Comparing biological characteristics of five kinds of mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.1834

Abstract

Abstract:

BACKGROUND: Various methods have been introduced to isolate mesenchymal stem cells of different origins. A full understanding of similarities and differences among mesenchymal stem cells of different origins will contribute to the rational and standardized application of mesenchymal stem cells in clinical practice.
OBJECTIVE: To compare the biological characteristics of mesenchymal stem cells of different origins.
METHODS: We collected umbilical cord, placenta and part of adipose tissues that were shed from parturients after cesarean delivery, and decidua basalis-derived mesenchymal stem cells, chorionic villi-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, mesenchymal stem cells at the junction between umbilical cord and placenta, and umbilical cord mesenchymal stem cells were cultured by trypsinization or tissue explant method. There were three kinds of stem cell culture media, including stem cell culture medium containing fetal bovine serum, stem cell culture medium containing cord blood lysate, and serum-free stem cell culture medium. Cell diameter, surface markers and multi-directional differentiation ability under different culture conditions were examined. Gene expression of different mesenchymal stem cells was analyzed by retrieval of the GEO database.
RESULT AND CONCLUSION: Under different culture conditions, there were no differences in cell size and cell cycle. The diameter of mesenchymal stem cells cultured in serum-free culture system was decreased, while the diameter of placenta-derived mesenchymal stem cells cultured in the stem cell culture medium containing cord blood lysate increased. Mesenchymal stem cells of five origins had similar surface markers, osteogenic, chondrogenic and adipogenic abilities, in which adipose-derived mesenchymal stem cells have stronger adipogenic ability. Moreover, adipose-derived mesenchymal stem cells are different from other kinds of stem cells in term of gene expression, and have complex extracellular matrix.

Key words: mesenchymal stem cells, tissue explant method, trypsin digestion, mesenchymal stem cells at the junction between umbilical cord and placenta, umbilical cord mesenchymal stem cells, adipose-derived mesenchymal stem cells, decidua basalis-derived mesenchymal stem cells, chorionic villi-derived mesenchymal stem cells

CLC Number:

Liu Chao, Zhang Lijun, Tao Hao, Du Xinjie, Wang Rui, Li Wenjing, Ding Shumei, Wang Yi, Jiang Lei, Huang Yuxiang. Comparing biological characteristics of five kinds of mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(33): 5334-5340.

Figures/Tables 3

2.3 流式分析间充质干细胞表面标志流式技术分析间充质干细胞表面标志物是鉴定间充质干细胞的重要指标，目前普遍认可间充质干细胞表达CD44、CD90、CD105和CD73，并且不表达CD45、CD34、CD14、CD79和HLA-DR[14]。实验选择了CD105和CD44作为干细胞标志物，选择HLA-DR、CD45和CD34为阴性参照，在不同培养条件下5种间充质干细胞的CD105和CD44阳性表达率均为95%以上，阴性参照则在2%以下，见图3B。流式数据的前向角散射值(forward scatter，FSC)参数反映细胞直径的大小，通过比较同一次实验每组前向角散射值平均荧光强度的大小，见图3A，进一步确认了不同培养条件下细胞直径的差异，其中含脐血裂解物干细胞培养基能够显著增加胎盘源间充质干细胞的直径。 2.4 间充质干细胞的细胞周期分析通过PI染色标记细胞内的DNA含量，进而实现对细胞周期分析。分析结果显示，这5种间充质干细胞在无血清添加物培养体系中G2期细胞所占的比例较大，见图4。 2.5 GEO数据库分析间充质干细胞蛋白表达的差异基因表达水平能够精确区分细胞间的差异，通过检索GEO数据库，检索到2套数据GSE72597[15](包含脐带、脐带胎盘连接处间充质干细胞)和GSE108511(包含脐带、脂肪以及胎盘间充质干细胞)，2组数据均采用GPL17586平台。以脐带间充质干细胞为参照，比较其他间充质干细胞与其蛋白表达差异，并将差异蛋白做功能注释，见图5，结果发现脂肪间充干细胞与脐带间充质干细胞有157个蛋白表达存在差异，其中76个高表达，81个低表达，见图6C，在高表达的基因中有许多细胞基质成分相关基因高表达如ABI3BP、Periostin、MFAP5等，这提示脂肪间充干细胞的细胞基质成分更复杂多样，在低表达基因中有许多细胞连接相关的膜蛋白如DSC2、PKP2等，这提示脂肪间充质干细胞相比于脐带间充质干细胞的细胞群体更疏松。脐带胎盘连接处间充质干细胞与脐带间充质干细胞有41个蛋白表达存在差异，其中21个高表达，31个低表达，见图6B，在高表达基因集合中没有显著富集的功能注释，在低表达基因集合中有许多细胞膜受体例如CXCL6、LY96等，这提示脐带胎盘连接处间充质干细胞相比于脐带间充质干细胞对一些信号刺激(如炎症因子)不敏感。胎盘间充干细胞(原GEO数据未区分底蜕膜和绒毛膜)与脐带间充质干细胞有57个蛋白表达存在差异，其中16个高表达，41个低表达，见图6A，在高表达基因中有ABI3BP、Tissue Factor、HGF、VCAM1等，在低表达基因中有DSC2、PKP2、NEDD4L等，这提示胎盘间充质干细胞的细胞群体更加疏松，细胞膜表面的离子通道更加开放。分析这3组差异蛋白的集合关系，可以看到脐带胎盘连接处间充质干细胞与脐带间充质干细胞的差异更小，见图6D。"

References

[1]Lou G, Chen Z, Zheng M, et al. Mesenchymal stem cell-derived exosomes as a new therapeutic strategy for liver diseases. Exp Mol Med. 2017;49(6):e346.

