一个新的人间充质干细胞特异性表面抗原的鉴定和间充质干细胞亚群生物学特性的研究
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摘要
间充质干细胞(Mesenchymal Stem Cells,MSCs)是一种具有多向分化潜能的成体干细胞,是当前干细胞研究领域的热点,在组织器官缺损性疾病、退行性疾病、自身免疫性疾病、遗传缺陷等疾病的治疗中有着巨大的临床应用前景。随着MSCs临床应用的进程,对MSCs生物学特性的研究和认识提出了更高的要求。一直以来,骨髓MSCs被认为是骨髓单个核细胞中具有贴壁的特性、并可在体外培养增殖和诱导多向分化的非造血系统的干细胞。值得注意的是,迄今为止MSCs尚未发现有特异性的表面分子。目前国际公认的MSCs表型特征为:细胞表面CD73、CD90、CD105等抗原呈阳性,而CD14、CD19、CD34、CD45、HLA-DR等造血谱系分子表达阴性。由于缺乏鉴定细胞的特异性标志,导致对MSCs的生物学特征缺乏严格统一的定义,制约了对MSCs生物学特性的研究和认识,使得MSCs的检测、分离纯化以及应用均面临着巨大的难题。
第1章人间充质干细胞特异性单克隆抗体及其抗原特征的鉴定
为发现新的MSCs特异的表面标志,以期深入研究MSCs的生物学特性,本研究以培养的人骨髓MSCs为免疫原免疫BALB/C小鼠,应用杂交瘤技术研究制备了具有自主知识产权的入骨髓MSCs特异性单克隆抗体ZUB1(专利号:ZL200510061034.2),并对ZUB1单克隆抗体及其识别抗原的生物学特性进行了深入研究。通过对抗体种属和组织细胞特异性鉴定,证实ZUB1单克隆抗体针对人MSCs有高度的特异性和敏感性,ZUB1可识别骨髓、脂肪组织、脐带组织和脐带血来源的MSCs,与大鼠、小鼠和兔骨髓MSCs均无交叉反应。Western-blot结果显示,ZUB1单克隆抗体识别一种分子量约为250KD的抗原分子,该种抗原在13种人血液系统恶性疾病细胞株和10种人实体组织细胞株均无表达。骨髓、脂肪组织、脐带组织和脐带血来源的MSCs在传代扩增过程中形态基本一致,流式细胞术检测传代扩增细胞表达ZUB1抗原的阳性率分别为97.58±0.68%、89.50±3.97%、96.63±1.23%、87.23±4.07%,此外CD29、CD44、CD105、CD166、CD146阳性标记出现单峰,CD3、CD14、CD19、CD34、CD117、CD133、CD45、CD235a、HLA-DR均表达阴性,传代细胞ZUB1抗原和其他各表面抗原表达均无显著性差异。当诱导骨髓MSCs向成骨细胞和脂肪细胞定向分化时,流式细胞检测显示ZUB1抗原表达相应减弱,提示ZUB1抗原可作为MSCs干细胞特征的标志分子,且可能参与了MSCs分化的细胞活动。
为进一步鉴定ZUB1单克隆抗体识别的抗原表位,我们通过Western-blot实验证实ZUB1单克隆抗体识别的抗原分子量约为250KD,应用ZUB1单克隆抗体通过免疫沉淀的方法从人骨髓MSCs细胞裂解液中获取与ZUB1抗体特异性结合的蛋白质,ZUB1抗体蛋白复合物经SDS-PAGE电泳分离后切取蛋白质条带,质谱分析提示ZUB1单克隆抗体识别的分子量为250KD的抗原肽段为非肌性肌球蛋白-9重链(non-muscle myosin heavy chain 9,NMMHCⅡa)。生物信息学分析提示该类细胞骨架蛋白的同型异构体在不同组织和干细胞的不同分化阶段有不同的异构体的表达,MSCs特异的形态特征和细胞骨架结构与其多向分化潜能是相适应的。该研究进一步丰富了对骨髓MSCs特异性分子特征的认识,也为进一步研究MSCs分化等细胞活动提供了一个重要的线索。
第2章ZUB1抗原阳性骨髓间充质干细胞亚群的鉴定和生物学特性的研究
随着对体外培养MSCs的生物学特性认识的深入,研究者利用不同的表面分子分离骨髓单个核细胞以期进一步研究和鉴定骨髓原始MSCs及其不同亚群的生物学特性。利用ZUB1单克隆抗体,我们建立了ZUB1~+骨髓MSCs的分离培养体系,并深入研究了ZUB1~+骨髓MSCs亚群的生物学特性。应用ZUB1单克隆抗体通过免疫磁珠分选骨髓单个核细胞,流式细胞仪检测分选后细胞纯度为61.52±6.69%,分选后获得的ZUB1~+骨髓单个核细胞体积较小、核质比大,符合干细胞的形态学特征。ZUB1~+骨髓单个核细胞培养3天后细胞贴壁呈梭形,5天后细胞扩增明显,梭形贴壁细胞增殖呈集落样分布,培养15天左右贴壁细胞达80%～90%汇合,细胞形态均一,且具有很好的增殖活性,细胞扩增至第8代细胞生长曲线无明显差异。流式细胞术分析显示ZUB1~+骨髓单个核细胞贴壁培养后具有MSCs的表型特性,且可诱导向成骨细胞、脂肪细胞和神经元样细胞。因此,ZUB1单克隆抗体可用于分离富集ZUB1~+骨髓MSCs亚群。
