Application Of Monoclonal Antibodies To Bone Marrow Mesenchymal Stem Cells In The Identification Of Bone Marrow Mesenchymal Stem Cells And Umbilical Cord Blood Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BM-MSCs), as a kind of non-hematopoietic tissue stem cells, have the capacity of self-renewal, high proliferation and multilineage differentiation.Under some certain conditions,MSCs can differentiate into lipocytes, chondrocytes, osteogenic precursor cells, astrocytes and neuron cells. Recent researches have shown that MSCs also have immunomodulatory effects.These characterizations may make them candidates for tissue regeneration,gene therapy and cellular therapy.Since 1990s,researchers have attempted to explore markers which are specific for MSCs and prepared some monoclonal antibodies(McAbs) to mesenchymal stem cells,such as SH2,SH3,SH4,STRO-1 .However none of these markers are specific for MSCs which hampers the identification and isolation of MSC populations.Our research group have generated and characterized four MSCs-specific McAbs,namely ZUB1,ZUB4,ZUC3,ZUF10,which were not found to be reactive with eight cell lines(HL-60, NB4, K562, U937, HEL, Jurkat, Raji, KM3) and eleven tissues ( ligament, tendon, nerve, fat, muscle, artery, epidermis, lung, liver, smallintestine,cholecyst).In this study,we have tried to use these McAbs to detect BM-MSCs and umbilical cord blood mesenchymal stem cells(UCB-MSCs) by immunocytochemistry staining, immunofluorescence staining, flow cytometry, and Western Blot and expected these antibodies could be used to develop human MSCs marker immunosensors.In this research,a bone marrow aspirate and umbilical cord blood were collected and mononuclear cells were taken by density gradient centrifugation, and plated in human MSC medium consisting of Dulbecco's Modified Eagle's Medium-Low Glucose (DMEM-LG), supplemented with 10% FBS. After allowing 48 hours for adherence to culture flask, nonadherent cells were removed. After a 14-day primary expansion period,BM-MSCs nearly reached confluency and these cells had a fibroblast-like morphology. Confluent cultures could be processed further by trypsinization and expansion through sequential passages to confluency. After a 28-day primary expansion period, UCB-MSCs nearly reached confluency and the shape was similar to BM-MSCs except smaller.Analyzed by flow cytometry, MSCs were demonstrated that they were uniformly positive for CD29,CD44,CD105 and CD 166, negative for CD14,CD34,CD45 and HLA-DR. No significant differences were presented between BM-MSCs and UCB-MSCs. In the other hand, we induced successfully diferentiation of MSCs into osteogenic precursor cells, lipocytes and neuron cell. These results verified that the cells we used in this study were MSCs.This study showed that four McAbs were reactive with MSCs by immunocytochemistry staining, immunofluorescence staining, flow cytometry, and Western Blot analysis.The specificity and sensitivity are high.In immunocytochemistry staining, four McAbs were reactive with BM-MSCs andUCB-MSCs . The positive rate of ZUB1,ZUB4,ZUC3 and ZUF10 on BM-MSCs was(97.44±1.39) %, (96.56±0.77) %, (98.67±0.58) %, (98.33±1.15) % respectivelyand the positive rate of ZUB1,ZUB4,ZUC3 and ZUF10 on UCB-MSCs was about94.5%,93.5%,95.84%,95.67%.Four McAbs exhibited bright staining of MSCs by immunofluorescence staining. About 95% of BM-MSCs and 90% of UCB-MSCs were stained.Analyzed by flow cytometry,the positive rate of ZUB1,ZUB4,ZUC3 and ZUF10 on BM-MSCs was (90.93±3.21) %, (82.35±4.14) %, (93.22±1.09) %, (87.10±3.83) % respectively,the positive rate of ZUB1,ZUB4, ZUC3 and ZUF10 on UCB-MSCs was about 75.89%, 66.95%, 77.37%, 77.53%.Western Blot was carried out with cultured BM-MSCs lysates and UCB-MSCs lysates.ALL of four McAbs showed single bands in BM-MSCs and UCB-MSCs. The antigens detected by ZUBK ZUB4^ ZUC3 and ZUF10 might have the size of about 72kDa,170kDa,33kDa and 72kDa.In conclusion(T)All of four McAbs were reactive with BM-MSCs with high specificity and sensitivity ,and any of them could be used to identify BM-MSCs instead of the routine means which identifies BM-MSCs by many markers.? Four McAbs can be utilized in the detection and identification of BM-MSCs by immunocytochemistry staining, immunofluorescence staining, flow cytometry, and Western Blot analysis.(3)ALL of four McAbs also can be utilized in the detection and identification of UCB-MSCs.