成人骨髓源性神经干细胞致瘤性的分子遗传学研究
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摘要
最近研究表明,成人骨髓基质细胞在体外可诱导分化成为神经干细胞,并且成人骨髓源性神经干细胞具有和自身脑源性神经干细胞相似的生物学特征。成人骨髓源性神经干细胞的应用可以避免诸如取材困难、免疫排斥及伦理学问题等困扰着脑源性、及胚胎源性神经干细胞应用所遇到的各种难题。骨髓源性神经干细胞自身具有的特征及优势使其在应用成体干细胞治疗人类神经系统疾病及损伤方面具有良好的临床应用前景。然而,在临床应用前对于成人骨髓源性神经干细胞在移植后的长期安全性问题是一有待深入探索的研究课题之一。
致瘤性是成体干细胞临床应用安全性方面一个重要的关注问题。有研究表明,体外培养的小鼠源性的骨髓基质细胞可表现出快速增殖、失去接触抑制及异常的细胞核表型改变等生物学特征,并且在将这些细胞移植于裸鼠或同系基因型的免疫缺陷小鼠体内后可导致肿瘤生成。再者,干细胞与肿瘤细胞有着很多相似的特征,有一理论假说认为肿瘤细胞中包含了少量被称为肿瘤干细胞的细胞,它们有着无限的自身增殖能力,并且可以导致肿瘤形成。因而,对临床应用的干细胞的致瘤性研究显得非常重要。目前为止,并没有明确的证据证明成人骨髓源性神经干细胞不存在致瘤性的可能。而对于成人骨髓源性神经干细胞的致瘤性问题,在前期工作中我们进行了染色体分析、体外凝集试验以及裸鼠成瘤试验等一系列研究,发现成人骨髓源性神经干细胞染色体形态、数目及结构正常,体外无凝集反应,接种裸鼠6个月无肿瘤形成,为评价其致瘤性提供了初步的实验依据。我们认为,对于成人骨髓源性神经干细胞的致瘤可能性不能仅依据体外实验或动物实验而完全排除,因为成人骨髓源性神经干细胞在人体内的生长条件和在培养皿中或动物体内存在很大的区别。在移植之后,成人骨髓源性神经干细胞在人体内可能存在一段难以估计的较长时期,在这段时期其可能发生很多变化,因而对于成人骨髓源性神经干细胞在人体内是否会发生恶性转变目前仍然还不能确定。对移植前的成人骨髓源性神经干细胞的致瘤性进行研究,能为评估其在移植后于人体内长期存在的过程中发生恶性转变的风险提供有意义的参考依据。
因为致瘤性可归因于遗传异常,在本研究中我们对成人骨髓源性神经干细胞中数百个肿瘤相关基因进行分析,目的是为了明确该细胞中是否存在某些基因特征可以有助于对其致瘤性的评价。
第一章成人骨髓源性神经干细胞的获得与鉴定
目的
建立一种快速有效的由成人骨髓基质细胞在体外诱导培养获得成人骨髓源性神经干细胞方法,并从细胞形态、标志性蛋白的表达及体外诱导分化成为神经系细胞的能力等方面对成人骨髓源性神经干细胞进行检测。
材料与方法
抽取正常成人志愿者全骨髓,应用密度梯度离心法分离成人骨髓基质细胞,体外培养纯化及扩增成人骨髓基质细胞,以神经干细胞培养基体外诱导培养成人骨髓基质细胞成为成人骨髓源性神经干细胞,以诱导培养基于体外诱导培养成人骨髓源性神经干细胞向神经系细胞分化。相差显微镜下观察成人骨髓基质细胞及成人骨髓源性神经干细胞的形态学特征;免疫组化法检测成人骨髓源性神经干细胞中神经干细胞标志性蛋白Nestin的表达,以及由成人骨髓源性神经干细胞诱导分化后得到的神经系细胞中神经胶质细胞标志性蛋白GFAP、少突胶质细胞标志性蛋白NG2及神经元标志性蛋白MAP2ab的表达。
结果
传代纯化后的成人骨髓基质细胞贴壁生长,呈梭形,贴壁生长,增殖旺盛,几乎不表达Nestin。经10～15天诱导培养,成人骨髓基质细胞分化为成人骨髓源性神经干细胞,成人骨髓源性神经干细胞由细胞克隆聚集成神经球样结构,不贴壁生长,增殖旺盛,高度表达Nestin。经7～10天诱导培养,成人骨髓源性神经干细胞体外分化成神经系细胞,细胞可表达GFAP、NG2及MAP2ab。
结论
1.成人骨髓基质细胞可在体外快速诱导分化成为成人骨髓源性神经干细胞。
2.成人骨髓源性神经干细胞具有神经分化的能力,在体外可进一步诱导分化成为神经系统的细胞类型。
3.成人骨髓基质细胞可为成人骨髓源性神经干细胞提供丰富的细胞来源。
第二章成人骨髓源性神经干细胞中重要抑癌基因的突变检测
目的
对成人骨髓源性神经干细胞中重要抑癌基因p53、Rb1和p16基因的常见突变点进行序列分析,观察是否存在异常的突变现象,从抑癌基因水平研究成人骨髓源性神经干细胞的致瘤性问题。
材料与方法