一直以来MSCs缺乏特异性分子标志,目前尚无有效的指标可以鉴定骨髓中不同亚群的原始MSCs的含量。CFU-F可用于评估骨髓单个核细胞中MSCs集落的形成情况,也是目前用于评估骨髓中原始MSCs的数量的主要指标之一。将未分选的骨髓单个核细胞、及磁珠分选后ZUB1~+和ZUB1~-骨髓单个核细胞按2×10~4/cm~2、1×10~3/cm~2、2×10~4/cm~2的细胞密度分别接种培养,细胞培养15d,ZUB1~+骨髓单个核细胞形成CFU-F的数量和直径明显多于未分选的骨髓单个核细胞,而ZUB1~-骨髓单个核细胞未见集落形成。按照每10~5单个核细胞计算ZUB1单克隆抗体分选骨髓单个核细胞的CFU-F富集指数(Enrichment Factor)为193.09±32.81,且ZUB1~+骨髓MSCs增殖较快。为进一步分析ZUB1~+骨髓MSCs的表型特征,对MSCs公认的一系列表面分子检测结果显示该类细胞的表型一致:CD29、CD105、CD166、CD146表达阳性,而CD3、CD14、CD19、CD33、CD235a、HLA-DR、CD45、CD34、CD133、CD117为阴性。由此,我们认为ZUB1单克隆抗体可有效地富集骨髓单个核细胞中的MSCs,ZUB1抗原可以作为研究骨髓原始MSCs的分子标志;ZUB1~+骨髓MSCs体外培养增殖较快,并可多向分化为成骨细胞、脂肪细胞和神经元样细胞。
本研究制备了具有自主知识产权的抗入骨髓MSCs特异性单克隆抗体,并证实ZUB1抗原针对人MSCs有高度的特异性,可用于人MSCs的检测、鉴定以及富集骨髓中原始MSCs。ZUB1抗原为一种在MSCs中特异的细胞骨架蛋白,参与了MSCs定向分化的生命活动。骨髓细胞中ZUB1~+骨髓单个核细胞作为一个新的骨髓MSCs亚群,丰富了对MSCs生物学特性的认识,并为MSCs的研究带来新契机。
Mesenchymal stem cells(MSCs) resemble a multipotent adult stem cell population capable of differentiating into different mesodermal cell lineages including osteoblasts, chondroblasts and adipocytes,and MSCs can also be differentiate into cells of nonmesodermal origin including hepatocytes and neurons.MSCs have generated a great deal of excitement as a potential source of cells for regenerative medicine and tissue engineering owing to their dramatic potential of proliferation and differentiation,and raise high hopes in clinical applications.Presently,MSCs are characterized by their adherence to plastic surface,a panel of cell-surface markers,and their in vitro and in vivo differentiation capacity.The heterogeneity of adherent cultured starting population renders comparison of results between different groups difficult may also in part account for the lack of reproducibility in some of the initial reports using MSCs.The surface markers,used alone or in combination,have not been specific for MSCs.There is just a minimal criteria proposed to define human MSCs,that the cells are positive for CD73,CD90,CD105 and negative for CD14,CD19,CD34,CD45,and HLA-DR. However,the lack of common standards and a precise definition of MSCs preparations remains a major obstacle in research and application of MSCs.The identification of a definitive marker allowing the prospective isolation of MSCs would be of the utmost importance.
