Objective: To explore the biological function methods of mesenchymal stem cells(MSCs) for quality analysis. Methods: The surface markers of MSCs were detected by flow cytometry. MSCs osteogenic differentiation was induced by ascorbic acid and β-glycerophosphate sodium, etc., followed by Alizarin Red S staining. MSCs adipogenic differentiation was induced by IBMX, Rosiglitazone, etc., followed by Oil Red O staining. MSCs could differentiate into chondrocytes with treatment of ITS and TGFβ3, etc., followed by Alcian Blue staining. Cell co-culture of THP-1-macrophage with MSCs and ELISA assay were applied to detect the effects of MSCs on macrophage polarization. The expression levels of IL-10 and TNF α in the cell co-culture supernatant were detected by ELISA. To observe the effects of MSCs on lymphocyte proliferation, MSCs cultured with PBMCs, which were labeled with CFSE and activated by CD3/CD28, followed by flow cytometry. Results: The expression of MSCs surface markers, CD105, CD73, and CD90, was more than 95% respectively, while the expression of CD45, CD34, CD14, CD19, and HLA-DR expression was less than 2%. MSCs osteogenic differentiation assay showed red calcium nodules. Red lipid vacuoles were observed in MSCs adipogenic induction differentiation. Furthermore, MSCs have the differentiation potential to chondrocyte spheroids, and typical cartilage pits were observed. Co-culture of MSCs with THP-1 macrophages, an increase in IL-10 expression and downregulate TNFα secretion were observed. MSCs played inhibitory effects on the proliferation of PBMC activated by CD3/CD28, with an inhibition rate of 76.4%. Conclusions: This study established some of biological activity detection methods for MSCs, including MSCs surface markers, differentiation abilities, promotion of macrophage polarization, and inhibitory effects on lymphocyte proliferation. It provides a potential application for MSCs products quality control.
[1] 韩晓燕,纳涛,张可华,等.人间充质干细胞生物学有效性的质量评价[J].中国新药杂志,2018,27(21): 2511
HAN XY, NA T, ZHANG KH, et al.Quality evaluation for biological effectiveness of human mesenchymal stem cells[J]. Chin J New Drugs, 2018, 27(21): 2511
[2] 王泽,宋扬,秦林伟.干细胞的研究进展和应用前景[J].沈阳师范大学学报(自然科学版),2021,39(6): 566
WANG Z, SONG Y, QIN LW.Research progress and application review of stem cells[J]. J Shenyang Norm Univ: Nat Sci Ed, 2021, 39(6): 566
[3] 马杰,史明霞,赵春华.间充质干细胞与血液系统疾病[J].基础医学与临床,2007,27(3): 346
MA J, SHI MX, ZHAO CH.Mesenchymal stem cells and hematological diseases[J]. Basic Clin Med, 2007, 27(3): 346
[4] 王友臣,史恩祥.间充质干细胞应用的研究进展[J].实用医学杂志,2011,27(20): 3806
WANG YC, SHI EX.Research progress on the application of mesenchymal stem cells[J]. J Pract Med, 2011, 27(20): 3806
[5] 何萍,程涛,郝莎.干细胞临床研究的现状及展望[J].中国医药生物技术,2020,15(3): 290
HE P, CHENG T, HAO S.The status quo and prospect of clinical study on stem cells[J]. Chin Med Biotech, 2020, 15(3): 290
[6] 袁宝珠. 干细胞产品生物学有效性评价的基本原则[J].中国生物制品年会暨第十六次全国生物制品学术研讨会,2016
YUAN BZ.Basic principles for evaluating the biological effectiveness of stem cell products[R]. China Bio Prod Annual Conference and 16th Nat Symposium Bio Prod(CBPC), 2016
[7] ULLAH I, SUBBARAO RB, RHO GJ.Human mesenchymal stem cells-current trends and future prospective[J]. Biosci Rep, 2015, 35(2): 1
[8] 吕林洁,王长生,王晓冰,等.间充质干细胞治疗膝骨性关节炎的临床研究进展[J].中华细胞与干细胞杂志(电子版),2021,11(6): 365