提取成人骨髓源性神经干细胞的基因组DNA,应用PCR-DNA测序技术,设计特定PCR引物,PCR扩增成人骨髓源性神经干细胞p53基因第5～9外显子、Rb1基因第19-21外显子以及p16基因第1～2外显子,并对扩增片段进行DNA测序。
结果
成人骨髓源性神经干细胞中p53基因第5～9外显子、Rb1基因第19～21外显子以及p16基因第1～2外显子的序列均与野生型一致,未见突变现象。
结论
由成人骨髓基质细胞体外诱导培养形成的成人骨髓源性神经干细胞未发现重要抑癌基因的常见突变现象,此结果为评价成人骨髓源性神经干细胞体内移植的致瘤性提供了有意义的参考依据。
第三章成人骨髓源性神经干细胞中肿瘤相关基因的表达检测
目的
检测成人骨髓源性神经干细胞中肿瘤相关基因的表达情况,为评价其移植后的致瘤性提供依据。
材料与方法
实验分组:实验组:成人骨髓源性神经干细胞;对照组:新鲜正常成人骨髓细胞去红细胞。提取总RNA;以肿瘤基因芯片Oligo GEArray Human CancerMicroarray OHS-802检测成人骨髓源性神经干细胞中440个肿瘤相关基因的表达水平;以实时定量RT-PCR的方法对成人骨髓源性神经干细胞中基因芯片检测结果显示呈高度表达的部分重要癌基因进行检测。
结果
基因芯片检测结果显示,与新鲜正常成人骨髓细胞去红细胞相比较,成人骨髓源性神经干细胞中有66个肿瘤相关基因呈高度表达(＞3倍),呈高度表达的基因中包含了促进细胞增殖、促进细胞侵袭和转移及一些重要致癌性信号传导通路的成员等基因。实时定量RT-PCR检测结果显示,与新鲜正常成人骨髓细胞去红细胞相比较,所检测的癌基因MYC、MMP2、Notch2、STC1、ITGA3、STAT5b、RhoC及Wnt1在成人骨髓源性神经干细胞中均呈高度表达,与基因芯片检测的结果相符,其表达倍数分别是: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)、Wnt1(3.64×10~0)。
结论
1.成人骨髓源性神经干细胞中存在大量的肿瘤相关基因的高度表达,其可能的生物学意义有待进一步深入研究。
2.高度表达的基因中包括了一些重要的癌基因,其激活与人类肿瘤的发生发展密切相关。
3.本研究的结果为评价成人骨髓源性神经干细胞体内移植的致瘤性提供了有意义的参考依据。
4.本研究的发现强调了对成人骨髓源性神经干细胞的长期致瘤性进行深入研究的重要性及必要性。
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 cells
Objective
To 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 methods
The 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.
Results
After 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).
Conclusion
1.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 cells
Objective
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 methods
The 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.
Results
The 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.
Conclusion
No 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 cells
Objective
To 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 methods
Grouping: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.
Results
Of 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.
Conclusion
1.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.
