New Monoclonal Antibodies Reactive with Human Bone Marrow-Derived Mesenchymal Stem Cells.
Authors: Lai Xiaoyu , Huang He , Luo Yi , Liao Zhixiong , Shen Jiangen 
Published: April 2020

Abstract
Human mesenchymal stem cells (hMSCs) could be well isolated and expanded from bone marrow and have been widely studied. In addition to hMSCs have ability to support hematopoiesis, they 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. However, the precise identity of hMSCs remains a challenge and the homogeneity of hMSCs is still controversial due in part to the lack of useful cell-specific markers. Thus, the objective of our study was to development monoclonal antibodies (MAbs) reactive with hMSCs surface antigens. hMSCs were isolated from bone marrow of volunteers on a density gradient solution by Ficoll-paque and cultured (LG-DMEM, 10%FBS) with their characteristic of adherence. hMSCs phenotype were analyzed by flow cytometry and demonstrated that they were uniformly positive for CD29, CD44, CD166 and CD105, negative for CD34, CD45 and HLA-DR. All hMSCs could be successfully induced to differentiate into osteogenic precursor cells, adipocytes and neuron cells when treated by correctly conditioned medium. There were detected to be no significant differences of phenotypes, growth characteristics and the potential of differentiation after either passage or programmed cryopreservation. Cultured and passaged hMSCs were used to immunized BALB/C mice at 1-week interval for 4 times, and their splenocytes subsequently harvested were fused with mouse SP 2/0 myeloma cells in the presence of PEG followed by culturing in the HAT selective medium. Indirect immunofluorescence assay (fluorescent antibody reacted with IgG and IgM isotypes) was utilized to preliminary screen the hybridoma colonies which synthesized and secreted antibodies having the specificity for cultured hMSCs. Out of 1248 wells initially plated, only 27 wells were tested positive for reactivity with living cultured hMSCs, and finally we got 5 hybridoma cell lines which could secreted MAbs steadily, named ZUB1, ZUB4, ZUC3, ZUE12, ZUF10. In order to identify hybridomas which secreted antibodies were only specific for hMSCs, peripheral blood cells, human malignant hematopoietic cell lines and rat marrow-derived mesenchymal stem cells were detected by indirect immunofluorescence analysis and subsequently a series of mesenchymal and non-mesenchymal derived tissues of human were performed by immunohistochemistry staining. The results demonstrated that all these 5 MAbs were specific for hMSCs except that ZUC3 had cross reaction with rat marrow-derived mesenchymal stem cells, and there were a few positive cells in the bone marrow. The subtype of monoclonal antibody ZUC3 was IgM, while other four MAbs were IgG1 and light chains of all MAbs were kappa. Using hybridoma cell lines, large amount of MAbs in ascites were generated then purified. MAbs ZUB1, ZUB4, ZUC3, ZUE12, ZUF10 were used to detected cultured hMSCs by flow cytometry, and percentage of positive cells are 87.39%, 88.07%, 88.12%, 69.89%, 83.67%, respectively. In conclusion, there 5 new MAbs that developed reactived with hMSCs surface antigens. Our study would further the understanding of hMSCs biological characterization and have potential application of new MAbs for identifying, sorting hMSCs derived from bone marrow. Further studies will focus on the characterization and epitopes of the MAbs to hMSCs.