

IntroductionCentral nervous system (CNS) has limited ability to repair itself and the lost neurons can not be regenerated. Cell replacement therapy is one of the strategies to repair the damaged CNS. Neural stem cells (NSCs) represent one of the candidate cell types for cell replacement because they have the ability to proliferate continuously in vitro and can differentiate into multiple neural lineage. Strategies based on NSC fall into two categories: stimulating the endogenesis NSC to proliferate and differentiate or transplantation of in vitro expanded NSC to the injury site. The present dissertation includes series of studies on the isolation and differentiation of NSC.Study 1: Isolation of neural stem cells from the spinal cords of low temperature preserved abortusesIn order to find the right source for transplantation, neural stem cells have been isolated from various regions of human fetal and adult brain except for human adult spinal cord. The neural tissues were obtained freshly used for cell culture immediately in most of the studies or after short preservation in culture medium. However, we usually failed to obtain fresh tissues for some delays. Therefore we explore the possibility to isolate spinal NSCs from 14 weeks old abortuses after the preservation in a refrigerator for postmortem intervals. The clonal ability and differentiation potential of the isolated stem cells were analyzed.Study 2: Motor neurons differentiating from neonatal mouse multipotent telencephalon precursor cellsSignals in the local environment play an important role in determining the fate of the neural stem cells after the transplantation to CNS. Neural stem cells are more restricted in damaged CNS and differentiate mainly into glia rather than neuronal cells. These problems need to be solved before the application of neural stem cells for CNS repair. In order to obtain the desired neuronal types, neuronal restricted precursors were tried for transplantation. The precursors failed to mature into neurons expressing neural transmitters although they did not differentiate into glia. Hence, it seems necessary to induce NSCs to differentiate into desired neuronal types. Motor neurons represent one CNS neuronal subtype for which pathways of neuronal specification have been defined and motor neuron differentiation is a hot topic at present. Recently, many studies have obtained motor neurons differentiating from in vitro cultured cells including embryonic stem cells and embryonic neural stem cells. The present study aims at exploring the possibility of motor differentiation from neonatal mouse multipotent telencephalon precursor cells, searching new methods to elevate the differentiation efficiency and providing a new cell source for motor neuron differentiation study.Study 3: The effect of OMgp on neural stem cell differentiationNeural stem cells differentiated mainly into glia rather than neuronal cells when they were transplanted into non-neurogenic or damaged adult CNS. But they could differentiate into neurons when they were implanted into developmental CNS. There is not a convincing explanation for the phenomenon. The matured CNS has well developmental myelin sheath comparing with developing CNS. We doubt that myelin sheath may affect negatively the transplanted NSCs to differentiate into neurons since it has been documented as an axon growth inhibitor. The present study explore whether of not OMgp (oligodendrocyte-myelin glycoprotein) which is main component of myelin sheath has an effect on NSCs differentiation as well on stem cell derived neuronal axon growth. We also test if the effect of OMgp can be reversed by NGF or dbcAMP (dibutyryl cAMP). Materials and MethodsStudy 1: Fourteen weeks old abortuses were stored in a refrigerator at 4℃for 2 h, 6 h and 12 h before use. Neural stem cells were isolated from cervical cord, thoracic cord and lumbar/sacral cord separately and induced to differentiate with fetal bovine serum. Clonal culture was carried out to demonstrate that the isolated cells met the standard of stem cells. Fluorescent immunocytochemistry was used to examine the expression of neural stem cell marker (nestin), neuronal marker (MAP2), astrocyte marker (GFAP) and cholinergic marker (ChAT). The stem cells in different cultures were compared.Study 2: Multipotent neural precursor spheres were isolated and expanded from neonatal mouse telencephalon in serum free medium. They were induced to differentiate by using 5% fetal bovine serum after 8-10 d culture and RA (retinoic acid), Shh (sonic hedgehog) and dbcAMP were employed to increase the efficiency of motor neuron differentiation. Motor neuron marker MAP2, ChAT and HB9 were detected by immunofluorescence. Immunofluorescence colocalization of MAP2 and N-AChR in precursor cell and myoblast coculture demonstrate the formation of motor endplate. Pharmacological approaches were used to prove that the synapse could transmit excitement.Study 3: Neural stem cells were isolated and expanded from neonatal mouse telencephalon. 