Chapter 1:Identification and characteristic of a novel monoclonal antibody against human mesenchymal stem cells
The phenotype of MSCs remains limited in spite of several decades of study,and several monoclonal antibodies to surface antigens have been used to identify and isolate MSCs.However,the precise identity of MSCs remains a challenge and the homogeneity of MSCs is still controversial due in part to the lack of useful cell-specific markers.We used the cultured and passaged human bone marrow(BM) MSCs to immunize BALB/C mouse,and a novel murine monoclonal antibody(mAb) ZUB1 to huaman MSCs was produced by hybridoma technology(Patent No:ZL 200510061034.2),and had no reactive with the BM MSCs from rat,mouse and rabbit. To further identify the specificity of ZUB1,western-blot analysis indicated the negative cross-reactivity when screened against a variety of human cell lines from hematopoietic and solid tissue.The ZUB1 mAb could been used to detect human MSCs from BM, adipose tissue(AT),umbilical tissue(UT) and umbilical cord blood(UCB),and the flow cytometry analysis show the percentage of positive cells are 97.58±0.68%, 89.50±3.97%,96.63±1.23%,87.23±4.07%,respectively.Overall,the ZUB1 mAb could be used detect human MSCs by western-blot,immunofluorescence,and flow cytometry, and the ZUB1 antigen was specific in MSCs.The expression of ZUB1 antigen had no difference after passaging,but it was significant decreased after differentiated into adipocytes and osteocytes,which indicated that the ZUB1 antigen may be an important marker of "stem" and was associated with the differentiation of MSCs.
To further identify the ZUB1 recognize distinct epitopes on human MSCs,we used the western-blot to demonstrate that the ZUB1 mAb recognized the antigen of molecular mass approximate 250 KD.A protein of molecular mass 250 KD was immunoprecipitated using the ZUB1 mAb,and was purified and identified by peptide sequencing analysis and mass spectrometry as non-muscle myosin heavy chain 9 (NMMHC Ha).Bioinformatics analysis showed that nonmuscle myosinⅡis one of the main motors interacting with cytoskeletal actin and is involved in regulating cytokinesis, cell motility and cell polarity,and been described for a number of different cultured cells and tissues with different informs.Depending on the cell type,there appear to contain a particular myosinⅡisoform,and interacts with specific proteins of the specific cells.The high expression of cytoskeletal proteins and their isoforms are associated with the multipotent differentiation of MSCs,and the expression of NMMHCⅡisoform in MSCs offerred an important cue for further study.
Chapter 2:Isolation and characteristic of ZUB1 positive human bone marrow mesenchymal stem cells population
MSCs represent a minor fraction of the total nucleated cell population in the BM with an approximate 1 MSCs in 10~4～10~5 mononuclear cells.In addition,there has been only scattered data on the phenotype of the native BM MSCs because different markers have been used for MSCs isolation in independent studies.ZUB1 antigen was strongly expressed by cultured human MSCs,we have used ZUB1 mAb to isolate MSCs from BM mononuclear cells directly by magnetic-activated cell sorting(MACS),and the purity of the recovered fractions for ZUB1 after MACS was 61.52±6.69%by flow cytometry.It was observed that the ZUB1~+ BM mononuclear cells was small,but had high nuclear-cytoplasmic ratio by Giemsa staining,which corresponded to the morphology of "stem cells".ZUB1 positive and negative cells were separated from BM mononuclear cells by MACS,and plated respectively in human MSCs medium consisting of 10%FBS,LG-DMEM.The positive cells have adhered to culture flask in vitro,and the quantity of adhered cells that had fibroblast-like morphology increased and proliferated during primary expansion period,while the negative cells were observed as round shape cells without any proliferation.It was demonstrated that ZUB1 positive cells continued growth with spindle-shape,extending beyond 8 passages in long-term culture,and the culture-expanded positive cells had the uniformly phenotype as MSCs.With proper medium,the ZUB1~+BM MSCs could be successfully induced to differentiate into adipocytes,osteoblasts,and neuro-like cells which were positive of neuron markers such as nestin,NSE and NF-M.So,ZUB1 mAb could be used to isolate the ZUB1~+ MSCs population from BM mononuclear cells.
