Tropism Of Neurally Differentiated Bone Marrow Mesenchymal Stem Cells To C6Glioma

In neurosurgery field, glioma is one of the most challenging diseases towhich neurosurgeon have to face. Gliomas, as the most common tumors incentral nervous system, account for45%of primary tumors are gliomas. Surgery,chemotherapy and radiotherapy are current clinical treatments of glioma.Although these treatments can produce some effect, the prognosis of glioma isstill not optimistic. Gene therapy is a prospective treatment, but a series problemssuch as toxicity of viral vectors during carrying the target gene, a short cycle oftarget gene expression, and targeting, immunogenic, and tropism to tumor cell,limit its application in clinical practice.Bone marrow mesenchymal stem cells (BMSCs) are adult stem cells withself-renewal and multi-differentiation potential, and act as a new carrier derivefrom the mesoderm, which has aroused the concern of scientists. Compared withneural stem cells (neural stem cell, the NSC) and embryonic stem cells(embryonic stem cell, the ESC), BMSCs have advantages, such as readilyavailable, easy cultivation, low immunogenicity, and no ethical issues, which has become a hot topic of research about stem cells in recent years. According to thebiological characteristics and tropism to tumor of BMSCs, researchers focus onits application in target therapy of gliomas. As the basis of the follow-up clinicalstudy, separation, cultivation, identification, and differentiation of BMSCs hasbeen the important issue in basic research.In the process of migration in vivo, BMSCs might be induced todifferentiate by surrounding microenvironment of the brain tissue, which affectsits migration and effects of therapy. We hypothesize that BMSCs differentiationin vitro by mimicing brain micro-environment would be a solution for aboveproblem. BMSCs differentiated neural cells (neurally differentiated bone marrowmesenchymal stem cells, ND-BMSCs) can partially simulate intracranialmicro-environment, but its tropism to glioma is still unclear. If N-D-of BMSCscan migrate to glioma, the mechanism underlying this process is unkown. Thus,on the basis of previous studies on NSC and BMSCs differentiation, presentstudy compared BMSCs differentiation to neural cells in vitro by nutrient factorsand chemicals, and then investigated the tropism of N-D-BMSCs to C6gliomacells and the involvement of VEGF and MMP in migration of BMSCs. In view ofthe above ideas, our study are as follows:Frist part:Bone marrow stromal stem cells into neural cell differentiationObjective: To observe the two methods of inducing rat bone marrowmesenchymal stem cells (BMSCs) to neural cells in vitro. Methods: BMSCswere culture and identified after obtained from SD rats femur bone, There weretwo different methods for induction: method one (induction by nutrientfactor):BMSCs were cultured with B27, basic fibroblast growth factor (bFGF),epidermal growth factor (EGF), brain-derived neurotrophic factor(BDNF), andalpha-minima essential medium(α-MEM) containing10%fetal bovine serum (FBS); method two(induction by chemicals): BMSCs were cultured with B27,ATRA, N5,O2’-dibutyryladenosine3’:5’-cyclic phosphate Cyclic AdenosineMonophosphate(Bt2CAMP) α-MEM containing10%FBS. Control group:α-MEM containing10%FBS of complete culture medium. The differentiatedBMSCs were identified by morphology and immunofluorescence staining.Results: After the induction by two methods, cell body retraction and synapticstretch of BMSCs were observed, which showed the nerve cell morphology.Immunofluorescence staining showed that NeuN (mature neural cell markers)-,NF-H (markers of neural cytoskeleton)-, and Nestin (precursor cellmarkers)-positive cells were observed after induction by two methods. Methodsof Expression of Nestin (16.37±0.31)%in method one was higher than that ofmethod two (10.58±0.16)%. Expression of NeuN,(43.53±1.34)%and NF-Hmethod (40.76±0.53)%in method one were lower than that of method two (NeuN,47.63±1.06%and NF-H,51.39±0.37%). Conclusion: Both methods can inducethe differentiation of bone marrow-derived mesenchymal stem cells into nervecells in vitro. The method one produce more neural precursor cells, which may becloser to intracranial micro-environment; method two produce more mature nervecells.Second part: The tropism of bone marrow stromal cells-derived neural cellstowards C6gliomaObjective: To investigate the tropism mechanism underlying bone marrowmesenchymal stem cell differentiation in nerve cells (ND-BMSCs) to C6gliomacells in vitro and in vivo. Methods: In vitro migration assay was employed bytranswell co-cultured with BMSCs-derived neural cells and C6glioma-conditioned medium (C6GCM), the number of cells which crossed thepores of polycarbonate membrane was analyzed by HE staining. In vivo migration assay was carried out with BrdU labeled BMSCs-derived neural cellstransplantation to the C6glioma model, immunohistochemistry was carried out toevaluate the tropism. Content of VEGF in C6GCM was assayed by using ELISAkit. After transwell co-cultured with N-D-BMSCs and C6glioma-conditionedmedium (C6GCM) with VEGF antibody, the number of cells which crossed thepores of polycarbonate membrane was analyzed by HE staining. MMP activity inBMSCs medium was assayed by gelatin zymography. Results: In vitroexperiments showed that the ND-of BMSCs significantly tend (P<0.01) in C6glioma conditioned medium; in vivo transplantation of BrdU-positive cells weremigrating to the tumor site and presented a time-specific; VEGF could bedetected in the C6GCM; tropism of N-D-BMSCs was reduced by GCM withVEGF antibody; MMP could be detected in the C6GCM. Conclusion: Thesignificant tropism of N-D-BMSCs to C6glioma is observed in vitro and in vivo.VEGF has a critical role in tropism of N-D-BMSCs to C6glioma. MMP might beinvolved in the migration of N-D-BMSCs to C6glioma.