| Mesenchymal stem cells (MSCs) are regarded as an ideal stem cell source for tissue repair and regeneration medicine, in view of their powerful self-renewal capacity, multipotential of differentiation and extremely weak immunogenicity. As a result, clinical trials are currently underway demonstrating efficacy of MSCs for a range of disorders. Besides human MSCs, studies have been carried out about many other species, such as cat, dog, baboon, rabbit, pig, goat, sheep, especially murine. As an important part of cell identification, specific surface markers of MSCs have been paid a lot of attention to for long, but no breakthrough as yet. Monoclonal antibodies (McAbs) against surface of certain cells have been used to characterize cell lineages. According to this idea, we raised a series of McAbs by immunizing mice with human mesenchymal stem cells, from which we isolated five hybridoma cell lines referred to as ZUB1, ZUB4, ZUC3, ZUE12 and ZUF10. Enzyme immunocytochemical analyses demonstrated that none of the five reacts with other human tissues except bone marrow, or other bone marrow derived cell strains except MSCs. However, ZUC3 was observed to react with bone marrow MSCs samples from both rat and bovine. The objective of our research is to observe the biological characteristics of rat MSCs, and try to find out the expression levels of the antigenic epitope which ZUC3 against after passage and differentiation, in order to tell weather ZUC3 isavailable for the identification of rat MSCs.In part I research, we compared the methods whole-marrow plastic adherence with density gradient centrifugation firstly, and then chose the former for MSCs isolation. Observed by inverted phase-contrast microscope, a few small adherent cells shaped spindle or triangle could be seen after 24 hours, situated around a cell-mass often. After several times of medium replacement, non-adherent hematopoietic cells were washed away, plastic adherent cells expanded exponentially and gradually reached confluence. They exhibited a homogeneous spindle morphology, with nucleus big and round, and one to several nucleoli clear inside. MSCs at early (PI and P2) passages have a similar form as the primary culture cells, while middle (P3) passage became wider and late passages (P4 and P5) show a larger, flatter phenotype, indicating an aging tendency. The growth curve of P3 MSCs could be divided into lag period in the first and second days, log period in third to eighth days, and plateau period hereafter. The doubling time (Td) of P3 MSCs was 78.9h±3.2h. Flow cytometry (FCM) provided evidence that rat MSCs were uniformly positive for adhesion molecules CD90 and CD44, negative for CD45, a marker of hematopoietic lineage.To demonstrate the multiple differentiate potential of MSCs, we induced differentiation of MSCs into neuron cells, osteogenic precursor cells and lipocytes successfully. Within the 24 hours incubated with lmM alpha-thioglycerol, there was no visible difference in shape. While exposed to 5mM alpha-thioglycerol for half an hour, morphological change appeared. The typical neuron-like cell was characterize for its dendrite or axon-like protrusions. After incubation for 3 to 5 hours, the majority of MSCs displayed a neuron-like appearance, with their dendrites and axons forming an interconnected network. Enzyme immunocytochemistry exhibited that rat MSCs express NF-M and NeuN while negative for GFAP. After exposed to osteogenic medium for approximately 10 days, scattered nodules in extracellular matrix could be find under the view of microscope, these nodules were then mineralizated by calcinosis, which was visualized by Von Kossa staining. MSCs treated with lipogenic medium produced tiny lipid droplets inside cytoplasma whichwere detectable after only 2 days, but another 2 weeks were necessary to accomplish a maximal lipid accumulation. Lipid droplet staining by Oil Red O coloured orange.In part II research, we initially confirmed that the antigenic epitope which ZUC3 bind to locates on the membrane of rat MSCs by enzyme immunocytochemistry and indirect immunofluorescence, thus evaluated the availability of ZUC3 as a McAb agaist MSCs surface markers. Then we did some exploratory research concerning the percentage of ZUC3-positive cells during passage and multidifferatiation. To this aim, we investigated FCM statistics from PI MSCs to P5, the outcome told that vast majority of MSCs (more than 85%) were ZUC3-positive. Analysing by multiple comparison we found some differences between P2 and PI (PO.01). The maximal percentage was reached at P3, presented a single symmetrical peak. Data of P2 and P4 population were slightly lower than P3, but the differences were not marked (P>0.05). By contrast, P5 MSCs showed a significant decline (PO.01) comparing with the former passages. Furthermore, there was to some extent a downward trend of FCM percentage after induced differentiation (PO.01). To mention the neuron differentiation, although nine tenths transformed to an neuron-like appearance after treated with alpha-thioglycerol ,the downward degree was not as much(97.77%-^80.07%). To analyse osteocytic differentiation, we could see an obvious down-regulation from the 10th day. The percentage of the 10th, 15th, 20th and 25th day were significant lower than their anterior time respectively (PO.05). The results of adipocytic differentiation after monolayer MSCs incubated with lipogenic medium were similar to what observed with osteocytic one. Distinct decrease of ZUC3 positive rate were detected in the 7th, 14th and 21st day compared with their anterior dots respectively (PO.01).In brief, several conclusions were reached:1. Homogeneous rat MSCs can be obtained in vitro after isolated by a single step of adhesion to cell culture plastic, and purified via replacement of medium and a serial of passage. Rat MSCs are uniformly positive for adhesion molecules CD90 and CD44, negative for CD45, a marker of hematopoietic lineage. Rat MSCs in vitro are capable of differentiating into neurons, osteoblasts andadipocytes once under appropriate stimuli.2. Anti-hMSCs-McAb-ZUC3 binds to the membrane of rat MSCs completely. Therefore can be used for identifiantion of rat MSCs.3. Significant decline of ZUC3 positive rate can be observed after inducing rat MSCs to differentiate along neuronal, osteoblastic and adipocytic pathways, indicating the antigenic apitope which ZUC3 against to be a primordial cell marker. |
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