[2]Karantalis V, Hare JM. Use of mesenchymal stem cells for therapy of cardiac disease. Circ Res. 2015;116(8):1413-1430.

[3]Wang J, Cen P, Chen J, et al. Role of mesenchymal stem cells, their derived factors, and extracellular vesicles in liver failure. Stem Cell Res Ther. 2017;8(1):137.

[4]Yang JX, Zhang N, Wang HW, et al. CXCR4 receptor overexpression in mesenchymal stem cells facilitates treatment of acute lung injury in rats. J Biol Chem. 2015;290(4):1994-2006.

[5]Panés J, García-Olmo D, Van Assche G, et al. Expanded allogeneic adipose-derived mesenchymal stem cells (Cx601) for complex perianal fistulas in Crohn's disease: a phase 3 randomised, double-blind controlled trial. Lancet. 2016;388(10051):1281-1290.

[6]Skyler JS, Fonseca VA, Segal KR, et al. Allogeneic Mesenchymal Precursor Cells in Type 2 Diabetes: A Randomized, Placebo-Controlled, Dose-Escalation Safety and Tolerability Pilot Study. Diabetes Care. 2015;38(9):1742-1749.

[7]Le Blanc K, Rasmusson I, Sundberg B, et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet.2004;363(9419):1439-1441.

[8]Caplan AI. Mesenchymal Stem Cells: Time to Change the Name! Stem Cells Transl Med. 2017;6(6):1445-1451.

[9]Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284(5411): 143-147.

[10]Friedman R, Betancur M, Boissel L, et al. Umbilical cord mesenchymal stem cells: adjuvants for human cell transplantation. Biol Blood Marrow Transplant.2007;13(12):1477-1486.

[11]严泉剑,郭金龙,刘恩靖,等.绘制细胞生长曲线及细胞群体倍增时间的简化计算[J].实用医药杂志, 2000,17(4):228-229.

[12]Russell AL, Lefavor R, Durand N, et al. Modifiers of mesenchymal stem cell quantity and quality. Transfusion. 2018;58(6):1434-1440.

[13]Yu G, Wang LG, Han Y, et al. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS. 2012; 16(5):284-287.

[14]Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8(4):315-317.

[15]Beeravolu N, Khan I, McKee C, et al. Isolation and comparative analysis of potential stem/progenitor cells from different regions of human umbilical cord. Stem Cell Res. 2016;16(3):696-711.

[16]Bateman ME, Strong AL, Gimble JM, et al. Concise Review: Using Fat to Fight Disease: A Systematic Review of Nonhomologous Adipose-Derived Stromal/Stem Cell Therapies. Stem Cells. 2018;36(9): 1311-1328.

[17]Steinberg GK, Kondziolka D, Wechsler LR, et al. Clinical Outcomes of Transplanted Modified Bone Marrow-Derived Mesenchymal Stem Cells in Stroke: A Phase 1/2a Study. Stroke. 2016;47(7):1817-1824.

[18]Gronthos S, Mankani M, Brahim J, et al. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A. 2000; 97(25):13625-13630.

[19]Wu M, Zhang R, Zou Q, et al. Comparison of the Biological Characteristics of Mesenchymal Stem Cells Derived from the Human Placenta and Umbilical Cord. Sci Rep. 2018;8(1): 5014.

[20]Kang E, Wang X, Tippner-Hedges R, et al. Age-Related Accumulation of Somatic Mitochondrial DNA Mutations in Adult-Derived Human iPSCs. Cell Stem Cell. 2016;18(5): 625-636.

[21]Rebelatto CK, Aguiar AM, Moretão MP, et al. Dissimilar differentiation of mesenchymal stem cells from bone marrow, umbilical cord blood, and adipose tissue. Exp Biol Med (Maywood). 2008;233(7):901-913.

[22]Cai J, Miao X, Li Y, et al. Whole-genome sequencing identifies genetic variances in culture-expanded human mesenchymal stem cells. Stem Cell Reports. 2014;3(2):227-233.

[23]Merkle FT, Ghosh S, Kamitaki N, et al. Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations. Nature. 2017;545(7653):229-233.

[24]Neurohr GE, Terry RL, Lengefeld J, et al. Excessive Cell Growth Causes Cytoplasm Dilution And Contributes to Senescence. Cell. 2019;176(5):1083-1097.e18.

[25]Ge J, Guo L, Wang S, et al. The size of mesenchymal stem cells is a significant cause of vascular obstructions and stroke. Stem Cell Rev. 2014;10(2):295-303.

[26]Du W, Li X, Chi Y, et al. VCAM-1+ placenta chorionic villi-derived mesenchymal stem cells display potent pro-angiogenic activity. Stem Cell Res Ther. 2016;7:49.

[27]Ulrich C, Abruzzese T, Maerz JK, et al. Human Placenta-Derived CD146-Positive Mesenchymal Stromal Cells Display a Distinct Osteogenic Differentiation Potential. Stem Cells Dev. 2015;24(13): 1558-1569.

[28]Liu H, Liu S, Li Y, et al. The role of SDF-1-CXCR4/CXCR7 axis in the therapeutic effects of hypoxia-preconditioned mesenchymal stem cells for renal ischemia/reperfusion injury. PLoS One. 2012;7(4):e34608.

[29]Marquez-Curtis LA, Janowska-Wieczorek A. Enhancing the migration ability of mesenchymal stromal cells by targeting the SDF-1/CXCR4 axis. Biomed Res Int. 2013;2013:561098.

[30]Zheng XB, He XW, Zhang LJ, et al. Bone marrow-derived CXCR4-overexpressing MSCs display increased homing to intestine and ameliorate colitis-associated tumorigenesis in mice. Gastroenterol Rep (Oxf). 2019;7(2):127-138.