Bone Marrow Derived Neural Progenitor Cells Differentiate Into Neurons And Promote Neurogenesis Of Brain Injury Rats

Background and purposeResearch on stem cells and regenerative medicine is the most popular in today's life science frontiers,stem cell transplant treatment of diseases of the central nervous injury in experimental study has made great progress at home and abroad.Mesenchymal stem cells(MSCs)is a kind of stem cells,the most studied 2014 global Clinical translational research on MSCs as many as 409 projects(WWW.Clinical Trial.gov),the United States approved more than 40 items in the brain injury in Clinical trials.In 2016,the China society for neuroscience branch nerve damage and repair brain damage nerve function damage and repair expert consensus,mesenchymal stem cells as a repair will be promising treatment strategies for the recovery of brain damage.One of the reasons is that MSCs has strong proliferation and multi-directional differentiation potential,in appropriate environment they can be differentiated into neurons and glial cells of ectoderm cells in vivo or in vitro,and play a role of nerve repair.However,there are some problems in the research of MSCs,such as stem cell proliferation ability is limited,the lack of MSCs in intracranial long-term survival and to participate in the direct evidence of nerve regeneration.In the study domain of MSCs,use the most is source of bone marrow mesenchymal stem cells(BM-MSCs).Although in recent years,many studies support that BM-MSCs can cross layer differentiate to neural cells,but less reseachers can detecte mature nerve cells and function nerve cells,especially in vivo study.Even many research sugeested,BM-MSCs transplantation will not be able to differentiate into functional neural cells,have not cell replacement effect,and the main role of promoting neural functional recovery is raised by inhibiting apoptosis,regulate the body's immune to reduce inflammation,and so on.Our previous study found that the nerve cell culture environment,in the process of inducing bone marrow derived nerve cells may exist a phase of bone marrow derived neural progenitor cells,BM-NPCs.BM-NPCs might be more suitable than BM-MSCs as seed cells of cell transplantation,give play to the role of cell replacement therapy in the central nervous injury disease.Therefore,how to better induce BM-MSCs directional differentiation into functional neural cells,looking the convincing proof for BM-NPCs,and observing the bone marrow derived neurons in intracranial survive for a long time,and participating in nerve regeneration,are problems to be solved in clinical cell transplantation for treating central nervous injury.Looking for BM-NPCs,discusing BM-NPCs purification culture scheme,proving that functional bone marrow derived neurons can survive for a long time in intracranial environment and participate in nerve regeneration,will provides valuable experimental data for BM-NPCs in the central nerve regeneration application.The first part Obtaining bone marrow derived neural progenitor cellsObjective According the method of neural stem cell suspension culture,obtain bone marrow neural progenitor cells(BM-NPCs),and analysis BM-NPCs cytological characteristics.Methods Using the whole bone marrow adherent culture method to separate rats BM-MSCs,observing the original generation and extending cell morphology and proliferation characteristics,using flow cytometry to detect the surface markers.Moving the third generation of BM-MSCs to serum-free medium of neural stem cells culture medium Neurobal-A with 1% N2-supplement,2% B27,2 mmol/L L-glutamine and 20 ng/ml b FGF&EGF in suspension culture bottles induction.After48 h,there were cells in suspended growth,using Accutase TM enzyme digestion batches,some of these cells has the ability of proliferation as a ball suspension growth.The third generation of bone marrow-derived sphere-like cells were prepared.To detect the source of bone marrow cells of the cell cycle by flow cytometry,using cell immunofluorescence and RT-PCR method for identification of cell differentiation potential and neural progenitor cells related gene protein expression,and then carried out a concomitant ability test.Results The project training of BM-MSCs in accordance with the international appraisal standard of MSCs,identified by the flow cytometry CD34/45/3/4/11b/14/133(-)and CD29/105(+).Through the method of neural stem cell suspension culture,represented amplification of bone marrow-derived sphere-like cells for several times,measured by the flow cytometry cycle,79.2% of the third generation of sphere-like cells was in G0/ G1 phase;Cell immunofluorescence appraisal protein Semi-quantitative RT-PCR detected sphere-like cells m RNA level,according to the results of pluripotent stem cell gene characteristics strong expression of c-myc / klf4 / sox2,express the Sca-1 /oct4,stronger expression Muashil1 / CD184 / CD133,expressing CD56 / Nestin /Muashil2 / Notch1 neural progenitor cells genetic characteristics,at the same time,a concomitant differentiation ability of lipid drops with staining positive.Conclusion Using the method of neural stem cell suspension culture,bone marrow sphere-like cells were obtained from the BM-MSCs,expressed neural progenitor cells gene and protein expression,have tendency to nerve cell differentiation and neural progenitor cells characteristic,but retained multi-directional differentiation potential.The second part Bone marrow neural progenitor cells differentiation into neuron-like cellsObjective Through direct adherent differentiation and neuron co-culture induction method,to analyze the differentiation ability of BM-NPCs to nerve cells,to detect genetic level changes in the process of cell differentiation,for providing the basis data for the functional differentiation of neurons.Methods The third generation of BM-NPCs from BM-MSCs cultured in neurons medium for 15 d,then by immunofluorescence