Molecular Genetics Study On Tumorigenicity Of Human Adult Bone Marrow-derived Neural Stem Cells

Recent studies have demonstrated that neural stem-like cells can be derived from human adult bone marrow stromal cells(hMSCs) and human adult bone marrow-derived neural stem-like cells(MDNSCs) have the similar characteristics with those of endogenous brain-derived neural stem cells(NSCs).MDNSCs can also potentially avoid many of the difficult issues surrounding the use of adult brain,fetal, or embryonic stem cell-derived NSCs.Their inherent characteristics and advantages have made them one of the most promising candidates in adult stem cell-based cell therapy for the treatment of human neurological disorders and injuries.However, before clinical application the long-term safety of this cell population upon transplantation must be thoroughly explored.Tumorigenicity of human adult stem cells is still a major concern.It was reported that cultured mouse bone marrow-derived stromal cells could show rapid proliferation,loss of contact inhibition and abnormal changes of karyotype,and transplantation of these cells into nude mice or syngeneic immunocompetent mice could induce tumor formations.Furthermore,there are many common characteristics between stem cells and cancer cells,and one hypothesis is that cancer cells may include 'cancer stem cells' - rare cells with indefinite potential for self-renewal that drive tumorigenesis.So,to evaluate the tumorigenicity of clinically applied stem cells seems to be quite necessary.Until now,there is still no confirmed evidence to exclude the possibility of malignant transformation in MDNSCs.In our pervious tumorigenic experiments on MDNSCs,no ConA-mediated agglutination or growth of cell colonies in double layers soft agar could be observed,and after implanted subcutaneously into BALB/c Nude Mice no tumor formation or any vegetation could be observed during the term of six months.We suppose that tumorigenicity of MDNSCs could not be excluded only by tumorigenic experiments in vitro or in animals because the survival conditions of MDNSCs in human body may be quite different from those in culture dishes or animals.After transplantation MDNSCs may survive in human body for an inestimably long time and undergo many changes. Whether they will undergo malignant transformation or not is still uncertain.To study the tumorigenic potentials of pre-transplanted MDNSCs may provide helpful information for evaluating their risk of malignant transformation during long-term survival in human body after transplantation.Since tumorigenesis could be attributed to genetic disorders,in the present study we investigated hundreds of cancer-related genes in MDNSCs in order to find out whether there are any characteristics that could help evaluate their tumorigenic potentials.ChapterⅠGeneration and identification of human adult bone marrow-derived neural stem cellsObjectiveTo explore the feasibility and efficiency of generation of human adult bone marrow-derived neural stem cells(MDNSCs) from human adult marrow stromal cell (hMSCs),and MDNSCs were identified by observing their morphology,expression of specific proteins and potency to differentiate into neural lingage cells. Materials and methodsThe hMSCs were harvested by density gradient centrifugation from human adult bone marrow cells obtained from normal adult volunteers,and cultured in the hMSCs medium to purify and proliferate,conversion of hMSCs into MDNSCs was conducted by culturing in the NSCs medium,MDNSCs were cultured in induction medium to differentiate in neural lineage cells,morphology of hMSCs and MDNSCs were observed by phase contrast microscope,Nestin staining was performed on MDNSCs,and GFAP,NG2 and MAP2ab stainings were performed on neural lingae cells differentiated from MDNSCs.ResultsAfter passages,hMSCs have a fusiformlike shape,adhere to the surface of tissue culture flasks with high proliferation ability,and do not significantly express the NSC marker Nestin.After 10-15 days of induction culture hMSCs were differentiated into MDNSCs.MDNSCs do not adhere to the surface of tissue culture flasks,form neurospherelike structures with high proliferation ability,and show high Nestin expression.After 10-15 days of induction culture MDNSCs were differentiated into astroglial,oligodendroglial,and neuronal cell types inviro.The cells were stained for markers for astrocytes(GFAP),oligodendrocytes(NG2),or neurons(MAP2ab).Conclusion1.hMSCs can be efficiantly differentaiated into MDNSCs in vitro.2.MDNSCs possess neural differentiation abilities and can be further differentaiated into neural lineage cells in vitro.3.hMSCs provide ample cell resources for MDNSCs.ChapterⅡMutational screening of important tumor suppressor genes in human adult bone marrow-derived neural stem cellsObjective To assess the tumorigenic potentials of adult human bone marrow-derived neural stem cells by analyzing the sequences of the frequent mutation sites of the important tumor suppressor genes p53,Rb1 and p16 in these cells.Materials and methodsThe sequence analysis of the exons 5～9 of p53 gene,the exons 19～21 of Rb1 gene and the exons 1～2 of p16 gene in the human adult bone marrow-derived neural stem cells were carried out by polymerase chain reaction(PCR) and DNA sequencing subsequently.ResultsThe sequences of the exons 5～9 of p53 gene,exon 19～21 of Rb1 gene and exons 1～2 of p16 gene in the adult human bone marrow-derived neural stem cells were all consistent with the wide types,without mutation found.ConclusionNo common mutation of the important tumor suppressor genes could be found in the adult human bone marrow-derived neural stem cells,which provided significant information for assessing the long-term tumorigenicity of these cells upon autologous transplantation.ChapterⅢInvestigation of the expression profile of cancer-related genes in human adult bone marrow-derived neural stem cellsObjectiveTo investigate the expression profile of cancer-related genes in MDNSCs in order to determine whether there were any characteristics that could help in the evaluation of their tumorigenic potential.Materials and methodsGrouping:the tested group:MDNSCs;the controlled group:fresh normal human adult bone marrow cells depleted of red blood cells(RBCs).The expression profle of 440 cancer-related genes were analysed by the Oligo GEArray Human Cancer Microarray OHS-802 and quantative real-time RT-PCR were performed on part of the important oncogenes with high expression as indicated by microarray test.ResultsOf the 440 cancer-related genes covered by Oligo GEArray Human Cancer Microarray OHS-802,63 were found to be significantly overexpressed in MDNSCs compared with that in fresh normal human adult bone marrow depleted of red blood cells(RBCs).In particular,the overexpressed genes included those promoting cell proliferation and cell invasion and metastasis and members of several oncogenic signaling pathways.The overexpression of MYC,MMP2,Notch2,STC1,ITGA3, STAT5b,RhoC,and Wnt1 in MDNSCs was also revealed by quantitative real-time RT-PCR,which was consistant with the reslts of microarray test,and the magnitude of the increased expression of these genes was:MYC,4.35×10~0;MMP2,2.84×10~0; Notch2,2.87×10~0;STC1,3.41×10~2;ITGA3,2.22×10~2;STAT5b,6.99×10~0;RhoC, 4.92×10~0;and Wnt1,3.64×10~0.Conclusion1.A number of cancer-related genes were found to be overexpressed in MDNSCs, and additional studies are needed to understand the possible biological significance of the molecular characteristics of this cell population.2.The overexpressed cancer-related genes include some important oncogenes, the activations of which have been proved to promote human tumorigenesis.3.Our findings provide important information for the evaluation of tumorigenicity in MDNSCs.4.Our findings highlight the need for further studies of long-term tumorigenicity in MDNSCs.