Study Of Isolating Human Bone Marrow Mesenchymal Stem Cells By Magnetic-activated Cell Sorting With Monoclonal Antibody ZUC3

Mesenchymal stem cells (MSCs) in bone marrow are non-hematopoietic stem cells and also called bone marrow stroma cells, because of its resulting from marrow matrix which is sustaining for the hematopoietic function of the marrow. MSCs is a kind of adult stem cell, which has the capacity of self-renewal, high proliferation and multilineage differentiation, under somecertain conditions, MSCs can differentiate into adipocytes, chondrocytes, osteoblasts, tenocytes, myoblasts, astrocytes, neuron cells, endothelial cells in vivo and in vitro. MSCs do not express MHC class II antigen and co-stimulatory molecules, therefore, would do not activate alloreactive T cells. What is more, MSCs have immuno-modulatory activity, then allogeneic MSCs could be widely used. More and more dramatic pre-clinical and clinical studies of MSCs in cell-based immunotherapy, regenerative medicine and tissue engineering illustrate their potential therapeutic value.In adult bone marrow, the content of MSCs is very low, about one in 10~4~10~5 bone marrow mononuclear cells. As a result, it is necessary to purificate, culture, andproliferate MSCs effectively in vitro to obtain large quantities of MSCs for clinical application. The property of plastic adherence itself is not sufficient to allow for the purification of MSCs, and the primary cultured MSCs are considered to be a heterogeneous population, which hampers the application of MSCs. With specific surface marker, MSCs could be highly purified from bone marrow mononuclear cells rapidly. Culture-expanded MSCs are uniformly positive for CD29, CD44, CD73, CD90, CD 105, CD 166, and do not express the typical hematopoietic antigens such as CD 14, CD34, CD45, HLA-DR, Glycophorin A and T- or B-cell-lineage surface markers. Although a great deal has been learned in recent years about the isolation and characterization of MSCs, little is known of the specific surface markers for definition of MSCs. Due to the lack of a single definitive marker and knowledge regarding the anatomical location and distribution of MSCs in vivo, the demonstration of their existence has relied primarily on retrospective assays. The gold standard assay, till now, utilized to identify MSCs is by its characteristics of morphology, phenotype, self-renewal and multilineage differentiation potential. The specific surface marker for selection, detection and testing of MSCs is still on researching.Our previous studies had preparation a new monoclonal antibody (McAb). named ZUC3, which had be identified as MSCs specific McAb. In this study, we would try to establish the method of isolation human MSCs from bone marrow mononuclear cells by magnetic-activated cell sorting (MACS) with McAb ZUC3, then demonstrate the proliferation and multiple differeniate potential of ZUC3 positive cells. Our novel selection protocol with McAb ZUC3 provides a means to generate purified populations of bone marrow derived MSCs for further research and application.The ZUC3 hybridoma cell line was resuscitated, cultured and injected into BALB/C mice abdomen (1.0*106 cells /mice) to inducing ascites. Collected ascitesof McAb ZUC3 was purified by chromatography and the valence was l:104 detceted by indirect immunofluorescence staining. There was no reactivity of McAb ZUC3 with 8 human malignant hematological cell lines (HL-60, NB4, K562, U-937, HEL, Jurkat, Raji, KM3). Flow cytometric analysis showed, in culture-expanded bone marrow MSCs, freshly isolated bone marrow mononuclear cells and peripheral blood mononuclear cells, the proportion of ZUC3 positive cells were 91.31±2.92%, 0.96±0.28%, 0.24±0.05% respectively, which demonstrated that McAb ZUC3 would be a specific probe for cell sorting which could be against human MSCs specifically.First, we establish the method of isolation ZUC3 postive cells from bone marrow mononuclear cells by MACS with McAb. In this research, samples of 20ml bone marrow were taken from the iliac crest of normal healthy adult volunteers, bone marrow mononuclear cells were separated by density gradient centrifugation and incubated with McAb ZUC3 (1:100 dilution) for 1 hour;washed twice to