To date,only little information exists about the features of BM MSCs in vivo,as a strict terminology to distinguish between the BM native MSCs and cultured MSCs as plastic-adherent cells is lacking.Classically,a subset of BM MSCs is designated as clonogenic if it is able to generate colonies of fibroblast-like cells from single cells when plated in culture.The clonogenic potential of the sorted cells is analyzed by scoring their ability to give rise to "colony-forming units-fibroblasts(CFU-F)".The unsorted BM mononuclear cells,ZUB1~+ and ZUB1~- BM mononuclear cells were seeded in the dishes at the densities of 2×10~4/cm~2,1×10~3/cm~2,2×10~4/cm~2,respectively.15 days after culture,the colonies were enumerated,and the number and size of CFU-Fs of ZUB1~+ BM mononuclear cells was more than ZUB1" cells.As CFU-F numbers were equalized to 105 plated cells,the CFU-F enrichment factor(number of colonies per 10~5 cells plated in the isolated fraction/number of colonies per 10~5 cells plated in the initial mononuclear cell fraction) was 193.09±32.81,and the ZUB1~+ BM MSCs had high capacity of proliferation.Phenotype of ZUB1~+ BM MSCs was analyzed by flow cytometry,the culture-expanded positive cells were uniformly positive for CD29, CD105,CD166 and CD146,and lack typical hematopoietic antigens such as CD3, CD14,CD19,CD33,CD235a,HLA-DR,CD34,CD45,CD133 and CD117.The ZUB1 antigen could be a specific surface marker to BM native MSCs. Overall,the current data indicated that the mAb ZUB1 was specific against human MSCs,and effective for identification and isolation MSCs from BM.ZUB1 antigen was a specific marker to MSCs,and ZUB1~+ BM MSCs was a subpopulation of BM MSCs with the capacity of high proliferation and multipotent differentiation.In addition, ZUB1 antigen was associated with the differentiation of MSCs,and may be an important marker of "stem".ZUB1 as a novel marker of human MSCs is promising.
第1章人间充质干细胞特异性单克隆抗体及其抗原特征的鉴定
为发现新的MSCs特异的表面标志,以期深入研究MSCs的生物学特性,本研究以培养的人骨髓MSCs为免疫原免疫BALB/C小鼠,应用杂交瘤技术研究制备了具有自主知识产权的入骨髓MSCs特异性单克隆抗体ZUB1(专利号:ZL200510061034.2),并对ZUB1单克隆抗体及其识别抗原的生物学特性进行了深入研究。通过对抗体种属和组织细胞特异性鉴定,证实ZUB1单克隆抗体针对人MSCs有高度的特异性和敏感性,ZUB1可识别骨髓、脂肪组织、脐带组织和脐带血来源的MSCs,与大鼠、小鼠和兔骨髓MSCs均无交叉反应。Western-blot结果显示,ZUB1单克隆抗体识别一种分子量约为250KD的抗原分子,该种抗原在13种人血液系统恶性疾病细胞株和10种人实体组织细胞株均无表达。骨髓、脂肪组织、脐带组织和脐带血来源的MSCs在传代扩增过程中形态基本一致,流式细胞术检测传代扩增细胞表达ZUB1抗原的阳性率分别为97.58±0.68%、89.50±3.97%、96.63±1.23%、87.23±4.07%,此外CD29、CD44、CD105、CD166、CD146阳性标记出现单峰,CD3、CD14、CD19、CD34、CD117、CD133、CD45、CD235a、HLA-DR均表达阴性,传代细胞ZUB1抗原和其他各表面抗原表达均无显著性差异。当诱导骨髓MSCs向成骨细胞和脂肪细胞定向分化时,流式细胞检测显示ZUB1抗原表达相应减弱,提示ZUB1抗原可作为MSCs干细胞特征的标志分子,且可能参与了MSCs分化的细胞活动。
