Identification And Characterization Of Human Mesenchymal Stem Cells Population By A Novel Monoclonal Antibody

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 cellsThe 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 populationMSCs 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.