remove unbound primary antibody, then resuspend cell pellet and incubated with 20ul rat antimouse IgM Microbeads (Miltenyi Biotec Inc.) per 107 cells for 40 min;finally, single-cell suspensionc was applied to MS columns and separated into positively-and negatively-labelled fractions by using Mini MACS?. The activity of positive cells was 97.9±0.39% dectected by trypan-blue staining and the purity was 76.82±6.32% by flow cytometry. The positive and negative cells were collected and plated in medium consisting of Dulbecco's Modified Eagle's Medium-Low Glucose (LG-DMEM), supplemented with 10% FBS, respectively. The positive cells had adhered to culture flask after 3-day culture period;the quantity of adhered cells increased obviously and proliferated as cluster shaped after 5-day;after a 18-day primary expansion period, the positive cells nearly reached confluency and these cells had a fibroblast-like morphology. While the negative cells were observed a large of round shape cells after 3-day culture period and it was still observed round shape cells and no proliferation after a 18-day primary expansion period.To demonstrate the proliferation potential of the positive cells by MACS with McAb ZUC3, the frist passage and the fourth passage cells cultured in vitro were used to be observed the growth characteristics. The results illustrated that passaged cells had adhered to culture flask completely during 12 hours, and all these ceils had a fibroblast-like morphology;the lag period and the log proliferative phase were 24-36 hours and 4-5 days respectively;after log proliferative phase these cells turned into plateau period;there was no significant difference of proliferation potential between the frist passage and the fourth passage (P>0.05). It was suggested that these ZUC3 postive cells have powerful proliferation potential in vitro. Analyzed by flow cytometry, the culture-expanded positive cells were uniformly positive for CD29 (81.25±4.53%), CD44 (89.59±5.83%), CD105 (88.80±5.81%), CD106 (92.14±4.51%) and negative for CD14 (2.21±0.97%), CD34 (1.42±0.61%), CD45 (3.33±1.05%), HLA-DR (2.04±0.67%). The results demonstrated that ZUC3 postive cells sorted from bone marrow mononuclear cells by McAb were MSCs and no hematopoietic cells in these cells.To demonstrate the multiple differeniate potential of the culture-expanded positive cells, we successfully induced differentiation of the positive cells into adipocytes and osteoblasts. A larger number of orange adipocytes were observed after the positive cells were treated with adipogenic medium (Stem Cell Inc.) and stained with oil red O. When the positive cells treated with osteogenic medium (LG-DMEM containing 10% FBS supplemented with 10~8M dexamethasone, 0.2 mM ascorbic acid, and 10 mM (5-glycerol phosphate) formed several scattered nodules and then became calcific nod can demonstrate by Von Kossa staining. The results illustrated that culture-expanded ZUC3 postive cells had the capacity of multilineage differentiation.Human primary MSCs could be well isolated from bone marrow with McAb ZUC3 and expanded in vitro.In brief, several conclusions were reached:1. ZUC3 McAb was a specific surface marker for cell sorting which can specificity against human MSCs.2. ZUC3 McAb could be used as a probe, and separated bone marrow mononuclear cells into positively- and negatively-labelled fractions by MACS. The positive cells had good activity and could culture-expanded in vitro.3. The primary ZUC'3 positive cells were adherent-capable cells displaying fibroblast-like morphology in vitro and demonstrated exciting proliferation potential during passage. While the negative cells by MACS with ZUC3 McAb demonstrated that they absent the capacity of proliferation.4. The culture-expanded ZUC3 positive cells were uniformly positive for CD29, CD44, CD105, CD106 and negative for hematopoietic antigens such as CD14, CD34, CD45, HLA-DR, and it could be considered as a homologous population.5. The culture-expanded ZUC3 positive cells remained the multiple differentiate potential in vitro that they could differentiate into adipocytes and osteoblasts.6. The novel MASC protocol with McAb ZUC3 provided an ideal means to generate purified populations of bone marrow derived MSCs.