为进一步鉴定ZUB1单克隆抗体识别的抗原表位,我们通过Western-blot实验证实ZUB1单克隆抗体识别的抗原分子量约为250KD,应用ZUB1单克隆抗体通过免疫沉淀的方法从人骨髓MSCs细胞裂解液中获取与ZUB1抗体特异性结合的蛋白质,ZUB1抗体蛋白复合物经SDS-PAGE电泳分离后切取蛋白质条带,质谱分析提示ZUB1单克隆抗体识别的分子量为250KD的抗原肽段为非肌性肌球蛋白-9重链(non-muscle myosin heavy chain 9,NMMHCⅡa)。生物信息学分析提示该类细胞骨架蛋白的同型异构体在不同组织和干细胞的不同分化阶段有不同的异构体的表达,MSCs特异的形态特征和细胞骨架结构与其多向分化潜能是相适应的。该研究进一步丰富了对骨髓MSCs特异性分子特征的认识,也为进一步研究MSCs分化等细胞活动提供了一个重要的线索。
第2章ZUB1抗原阳性骨髓间充质干细胞亚群的鉴定和生物学特性的研究
随着对体外培养MSCs的生物学特性认识的深入,研究者利用不同的表面分子分离骨髓单个核细胞以期进一步研究和鉴定骨髓原始MSCs及其不同亚群的生物学特性。利用ZUB1单克隆抗体,我们建立了ZUB1~+骨髓MSCs的分离培养体系,并深入研究了ZUB1~+骨髓MSCs亚群的生物学特性。应用ZUB1单克隆抗体通过免疫磁珠分选骨髓单个核细胞,流式细胞仪检测分选后细胞纯度为61.52±6.69%,分选后获得的ZUB1~+骨髓单个核细胞体积较小、核质比大,符合干细胞的形态学特征。ZUB1~+骨髓单个核细胞培养3天后细胞贴壁呈梭形,5天后细胞扩增明显,梭形贴壁细胞增殖呈集落样分布,培养15天左右贴壁细胞达80%～90%汇合,细胞形态均一,且具有很好的增殖活性,细胞扩增至第8代细胞生长曲线无明显差异。流式细胞术分析显示ZUB1~+骨髓单个核细胞贴壁培养后具有MSCs的表型特性,且可诱导向成骨细胞、脂肪细胞和神经元样细胞。因此,ZUB1单克隆抗体可用于分离富集ZUB1~+骨髓MSCs亚群。
一直以来MSCs缺乏特异性分子标志,目前尚无有效的指标可以鉴定骨髓中不同亚群的原始MSCs的含量。CFU-F可用于评估骨髓单个核细胞中MSCs集落的形成情况,也是目前用于评估骨髓中原始MSCs的数量的主要指标之一。将未分选的骨髓单个核细胞、及磁珠分选后ZUB1~+和ZUB1~-骨髓单个核细胞按2×10~4/cm~2、1×10~3/cm~2、2×10~4/cm~2的细胞密度分别接种培养,细胞培养15d,ZUB1~+骨髓单个核细胞形成CFU-F的数量和直径明显多于未分选的骨髓单个核细胞,而ZUB1~-骨髓单个核细胞未见集落形成。按照每10~5单个核细胞计算ZUB1单克隆抗体分选骨髓单个核细胞的CFU-F富集指数(Enrichment Factor)为193.09±32.81,且ZUB1~+骨髓MSCs增殖较快。为进一步分析ZUB1~+骨髓MSCs的表型特征,对MSCs公认的一系列表面分子检测结果显示该类细胞的表型一致:CD29、CD105、CD166、CD146表达阳性,而CD3、CD14、CD19、CD33、CD235a、HLA-DR、CD45、CD34、CD133、CD117为阴性。由此,我们认为ZUB1单克隆抗体可有效地富集骨髓单个核细胞中的MSCs,ZUB1抗原可以作为研究骨髓原始MSCs的分子标志;ZUB1~+骨髓MSCs体外培养增殖较快,并可多向分化为成骨细胞、脂肪细胞和神经元样细胞。
本研究制备了具有自主知识产权的抗入骨髓MSCs特异性单克隆抗体,并证实ZUB1抗原针对人MSCs有高度的特异性,可用于人MSCs的检测、鉴定以及富集骨髓中原始MSCs。ZUB1抗原为一种在MSCs中特异的细胞骨架蛋白,参与了MSCs定向分化的生命活动。骨髓细胞中ZUB1~+骨髓单个核细胞作为一个新的骨髓MSCs亚群,丰富了对MSCs生物学特性的认识,并为MSCs的研究带来新契机。
Mesenchymal stem cells(MSCs) resemble a multipotent adult stem cell population capable of differentiating into different mesodermal cell lineages including osteoblasts, chondroblasts and adipocytes,and MSCs can also be differentiate into cells of nonmesodermal origin including hepatocytes and neurons.MSCs have generated a great deal of excitement as a potential source of cells for regenerative medicine and tissue engineering owing to their dramatic potential of proliferation and differentiation,and raise high hopes in clinical applications.Presently,MSCs are characterized by their adherence to plastic surface,a panel of cell-surface markers,and their in vitro and in vivo differentiation capacity.The heterogeneity of adherent cultured starting population renders comparison of results between different groups difficult may also in part account for the lack of reproducibility in some of the initial reports using MSCs.The surface markers,used alone or in combination,have not been specific for MSCs.There is just a minimal criteria proposed to define human MSCs,that the cells are positive for CD73,CD90,CD105 and negative for CD14,CD19,CD34,CD45,and HLA-DR. However,the lack of common standards and a precise definition of MSCs preparations remains a major obstacle in research and application of MSCs.The identification of a definitive marker allowing the prospective isolation of MSCs would be of the utmost importance.