technique analysis of neuronal markers Tuj-1 /NF200 and glial cell marker GFAP,using semi-quantitative RT-PCR and quantitative q PCR to detect m RNA expression level before and after inducing BM-NPCs about neural stem cell marker genes(Nestin / NCAM1 / CD133),the nerve cells marker genes(the beta-III-tubulin / Neun / 5-HT / ACHE / GABA,and CNPase and neurotrophic factor gene NGF / BDNF / GDNF gene.In addition,the CM-Dil cells tracer tagged BM-NPCs cocultured with the original generation cortex neurons for 15 d,using inverted microscope and immunofluorescence observations to detect neuronal markers Tuj-1 expression.Results BM-NPCs stick wall directly induced bone marrow derived nerve cells,neuron-like cells can be observed after 10 d,some of the cells as glial cells,and linked with each other to grow;continue to induce 5 d,a typical form of nerve cells,similar to normal cortex neuron cells form and completely different from BM-MSCs can be observed.These nerve-like cells can express some nerve cell phenotype Tuj-1(+)/ NF200(-)and GFAP(+)/ S100(+),and the expression of beta-III-tubulin(+)/ Neu N /5-HT(+)/ ACHE(+)/ GABA(-)and CNPase(+ +)/ NGF(+ + +)/ BDNF(+)/GDNF(+)genetic characteristics.Quantitative gene expression results shew BM-NPCs were higher expression of neural progenitor cell gene NCAM1 and CD133 compared to BM-MSCs.After differentiation of stem cells,the Nestin,NCAM1 and CD133 gene expression decreased obviously,as the sample cells to differentiate into different nerve cells,beta-III-tubulin / Neu N / 5-HT / ACHE gene expression also decreased obviously,which suggested in our experiment stick wall differentiation environment was not conducive to neuron differentiation,so,unable to form mature neurons with neurotransmitter expression,cells cultivation more longer may be to more easy to glial cell differentiation,because glial cell were more easy survival and proliferation,then with higher CNPase expression and NGF nutrition factor gene level increased significantly.CM-Dil cells after tracer tagged BM-NPCs in the more suitable environment for neuronal growth,co-culture with the original generation of cortex neuron cell,B-NPCs can be differentiated into more typical neuron morphological characteristics,with Tuj1 fluorescent protein positive expression,and normal neural network connected into the growth cells.It suggested the more appropriate growth environment for neurons,BM-NPCs might have the capacity to differentiation into mature functional neurons.Conclusion The experiment in vitro induced neuronal cells of bone marrow cells,the cells similar to fully mature neurons cell form,but there still exists certain different in the gene expression.BM-NPCs cultured with suspension method have more ability to differentiate into nerve cells compared with BM-MSCs.BM-NPCs in the suitable environment may have the ability to functional neural differentiation,and play a role in the central nervous system injury disease.The third part The evidences of long-term survival of bone marrow derived neurons involved in nerve regenerationObjective To search for the evidences about bone marrow derived neurons can long-term survival in the brain injury rats,and participate in nerve regeneration of brain damage,to provide valuable experimental basis data for BM-NPCs transplantation treatment of central nervous injury disease.Methods Brain injury rat models after 7 days,random group into cell group(n=20)and control group(n=20),cell group rats tracer injected with CD-Dil tagged BM-NPCs10ul(1 million)through the micro syringe transplantation to cerebral injury rats,under the condition of same set as control group with injecting imedium.Movement function Wayne clark test and grooming test were carried out,respectively after transplantation of 1 d,3 d,7 d and 30 d and 60 d.At the same time,7 d,30 d,60 d and 90 d after transplantation,brain tissue pathological conditions were detected, using immunofluorescence test to analayze CM-Dil tagged BM-NPCs migration in brain injury and differentiation nerve cell markers Neu N and GFAP.Results(1)HE staining showed: building 7 d,groups of rats damage focal fractured surrounding tissue,and blood vessel compression deformation,reduce blood flow,swelling of the nerve cells necrosis,degeneration.7 d cell transplantation,the control group damage surrounding tissue with edema,oven visible cystic cavity,significantly reduced the number of nerve cells,inflammatory cells infiltration around,cell group edema,lighter,cystic cavity range limit,glial cells.Transplantation for 30 d,focal brain injury recovered from the surrounding tissue,compared with the control group,cell group cystic cavity was small,and the surrounding cells row of class was neat,tissue edema and inflammatory cells dispeared.(2)the organization immunofluorescence results: 7 d of transplantation,the Dil labeled cells transplanted into the area of injury around brain tissue damage,but did not see Dil+ Neu N cells.Transplantation for 30 d,brain damage tissue around GFAP positive astrocytes,some Dil+ cells,in the region of the hippocampus and cerebral cortex neurons,with normal nerve cells express integration expressed Neu N.After 90 days,cell group was still visible CM-Dil tagged positive cells expressed Neu N,which integrated in normal nerve cells in the brain tissue,and the damage zone of the surrounding tissue growth.(3)the behavioral science score,1 d,two groups of Wayne clark and grooming score results comparison were no significant difference(P > 0.05);Transplantation for 3 d,7 d,30 d,60 d,Wayne clark,grooming score results had significant(P <0.05),cell group had better functional recovery.Conclusion BM-NPCs transplantation can promote brain injury of limb motor function recovery in rats.We successfully find evidences of the bone marrow derived nerve cells long-term survival in intracranial,the bone marrow derived neurons can integrate into damage brain and participate in nerve regeneration.