5% fetal bovine serum was used to induce stem cell differentiation. The differentiation cultures were grouped as follows: group 1 as negative control, group 2 supplemented with 50 ng/ml OMgp, group 3 supplemented with 50 ng/ml OMgp +100 ng/ml NGF, group 4 supplemented with 50 ng/ml OMgp + 1000μM dbcAMP. Fluorescent immunocytochemistry was used to examine the expression of neuronal marker MAP2 and astrocyte marker GFAP. The percentage of neurons and glia and the length of neuronal axons in different groups were compared. ResultsStudy 1: Neural stem cells were obtained from all the spinal cord segments with different postmortem intervals. The lumbar/sacral cord cultures gave rise to the most abundant primary neurospheres. When the preservation was prolonged to 12 h, the number of primary neurospheres decreased sharply. Neurospheres in all cultures showed nestin positive immunoreactivity and could yield GFAP positive astrocytes and MAP2 positive neurons including ChAT positive cholinergic neurons in differential cultures. The clonal formation and phenotype capacity were similar in all cultures.Study 2: Multipotent neural precursor spheres were isolated from neonatal mouse telencephalon and could proliferate continuously. They could differentiate into MAP2~+ neurons and GFAP~+ astrocytes. 4% of the differentiated cells were ChAT~+ cholinergic neurons. 1% of the differentiated cell expressed specified motor neuron marker HB9. The percentage increased to 5% when the inducing factors were added in. The differentiated motor neurons could develop neuromuscular junctions when cocultured with myoblasts and the synapse had the ability to transmit excitements.Study 3: The ratio of MAP2 positive neurons decreased from 13.85% to 3.40% and the ratio of GFAP positive astrocyte increased from 3.15% to 6.01% when OMgp was added into the differentiation cultures. The growth of neuronal axons was also depressed by OMgp. The effect of OMgp could be reversed by NGF or dbcAMP.DiscussionStudy 1: Clonal culture demonstrated the isolated cells in the present study were genuine neural stem cells as they were self-renewal, expandable and could differentiate into the main cell types in the CNS. The fact that more primary neurospheres were obtained from sacral/lumber segment indicates that more responsive stem cells exist in this segment or survived the preservation. The number of neurospheres decreased as the preservation period was prolonged, which indicates that more stem cell died. The cells obtained from all the spinal cord segments may be the same cells in essence as they had very similar clonal and differentiation capacity.Study 2: The isolated and expanded cells met the standards of multipotent neural precursor because they could proliferate continuously and differentiate into neurons and astrocytes. Some of the differentiated cells expressed specified motor neuron marker HB9, which indicated motor neuron differentiation. The differentiated motor neurons were functional because they could develop neuromuscular junctions when cocultured with myoblasts and the synapse had the ability to transmit excitements.Study 3: OMgp had an effect on neural stem cell differentiation by increasing astrocyte lineage differentiation while depressing neuron lineage differentiation. NGF could reverse the OMgp effect. The fact that dbcAMP had antagonism action similar to NGF indicated that NgR might be involved in the pathway.ConclusionsStudy 1: Spinal neural stem cells can be isolated from low temperature preserved abortuses. Sacral/lumber segments give rise to the most abundant stem cells and the number of neurospheres decreases as preservation time goes on.Study 2: Neonatal mouse multipotent telencephalon precursor cells can give rise to functional motor neurons. Inducing factors can elevate the efficiency of motor neuron differentiation.Study 3: OMgp has an effect on neural stem cell differentiation by increasing astrocyte lineage differentiation while depressing neuron lineage differentiation. NGF or dbcAMP could reverse the OMgp effect.