Chapter 1:Identification and characteristic of a novel monoclonal antibody against human mesenchymal stem cells
The phenotype of MSCs remains limited in spite of several decades of study,and several monoclonal antibodies to surface antigens have been used to identify and isolate MSCs.However,the precise identity of MSCs remains a challenge and the homogeneity of MSCs is still controversial due in part to the lack of useful cell-specific markers.We used the cultured and passaged human bone marrow(BM) MSCs to immunize BALB/C mouse,and a novel murine monoclonal antibody(mAb) ZUB1 to huaman MSCs was produced by hybridoma technology(Patent No:ZL 200510061034.2),and had no reactive with the BM MSCs from rat,mouse and rabbit. To further identify the specificity of ZUB1,western-blot analysis indicated the negative cross-reactivity when screened against a variety of human cell lines from hematopoietic and solid tissue.The ZUB1 mAb could been used to detect human MSCs from BM, adipose tissue(AT),umbilical tissue(UT) and umbilical cord blood(UCB),and the flow cytometry analysis show the percentage of positive cells are 97.58±0.68%, 89.50±3.97%,96.63±1.23%,87.23±4.07%,respectively.Overall,the ZUB1 mAb could be used detect human MSCs by western-blot,immunofluorescence,and flow cytometry, and the ZUB1 antigen was specific in MSCs.The expression of ZUB1 antigen had no difference after passaging,but it was significant decreased after differentiated into adipocytes and osteocytes,which indicated that the ZUB1 antigen may be an important marker of "stem" and was associated with the differentiation of MSCs.
To further identify the ZUB1 recognize distinct epitopes on human MSCs,we used the western-blot to demonstrate that the ZUB1 mAb recognized the antigen of molecular mass approximate 250 KD.A protein of molecular mass 250 KD was immunoprecipitated using the ZUB1 mAb,and was purified and identified by peptide sequencing analysis and mass spectrometry as non-muscle myosin heavy chain 9 (NMMHC Ha).Bioinformatics analysis showed that nonmuscle myosinⅡis one of the main motors interacting with cytoskeletal actin and is involved in regulating cytokinesis, cell motility and cell polarity,and been described for a number of different cultured cells and tissues with different informs.Depending on the cell type,there appear to contain a particular myosinⅡisoform,and interacts with specific proteins of the specific cells.The high expression of cytoskeletal proteins and their isoforms are associated with the multipotent differentiation of MSCs,and the expression of NMMHCⅡisoform in MSCs offerred an important cue for further study.
