The Research On Stem Cells Differentiate Into Neural Cells

ObjectiveThe study on induction of embryonic stem cells (ESCs) into neurons is valuable for the future application of cell transplantation, which may be a useful treatment for the patients of central nervous system disorders, such as the Pakin-son's disease, stroke, myeleterosis. It's also a perfect model in vitro of the mammal neural system development. Because of its easy genetic manipulation, gene expression and regulation can be studied conveniently.It was known that neural cells are terminal differentiation cells, which can not be supplied by neural stem cells once they are injured. Therefore, the neuron of central nervous system does not regenerate once it is injured, and especially neuron in the mammalian animals is lack of the capability of regeneration.But in the 1980's, studies were reported that transplantation of peripheral nerves and fetal spinal cord could cause injured axons of spinal cord to regeneration. Then, many studies have showed that regeneration of the injured spinal cord might really be possible. Recently, the study shows that neural stem cell can replace the injured neuron of central nervous system. The stem cells from other resource, such as mesenchymal stem cells (MSCs) , embryonic stem cells (ESCs), under specific conditions differentiate into neurons. But the proportion of neural differentiation in vitro is still very low.Transplantation of MSCs into the spinal cord after a contusion injury was also reported to enhance functional recovery. MSGs were injected into the spinal cord immediately after injury directly or after cocultured with neurosphere cells. Treated animals generally showed better performance of gait than control animals. MSCs at the centre of the injury appeared to pull the surrounding host tis-sues toward the center, preventing the formation of cavities. Although too many studies have shown that MSCs have ability to partly fix up anatomy connection of injured spinal cord, at present, there isn't enough evidence to show that whether MSCs can promote recovery of conduction function in the injured spinal cord or not.Here we report a simple effective and nontoxic method to induce ESCs into neurons, and through transplantation of stem cell, invest how to induce ESCs into nerve cells in vivo. With the help of gene chip analysis we can confirm the result of induction and find some master genes in neural differentiation and research mechanism of how to induce ESCs into nerve cells.Methods1. Isolation, culture, identification and neural differentiation of MSCs1.1 Under sterile conditions collect bone marrow cells from the tibias and femurs of the Wistar rats as primary culture.1.2 According to ability of adherence, sieve easily adherent but not stable cells to subculture.1. 3 Immunocytochemistry identification of the expression of CD45 and CD90 when subculture.1.4 Inducing MSCs to differentiate into neural cells: |3 - mercaptoethanol + DMSO + lower concentration of serum.2. Culture, identification and neural differentiation of ESCs.2.1 Alkaline phosphatase test of undifferentiated ESCs by Ca - Co.2.2 Immunostaining of neural specific antigens: Nestin, NSE, NCAM1, NF-LandGFAP.2.3 ESCs line (AB2.1) was routinely cultured on feeder cells. When induced differentiation, the cells are removed from feeder layer cells and replanted on none - coated tissue culture plastic dishes in NSC medium. The medium was gradually changed into serum - free NSC medium.3. MSCs were induced to differentiate into neural cells in vivo. 3.1 MSCs are labeled with BrdU in vitro before transplantation.3.2 Transplantation of MSCs3.2.1 Preparations of rats model of spinal cord half - transection injury3.2.2 Inject MSCs into the spinal cord injury site immediately after injury in experimental groups (12 rats) , but PBS are injected in control groups (12 rats).3.3 Result assessments3.3.1 Functional evaluation using the open-field BBB scoring system3.3.2 Fixation of the rat's spinal cord by heart perfusion3.3.3 Immunohistochemistry identification of BrdU to show whether MSCs survive or not after transplantation3.3.4 HRP antitracting 48 hours before collect spinal cord sample3.3.5 Examine the formation of neuronal circuits by HRP staining.3.3.6 Examination of the reconstruction of neuronal circuits by CSEP 4. Expressing gene chip analysis.Using Affymetrix MOE430A mouse expressing gene chip, on the state of undifferentiating, the fourth and tenth day after inducing, analyze gene expression spectra.5- mRNA level identification of ESCsN neural stem cells and neural related genes by semi - quantities RT - PCR analysis.6. TRAP assays.7. Statistics analysis.Results1. Isolation, culture, identification and neural differentiation of MSCs1.1 Immunocytochemistry showed that MSCs express CD45( - ) and 70% CD90( +).1.2 After preliminary induction of MSCs and serum - free medium to be replaced, the cells were similar to nerve cell emerge, which express NSE at 80 percent.2. Inducing MSCs differentiate into neural cells in vivo2.1 Treated rats generally showed better motor function recovery of hindlimb than control rats. After 1 week> 2 weeks%3 weeksS4 weeksX8 weeks, BBB scoring of treated rats is much higher than control rats.2.2 BrdU positive cells could be found in the spinal cord injury site one week after transplantation..2.3 Two months after transplantation, HRP positive cells could be found at rostral of the spinal cord injury site of treated rats, but not be found in control rats.2.4 CSEP could be evoked from treated rats two months after transplantation, but not be obtained from control rats.3. ESCs can be induced into neuron -like cells in vitro.The undifferentiated ESCs grew in colonies, with clear bound and compact arrangement.Alkaline phosphatase staining show dark brown hadro - positive particles were distributed. After the sequential neural induction, the cells converted into neuron - like cells, with homogeneous forms, which had round light cell bodies and thin long bipolar or multipolar processes. The differentiated cells were im-munopositive for many neural markers, and over 80% induced cells were NSE and NCAM -positive. Less than 1% GFAP - positive cells were detected.4. Research mechanism of how to induce ESCs into neuron - like cells in vitro10 days after neural induction, gene expression pattern was analyzed by gene chips, which showed the expression of some neural specific and related genes, such as nestin%sox2>cdk5>Dnm6AxNSExNF - M%NCAM1XMAP2, et al. Especially the expression of GABAr and Glutamate dehydrogenase (GAD) , which meant the induced cells could be GABAnergic neurons. The astrocyte marker GFAP and oligodendrocyte marker Mbp were not detected. No the endo-derm and mesoderm markers. Semi - quantities RT - PCR also confirmed the a-bove results.The undifferentiated ESCs showed strongest telomerase activity. It decreased during differentiation. Until on the tenth day, it was almost invisible, but the telomerase activity of the PMEF - P3 was still lower than it.Conclusions1. Using beta - mercaptoethanol and serum - free medium which induce MSCs differentiation to nerve cell, we have differentiated rate rising to 80%.2. MSCs may survive in the spinal cord injury site by local injection immediately after spinal cord injury and then differentiate into neurons to form neuro-nal circuits in the injury site, set up connection between distal end and proximal end in the injury site to recover the conduction function of spinal nerve fiber, and may promote regeneration of the injured axons.3. The sequential neural induction can convert ESCs into neurons effective-ly.4. With the help of gene chip analysis, more neural differentiation related or specific genes can be discovered.