致瘤性是成体干细胞临床应用安全性方面一个重要的关注问题。有研究表明,体外培养的小鼠源性的骨髓基质细胞可表现出快速增殖、失去接触抑制及异常的细胞核表型改变等生物学特征,并且在将这些细胞移植于裸鼠或同系基因型的免疫缺陷小鼠体内后可导致肿瘤生成。再者,干细胞与肿瘤细胞有着很多相似的特征,有一理论假说认为肿瘤细胞中包含了少量被称为肿瘤干细胞的细胞,它们有着无限的自身增殖能力,并且可以导致肿瘤形成。因而,对临床应用的干细胞的致瘤性研究显得非常重要。目前为止,并没有明确的证据证明成人骨髓源性神经干细胞不存在致瘤性的可能。而对于成人骨髓源性神经干细胞的致瘤性问题,在前期工作中我们进行了染色体分析、体外凝集试验以及裸鼠成瘤试验等一系列研究,发现成人骨髓源性神经干细胞染色体形态、数目及结构正常,体外无凝集反应,接种裸鼠6个月无肿瘤形成,为评价其致瘤性提供了初步的实验依据。我们认为,对于成人骨髓源性神经干细胞的致瘤可能性不能仅依据体外实验或动物实验而完全排除,因为成人骨髓源性神经干细胞在人体内的生长条件和在培养皿中或动物体内存在很大的区别。在移植之后,成人骨髓源性神经干细胞在人体内可能存在一段难以估计的较长时期,在这段时期其可能发生很多变化,因而对于成人骨髓源性神经干细胞在人体内是否会发生恶性转变目前仍然还不能确定。对移植前的成人骨髓源性神经干细胞的致瘤性进行研究,能为评估其在移植后于人体内长期存在的过程中发生恶性转变的风险提供有意义的参考依据。
因为致瘤性可归因于遗传异常,在本研究中我们对成人骨髓源性神经干细胞中数百个肿瘤相关基因进行分析,目的是为了明确该细胞中是否存在某些基因特征可以有助于对其致瘤性的评价。
第一章成人骨髓源性神经干细胞的获得与鉴定
目的
建立一种快速有效的由成人骨髓基质细胞在体外诱导培养获得成人骨髓源性神经干细胞方法,并从细胞形态、标志性蛋白的表达及体外诱导分化成为神经系细胞的能力等方面对成人骨髓源性神经干细胞进行检测。
材料与方法
抽取正常成人志愿者全骨髓,应用密度梯度离心法分离成人骨髓基质细胞,体外培养纯化及扩增成人骨髓基质细胞,以神经干细胞培养基体外诱导培养成人骨髓基质细胞成为成人骨髓源性神经干细胞,以诱导培养基于体外诱导培养成人骨髓源性神经干细胞向神经系细胞分化。相差显微镜下观察成人骨髓基质细胞及成人骨髓源性神经干细胞的形态学特征;免疫组化法检测成人骨髓源性神经干细胞中神经干细胞标志性蛋白Nestin的表达,以及由成人骨髓源性神经干细胞诱导分化后得到的神经系细胞中神经胶质细胞标志性蛋白GFAP、少突胶质细胞标志性蛋白NG2及神经元标志性蛋白MAP2ab的表达。
结果
传代纯化后的成人骨髓基质细胞贴壁生长,呈梭形,贴壁生长,增殖旺盛,几乎不表达Nestin。经10～15天诱导培养,成人骨髓基质细胞分化为成人骨髓源性神经干细胞,成人骨髓源性神经干细胞由细胞克隆聚集成神经球样结构,不贴壁生长,增殖旺盛,高度表达Nestin。经7～10天诱导培养,成人骨髓源性神经干细胞体外分化成神经系细胞,细胞可表达GFAP、NG2及MAP2ab。
结论
1.成人骨髓基质细胞可在体外快速诱导分化成为成人骨髓源性神经干细胞。
2.成人骨髓源性神经干细胞具有神经分化的能力,在体外可进一步诱导分化成为神经系统的细胞类型。
3.成人骨髓基质细胞可为成人骨髓源性神经干细胞提供丰富的细胞来源。
第二章成人骨髓源性神经干细胞中重要抑癌基因的突变检测
目的
对成人骨髓源性神经干细胞中重要抑癌基因p53、Rb1和p16基因的常见突变点进行序列分析,观察是否存在异常的突变现象,从抑癌基因水平研究成人骨髓源性神经干细胞的致瘤性问题。
材料与方法
提取成人骨髓源性神经干细胞的基因组DNA,应用PCR-DNA测序技术,设计特定PCR引物,PCR扩增成人骨髓源性神经干细胞p53基因第5～9外显子、Rb1基因第19-21外显子以及p16基因第1～2外显子,并对扩增片段进行DNA测序。
结果
成人骨髓源性神经干细胞中p53基因第5～9外显子、Rb1基因第19～21外显子以及p16基因第1～2外显子的序列均与野生型一致,未见突变现象。
结论
由成人骨髓基质细胞体外诱导培养形成的成人骨髓源性神经干细胞未发现重要抑癌基因的常见突变现象,此结果为评价成人骨髓源性神经干细胞体内移植的致瘤性提供了有意义的参考依据。
第三章成人骨髓源性神经干细胞中肿瘤相关基因的表达检测
目的
检测成人骨髓源性神经干细胞中肿瘤相关基因的表达情况,为评价其移植后的致瘤性提供依据。
材料与方法
实验分组:实验组:成人骨髓源性神经干细胞;对照组:新鲜正常成人骨髓细胞去红细胞。提取总RNA;以肿瘤基因芯片Oligo GEArray Human CancerMicroarray OHS-802检测成人骨髓源性神经干细胞中440个肿瘤相关基因的表达水平;以实时定量RT-PCR的方法对成人骨髓源性神经干细胞中基因芯片检测结果显示呈高度表达的部分重要癌基因进行检测。
结果
基因芯片检测结果显示,与新鲜正常成人骨髓细胞去红细胞相比较,成人骨髓源性神经干细胞中有66个肿瘤相关基因呈高度表达(＞3倍),呈高度表达的基因中包含了促进细胞增殖、促进细胞侵袭和转移及一些重要致癌性信号传导通路的成员等基因。实时定量RT-PCR检测结果显示,与新鲜正常成人骨髓细胞去红细胞相比较,所检测的癌基因MYC、MMP2、Notch2、STC1、ITGA3、STAT5b、RhoC及Wnt1在成人骨髓源性神经干细胞中均呈高度表达,与基因芯片检测的结果相符,其表达倍数分别是: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)、Wnt1(3.64×10~0)。
结论
1.成人骨髓源性神经干细胞中存在大量的肿瘤相关基因的高度表达,其可能的生物学意义有待进一步深入研究。
2.高度表达的基因中包括了一些重要的癌基因,其激活与人类肿瘤的发生发展密切相关。
3.本研究的结果为评价成人骨髓源性神经干细胞体内移植的致瘤性提供了有意义的参考依据。
4.本研究的发现强调了对成人骨髓源性神经干细胞的长期致瘤性进行深入研究的重要性及必要性。
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 cells
Objective
To 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 methods
The 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.
Results
After 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).
Conclusion