Chapter 2:Isolation and characteristic of ZUB1 positive human bone marrow mesenchymal stem cells population
MSCs represent a minor fraction of the total nucleated cell population in the BM with an approximate 1 MSCs in 10~4～10~5 mononuclear cells.In addition,there has been only scattered data on the phenotype of the native BM MSCs because different markers have been used for MSCs isolation in independent studies.ZUB1 antigen was strongly expressed by cultured human MSCs,we have used ZUB1 mAb to isolate MSCs from BM mononuclear cells directly by magnetic-activated cell sorting(MACS),and the purity of the recovered fractions for ZUB1 after MACS was 61.52±6.69%by flow cytometry.It was observed that the ZUB1~+ BM mononuclear cells was small,but had high nuclear-cytoplasmic ratio by Giemsa staining,which corresponded to the morphology of "stem cells".ZUB1 positive and negative cells were separated from BM mononuclear cells by MACS,and plated respectively in human MSCs medium consisting of 10%FBS,LG-DMEM.The positive cells have adhered to culture flask in vitro,and the quantity of adhered cells that had fibroblast-like morphology increased and proliferated during primary expansion period,while the negative cells were observed as round shape cells without any proliferation.It was demonstrated that ZUB1 positive cells continued growth with spindle-shape,extending beyond 8 passages in long-term culture,and the culture-expanded positive cells had the uniformly phenotype as MSCs.With proper medium,the ZUB1~+BM MSCs could be successfully induced to differentiate into adipocytes,osteoblasts,and neuro-like cells which were positive of neuron markers such as nestin,NSE and NF-M.So,ZUB1 mAb could be used to isolate the ZUB1~+ MSCs population from BM mononuclear cells.
To date,only little information exists about the features of BM MSCs in vivo,as a strict terminology to distinguish between the BM native MSCs and cultured MSCs as plastic-adherent cells is lacking.Classically,a subset of BM MSCs is designated as clonogenic if it is able to generate colonies of fibroblast-like cells from single cells when plated in culture.The clonogenic potential of the sorted cells is analyzed by scoring their ability to give rise to "colony-forming units-fibroblasts(CFU-F)".The unsorted BM mononuclear cells,ZUB1~+ and ZUB1~- BM mononuclear cells were seeded in the dishes at the densities of 2×10~4/cm~2,1×10~3/cm~2,2×10~4/cm~2,respectively.15 days after culture,the colonies were enumerated,and the number and size of CFU-Fs of ZUB1~+ BM mononuclear cells was more than ZUB1" cells.As CFU-F numbers were equalized to 105 plated cells,the CFU-F enrichment factor(number of colonies per 10~5 cells plated in the isolated fraction/number of colonies per 10~5 cells plated in the initial mononuclear cell fraction) was 193.09±32.81,and the ZUB1~+ BM MSCs had high capacity of proliferation.Phenotype of ZUB1~+ BM MSCs was analyzed by flow cytometry,the culture-expanded positive cells were uniformly positive for CD29, CD105,CD166 and CD146,and lack typical hematopoietic antigens such as CD3, CD14,CD19,CD33,CD235a,HLA-DR,CD34,CD45,CD133 and CD117.The ZUB1 antigen could be a specific surface marker to BM native MSCs. Overall,the current data indicated that the mAb ZUB1 was specific against human MSCs,and effective for identification and isolation MSCs from BM.ZUB1 antigen was a specific marker to MSCs,and ZUB1~+ BM MSCs was a subpopulation of BM MSCs with the capacity of high proliferation and multipotent differentiation.In addition, ZUB1 antigen was associated with the differentiation of MSCs,and may be an important marker of "stem".ZUB1 as a novel marker of human MSCs is promising.
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