LÜ LJ, WANG CS, WANG XB, et al.Clinical progression of mesenchymal stem cells in the treatment of knee osteoarthritis[J]. Chin J Cell Stem Cell(Elect Ed), 2021, 11(6): 365
[9] 闫小滨,郭志坤.脂肪来源间充质干细胞的生物学特性及临床应用前景[J].解剖科学进展,2009,15(4): 416
YAN XB, GUO ZK.Bionomics and clin icalapplication prospect of ad ipose derivedm esenchym alstem cells[J]. Prog Anatom Sci, 2009, 15(4): 416
[10] 杨莹,李政楠,王秀娟,等.脐带来源间充质干细胞的免疫抑制功能研究[J].药物评价研究,2020,43(12): 2385
YANG Y, LI ZN, WANG XJ, et al.Research on immunosuppressive function of umbilical cord mesenchymal stem cells[J]. Drug Eval Res, 2020, 43(12): 2385
[11] CHEN YH, YANG LH, LI XH.Advances in mesenchymal stem cells regulating macrophage polarization and treatment of sepsis-induced liver injury[J]. Front Immunol, 2023, 14: 1238972
[12] LUZ-CRAWFORD P, KURTE M, BRAVO-ALEGRÍA J, et al. Mesenchymal stem cells generate a cd4+cd25+foxp3+ regulatory T cell population during the differentiation process of th1 and th17 cells[J]. Stem Cell Res Ther, 2013, 4(3): 65
[13] 王方迪,侯瑞霞,李俊琴,等.间充质干细胞对T细胞免疫调节机制的研究进展[J].中国免疫学杂志,2021,37(22): 2715
WANG FD, HOU RX, LI JQ, et al.Research progress of mesenchymal stem cells on immunomodulatory mechanism of T lymphocyte[J]. Chin J Immunol, 2021, 37(22): 2715
[14] 汪健芳,莫春阳,许艳华,等.骨髓间充质干细胞的临床应用研究[J].中国细胞生物学学报,2018,40(13): 2145
WANG JF, MO CY, XU YH, et al.Bone marrow mesenchymal stem cells and its clinical applications[J]. Chin J Cell Biol, 2018, 40(13): 2145
[15] 张可华,纳涛,韩晓燕,等.基于免疫调控功能的间充质干细胞生物学有效性质量评价策略[J].中国新药杂志,2016,25(3): 283
ZHANG KH, NA T, HAN XY, et al.Immunomodulatory properties-based strategy for the assessmentof biological effectiveness of mesenchymalstem cells[J]. Chin J New Drugs, 2016, 25(3): 283
[16] 胡资兵,曾荣,郭伟韬,等.骨髓间充质干细胞诱导分化特征[J].中国组织工程研究与临床康复,2008,12(43): 8561
HU ZB, ZENG R, GUO WT, et al.Induction and differentiation of bone marrow mesenchymal stem cells[J]. J Clin Rehab Tissue Engin Res, 2008, 12(43): 8561
[17] 人源干细胞产品药学研究与评价技术指导原则(试行)[S]. 2023: 4
Guiding Principles for Pharmaceutical Research and Evaluation of Human Stem Cell Products (Trial)[S]. 2023: 4
[18] 张可华,刘静,纳涛,等.人间充质干细胞体外成软骨分化能力综合评价策略研究[J].中国新药杂志,2017,26(18): 2196
ZHANG KH, LIU J, NA T, et al.A comprehensive evaluation strategy for chondrogenic differentiation of human mesenchymal stem cells[J]. Chin J New Drugs, 2017, 26(18): 2196
[19] FENG L, YANG ZM, LI YC, et al.Linc-ror promotes mesenchymal stem cells chondrogenesis and cartilage formation via regulating sox9 expression[J]. Osteoarthritis Cartilage, 2021, 29(4): 568
[20] 尹学红,庞春燕,白力,等.脂肪间充质干细胞促进M1型巨噬细胞向M2型巨噬细胞转化[J].细胞与分子免疫学杂志,2016,32(3): 332
YIN XH, PANG CY, BAI L, et al.Adipose-derived stem cells promote the polarization from M1 macrophages to M2 macrophages[J]. Chin J Cell Mol Immunol, 2016, 32(3): 332
[21] KRAMPERA M, GALIPEAU J, SHI Y, et al.Immunological characterization of multipotent mesenchymal stromal cells--the international society for cellular therapy (ISCT) working proposal[J]. Cytotherapy, 2013, 15(9):1054
[22] 张可华,纳涛,韩晓燕,等.人间充质干细胞生物学有效性质量评价用标准细胞株CCRC-HMSC-S1的建立及评价[J].中国新药杂志,2020,29(21): 2502
ZHANG KH, NA T, HAN XY, et al.Establishment of a reference cell line, CCRC-hMSC-S1, for the evaluationof biological effectiveness of human mesenchymal stem cells[J]. Chin J New Drugs, 2020, 29(21): 2502