1.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 cells
Objective
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 methods
The 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.
Results
The 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.
Conclusion
No 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 cells
Objective
To 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 methods
Grouping: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.
Results
Of 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.
Conclusion
1.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.
引文
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[1]Lee J,Elkahloun AG,Messina SA,et al.Cellular and Genetic Characterization of Human Adult Bone Marrow-Derived Neural Stem-Like Cells:A Potential Antiglioma Cellular Vector.Cancer Res.2003;63(24):8877-8889.
[2]Hermann A,Gastl R,Liebau S,et al.Efficient generation of neural stem cell-like cells from adult human bone marrow stromal cells.J Cell Sci.2004;117(Pt 19):4411-4422.
[3]Hermann A,Liebau S,Gastl R,et al.Comparative analysis of neuroectodermal differentiation capacity of human bone marrow stromal cells using various conversion protocols.J Neurosci Res.2006;83(8):1502-1514.
[4]McKay R.Stem cells in the central nervous system.Science.1997;276(5309):66-71.
[5] Weissman IL. Translating stem and progenitor cell biology to the clinic: barriers and opportunities. Science. 2000; 287 (5457): 1442-1446.
[6] Donovan PJ, Gearhart J. The end of the beginning for pluripotent stem cells. Nature. 2001; 414 (6859): 92-97.
[7] Gage FH. Mammalian neural s tern cells. Science 2000; 287 (5457):1433-1438.
[8] Ben-Hur T, Idelson M, Khaner H, et al. Transplantation of human embryonic stem cell-derived neural progenitors improves behavioral deficit in Parkinsonian rats. Stem Cells. 2004; 22(7): 1246-1255.
[9] Wang QH, Xu RX, Nagao S. Transplantation of cholinergic neural stem cells in a mouse model of Alzheimer's disease. Chin Med J (Engl). 2005; 118(6):508-511.
[10] McBride JL, Behrstock SP, Chen EY, et al. Human neural stem cell transplants improve motor function in a rat model of Huntington's disease. J Comp Neurol. 2004; 475(2):211-219.
[11] Bosch M, Pineda JR, Sunol C, et al. Induction of GABAergic phenotype in a neural stem cell line for transplantation in an excitotoxic model of Huntington's disease. Exp Neurol. 2004; 190(1):42-58.
[12] Shihabuddin LS, Numan S, Huff MR, et al. Intracerebral transplantation of adult mouse neural progenitor cells into the Niemann-Pick-A mouse leads to a marked decrease in lysosomal storage pathology. J Neurosci. 2004; 24(47): 10642-10651.
[13] Nonaka M, Yoshikawa M, Nishimura F, et al. Intraventricular transplantation of embryonic stem cell-derived neural stem cells in intracerebral hemorrhage rats. Neurol Res. 2004; 26(3):265-272.
