Biological Characteristics of ZUC3 Antigen Expressed on Rat Bone Marrow Mesenchymal Stem Cells.
Authors: Huang He , Zheng Jing , Lai Xiaoyu , Cao Junli , Fan Jianling 
Published: April 2020

Abstract
Objective: Bone marrow mesenchymal stem cells (MSCs) are widely studied in recent years. 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 (McAb) against surface of certain cells have been used to characterize cell lineages. ZUC3, a novel murine McAb was produced by hybridoma technology previously, which was specifically reactive with both human MSCs and rat MSCs. Studying the expression of ZUC3 antigen on rat MSCs after passage and differentiation, it was to define whether ZUC3 antigen would be available for the identification of rat MSCs or their differentiation lineages.
Methods: Rat MSCs isolated by a single step of adhesion to cell culture plastic, and purified via replacement of medium and a serial of passage, then the cells were identified by surface molecules CD90, CD44 and CD45 by flow cytometry. Enzyme immunocytochemistry and indirect immunofluorescence were used to evaluate the availability of ZUC3 expression by rat MSCs as a surface marker. Then further exploratory researches were carried out concerning ZUC3 expression by rat MSCs during passages (P1 to P5) and multiple differentiation (neuron, osteoblasts and adipocytes) in the certain condition.
Results: Homogeneous rat MSCs could be obtained in vitro, which were uniformly positive for adhesion molecules CD90, CD44, and negative for CD45. The McAb was specifically reactive with rat MSCs as the positive cells were more than 99% by immunohistochemistry and immunofluorescence staining, and ZUC3 antigen located on the membrane of rat MSCs. The flow cytometric analysis show ZUC3 antigen expression by rat MSCs from P1 to P5 were all more than 85%. Analysis by multiple comparison, it was found some differences between P2 and P1 (93.95±2.44% v.s. 86.90±1.80%, P&lt;0.01). The maximal expression was reached at P3 (97.10±1.25%), and the flow cytometric analysis showed a single symmetrical peak. Data of P4 (94.50±2.23%) population were slightly lower than P3 (P&gt;0.05). By contrast, P5 (88.35±2.99%) showed a significant decline comparing with the former passages (P&lt;0.01). Furthermore, rat MSCs could be successfully induced to differentiate into neuron-like cells, osteoblasts, and adipocytes and there was to some extent a downward trend of ZUC3 expression after differentiation (P&lt;0.01). More than 90% rat MSCs could transform to an neuron-like appearance which were positive for NeuN, NF-M after treated with alpha-thioglycerol, and there was some downward degree of ZUC3 expression (97.77±1.03% to 80.07±2.70%, P&lt;0.01). During the osteoblastic differentiation, it was observed an obvious down-regulation of ZUC3 expression from the 10th day (96.63±1.03% to 90.07±2.40%, P&lt;0.01 ) and percentage on the 10th (90.07±2.40%), 15th (84.43±2.80%), 20th (64.53±7.63%) and 25th (53.40±10.02%) day were significantly lower than their anterior time respectively (P&lt;0.05). The results of adipogenic differentiation after MSCs incubated with proper medium were similar to what observed during osteoblastic differentiation and ZUC3 expression were down-regulation on the 7th (84.33±2.70%), 14th (75.90±2.00%) and 21st (70.57±0.47%) day compared with their anterior dots respectively (P&lt;0.01).
Conclusion: ZUC3 antigen could be used for identification of rat MSCs. Significant decline of ZUC3 expression had be observed after rat MSCs were induced to differentiate along neuronal, osteoblastic and adipogenic pathways, which indicated that ZUC3 antigen would be a marker of progenitor.