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The Effect of Subculture Passage on the Biological Characteristics of Menstrual Blood Derived Endometrial Stem Cells

REN Yakun^1,2, SUN Yuliang², HE Yanan², ZHANG Shenghui^2,3, ZHU Xinxing^2,4, LIU Yanli^1,2*, CHENG Hongbin^4,5, FENG Huigen², LIN Juntang^1,2,4

(¹Henan Key Laboratory of Medical Tissue Regeneration, Xinxiang 453003, China; ²Stem Cell and Biotherapy Technology Research Center, College of Life Science and Technology, Xinxiang Medical University, Xinxiang 453003, China; ³The Third Affiliated Hospital of Xinxiang Medical University, Xinxiang 453003, China; ⁴College of Biomedical Engineering, Xinxiang Medical University, Xinxiang 453003, China; ⁵The Third Medical Center of Chinese PLA General Hospital, Beijing 100039, China)

Abstract:

The biological characteristics of MenSCs （menstrual blood derived endometrial stem cells) cultured in vitro at different passages (passage 3, 9 and 15) were examined, in order to provide support for further studies on the potential clinical applications of MenSCs. Therefore, in this study, the morphology of MenSCs was observed after Calcein-AM staining. The senescence of MenSCs was detected by β-galactosidase staining. The production of ROS (reactive oxygen species) in MenSCs was detected by ROS probe staining. The activity of mouse derived spleen lymphocytes co-cultured with MenSCs was determined by conventional MTT assay and Live/Dead staining, respectively. Additionally, both the cell cycle of mouse derived spleen lymphocytes co-cultured with MenSCs and the activation of CD3+ and CD19+ lymphocytes in mouse derived spleen lymphocytes co-cultured with MenSCs were analyzed by flow cytometry. Consequently, our results showed that no matter the cell size of MenSCs, or the degree of senescence and ROS production in MenSCs had significant increases as the subculture passage increased in vitro, also a large number of filamentous pseudopodia was observed in P15 MenSCs. Subsequently, the mouse derived spleen lymphocytes co-cultured with MenSCs exhibited a superior metabolic activity than the lymphocytes cultured alone, the following Live/Dead staining results also confirmed a lower death rate in the mouse derived spleen lymphocytes co-cultured with MenSCs, and P3 MenSCs exhibited the optimal viabilitykeeping capacity. Additionally, the further cell cycle examination showed no influence on the proliferation of mouse derived spleen lymphocytes, no matter co-cultured with or without MenSCs, but the percentage of debris in lymphocytes co-cultured with MenSCs had a significant decrease, which suggested MenSCs were capable of decreasing the death rate of co-cultured mouse derived spleen lymphocytes. Finally, there was no significant change in the percentage of CD3+ and CD19+ lymphocytes in mouse derived spleen lymphocytes, no matter co-cultured with or without MenSCs, which suggested the low immunogenicity of MenSCs. In summary, with the increased subculture passages in vitro, MenSCs not only exhibited a significant decrease in their viability, but also the MenSCs derived viability-keeping capacity for lymphocytes was significantly down-regulated. These results will contribute to balance the quality and quantity of MenSCs used in clinic, and then guarantees the therapeutic effect of MenSCs based therapies.

CSTR: 32200.14.cjcb.2020.02.0005