[14] Iwanami A, Kaneko S, Nakamura M, et al. Transplantation of human neural stem cells for spinal cord injury in primates. J Neurosci Res. 2005; 80(2): 182-190.
[15] Li S, Tokuyama T, Yamamoto J, et al. Bystander effect-mediated gene therapy of gliomas using genetically engineered neural stem cells. Cancer Gene Ther. 2005; 12(7):600-607.
[16]Totoiu MO, Nistor GI, Lane TE, et al. Remyelination, axonal sparing, and locomotor recovery following transplantation of glial-committed progenitor cells into the MHV model of multiple sclerosis. Exp Neurol. 2004; 187(2):254-265.
[17] McKay R. Stem cells in the central nervous system. Science. 1997; 276 (5309):66-71.
[18] Weissman IL. Translating stem and progenitor cell biology to the clinic: barriers and opportunities. Science. 2000; 287 (5457):1442-1446.
[19] Donovan PJ, Gearhart J. The end of the beginning for pluripotent stem cells. Nature. 2001; 414 (6859):92-97.
[20] Friedenstein AJ, Gorskaja JF, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol. 1976; 4(5):267-274.
[21]Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science. 1997; 276(5309):71-74.
[22] Prockop DJ, Sekiya I, Colter DC. Isolation and characterization of rapidly self-renewing stem cells from cultures of human marrow stromal cells. Cytotherapy. 2001; 3(5):393-396.
[23] Woodbury D, Schwarz EJ, Prockop DJ, et al. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res. 2000; 61(4):364-370.
[24] Sanchez-Ramos J, Song S, Cardozo-Pelaez F, et al. Adult bone marrow stromal cells differentiate into neural cells in vitro.Exp Neurol. 2000; 164(2):247-256.
[25] Kopen GC, Prockop DJ, Phinney DG. Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc Natl Acad Sci U S A. 1999; 96(19): 10711-10716.
[26] Li Y, Chopp M, Chen J, et al. Intrastriatal transplantation of bone marrow nonhematopoietic cells improves functional recovery after stroke in adult mice.J Cereb Blood Flow Metab.2000;20(9):1311-1319.
[27]Li Y,Chen J,Wang L,et al.ntracerebral transplantation of bone marrow stromal cells in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease.Neurosci Lett.2001;316(2):67-70.
[28]Hou LL,Zheng M,Wang DM,et al.Migration and differentiation of human bone marrow mesenchymal stem cells in the rat brain.Sheng Li Xue Bao.2003;55(2):153-159.
[29]Chen J,Li Y,Wang L,et al.Therapeutic benefit of intracerebral transplantation of bone marrow stromal cells after cerebral ischemia in rats.J Neurol Sci.2001;189(1-2):49-57.
[30]娄晓辉,张亚卓,孙梅珍,等.应用人骨髓基质细胞治疗大鼠缺血性脑梗塞的实验研究.中华神经外科杂志.2002;18(5):302-305.
[31]Urdzikova L,Jendelova P,Glogarova K,et al.Transplantation of bone marrow stem cells as well as mobilization by granulocyte-colony stimulating factor promotes recovery after spinal cord injury in rats.J Neurotrauma.2006;23(9):1379-1391.
[32]Kim KN,Oh SH,Lee KH,et al.Effect of human mesenchymal stem cell transplantation combined with growth factor infusion in the repair of injured spinal cord.Acta Neurochir Suppl.2006;99:133-136.
[33]Chen Q,Long Y,Yuan X,et al.Protective effects of bone marrow stromal cell transplantation in injured rodent brain:synthesis of neurotrophic factors.J Neurosci Res.2005;80(5):611-619.
[34]Mahmood A,Lu D,Chopp M.Intravenous administration of marrow stromal cells(MSCs) increases the expression of growth factors in rat brain after traumatic brain injury.J Neurotrauma.2004;21(1):33-39.
[35] Mahmood A, Lu D, Chopp M. Marrow stromal cell transplantation after traumatic brain injury promotes cellular proliferation within the brain. Neurosurgery. 2004; 55(5):1185-1193.
[36] Sato H, Kuwashima N, Sakaida T, et al. Epidermal growth factor receptor-transfected bone marrow stromal cells exhibit enhanced migratory response and therapeutic potential against murine brain tumors.Cancer Gene Ther. 2005; 12(9):757-768.
[37]Nakamizo A, Marini F, Amano T, Khan A, Studeny M, Gumin J, Chen J, Hentschel S, Vecil G, Dembinski J, Andreeff M, Lang FF. 2005. Human bone marrow-derived mesenchymal stem cells in the treatment of gliomas. Cancer Res. 2005; 65(8):3307-3318.
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