3D-cultured umbilical cord mesenchymal stem cells for treatment of type 1 diabetes: therapeutic effects and immunomodulatory mechanism

doi:10.3969/j.issn.2095-4344.0437

Abstract

Abstract:

BACKGROUND: Umbilical cord mesenchymal stem cells (UC-MSCs) can differentiate into islet beta cells for type 1 diabetes mellitus therapy; however, it is necessary to further improve the therapeutic effect and study its immunomodelatory mechanism.
OBJECTIVE: To investigate the therapeutic effect and immunomodulatory mechanism of 3D cultured UC-MSCs in type 1 diabetes mellitus mice.
METHODS: UC-MSCs were separated by tissue-attached method and cultured in 3D system. Cell morphology was detected by scanning electron microscope and surface markers were assayed by flow cytometry. A mouse model of type 1 diabetes mellitus was made via injection of streptozotocin. Mice in stem cell transplantation group were given injection of UC-MSCs on the 7^th day after modeling, and those in model group and control group were injected the same volume of normal saline. Fasting blood glucose level in each group was detected once a week, for continuous 4 weeks. Mouse spleen mononuclear cells suspension was prepared at the 30^th day after injection, T cell subset changes were detected by flow cytometry, and expression levels of serum cytokines, interleukin (IL)-4, IL-10, IL-2 and interferon-γ were measured by ELISA kits.
RESULTS AND CONCLUSION: (1) UC-MSCs cultured in the 3D system grew well and highly expressed surface markers CD44, CD73, CD90, CD105. (2) The blood glucose level was reduced significantly at 2-4 weeks after stem cell injection, and there was a significant difference compared with the model group (P < 0.05). The blood glucose level in the 3D cultured stem cell transplantation group was lower than that in the 2D cultured stem cell transplantation group, and there was significant difference at the 4^th week (P < 0.05). (3) Compared with the model group, Th1 cell percentage declined, Th2 cell and Treg percentages increased significantly in the stem cell transplantation group (P < 0.01); compared with the 2D cultured stem cell transplantation group, Th1 cell percentage declined, Th2 cell and Treg percentages increased significantly in the 3D cultured stem cell transplantation group (P < 0.05). (4) Compared with the model group, the levels of IL-2 and interferon-γ in the stem cell transplantation group decreased, while the levels of IL-4 and IL-10 increased significantly; compared with the 2D system stem cell transplantation group, the IL-2 level in 3D system stem cell transplantation group decreased, while the levels of IL-4 and IL-10 increased significantly (P < 0.05). These results indicate that 3D cultured stem cell transplantation in the treatment of type 1 diabetes mice has better curative effects than 2D cultured stem cell transplantation, and the possible mechanism may be related to upregulate Treg cells and maintain Th1/Th2 balance in the body.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Umbilical Cord, Mesenchymal Stem Cell Transplantation, Diabetes Mellitus, Type 1, Blood Glucose, Immunomodulation, Tissue Engineering

CLC Number:

R318

Liu Ke-na, Li Dong, Yang Guang-sheng. 3D-cultured umbilical cord mesenchymal stem cells for treatment of type 1 diabetes: therapeutic effects and immunomodulatory mechanism[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(5): 692-697.

Figures/Tables 2

2.2 脐带间充干细胞表面标志物表达流式细胞仪结果显示3D培养的脐带间充质干细胞表面标志物CD44、CD73、CD90和CD105呈高表达，CD34和CD45呈低表达，与2D培养的脐带间充质干细胞相比差异有显著性意义(P < 0.05)，见表1。 2.3 各组小鼠血糖水平模型组小鼠出现饮水量、尿量、进食量明显增加的现象，而且体毛无光泽，活动减少，体质量下降，说明造模成功。模型组与对照组相比，血糖明显升高，差异具有显著性意义(P < 0.05)。移植1周后，2D和3D干细胞移植组小鼠血糖与模型组相比差异不明显(P < 0.05)，移植后2-4周，小鼠血糖水平下降明显，与模型组相比差异有显著性意义(P < 0.05)。3D干细胞移植组血糖水平从2周起低于2D干细胞移植组，4周出现显著性差异(P < 0.05)，见表2。 2.4 各组小鼠T细胞亚群变化流式细胞术检测各组小鼠T细胞亚群百分比，结果见表3和图2。模型组与对照组相比，Th1细胞亚群(CD4+IFNγ+)百分比明显升高(P < 0.01)，Th2细胞亚群(CD4+IL-4+)和Treg细胞(CD4+CD25+)百分比均显著下降(P < 0.01)。2D和3D干细胞移植组与模型组相比，Th1细胞亚群百分比明显降低(P < 0.05)，Th2细胞亚群和Treg细胞百分比均显著升高(P < 0.01)，说明脐带间充质干细胞可以上调糖尿病小鼠的Treg细胞表达。3D干细胞移植组与2D干细胞移植组相比，Th1细胞亚群百分比降低，Th2细胞亚群和Treg细胞百分比明显升高(P < 0.05)。 2.5 各组小鼠血清细胞因子水平如表4所示，模型组与对照组相比，白细胞介素2、γ-干扰素、白细胞介素4和白细胞介素10的表达水平均显著升高，差异有显著性意义(P < 0.05)。2D和3D干细胞移植组Th1型细胞因子白细胞介素2和γ-干扰素的表达水平与模型组相比显著降低，而Th2型细胞因子白细胞介素4和白细胞介素10的表达水平显著升高，差异有显著性意义(P < 0.05)。3D干细胞移植组与2D干细胞移植组相比，白细胞介素2表达水平显著降低，白细胞介素4和白细胞介素10的表达水平显著升高(P < 0.05)，γ-干扰素的变化不明显。"

References

[1] Handorf AM, Sollinger HW, Alam T. Insulin gene therapy for type 1 diabetes mellitus. Exp Clin Transplant. 2015;13 Suppl 1:37-45.

[2] Morgan E, Halliday SR, Campbell GR, et al. Vaccinations and childhood type 1 diabetes mellitus: a meta-analysis of observational studies. Diabetologia. 2016;59(2):237-243.

[3] American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care. 2004;27 Suppl 1:S5-S10.

[4] 郝永蕾,朱旅云,王更银,等.体外诱导人脐带间充质干细胞分化为胰岛样细胞实验研究[J].临床误诊误治,2010,23(9):804-807.

[5] 申义,王意忠,时瀚,等.人脐带间充质干细胞分化胰岛样细胞过程中胰岛素和巢蛋白的表达[J].中国组织工程研究, 2016,20(50): 7500-7506.

[6] Aguayo-Mazzucato C, Bonner-Weir S. Stem cell therapy for type 1 diabetes mellitus. Nat Rev Endocrinol. 2010;6(3):139-148.

[7] Hu J, Yu X, Wang Z, et al. Long term effects of the implantation of Wharton's jelly-derived mesenchymal stem cells from the umbilical cord for newly-onset type 1 diabetes mellitus. Endocr J. 2013;60(3):347-357.

[8] 贾晓蕾,沈山梅,李莉蓉,等. 异体间充质干细胞移植治疗酮症起病1型糖尿病的临床疗效[J].中华糖尿病杂志, 2015, 7(7):425-430.

[9] He B, Li X, Yu H, et al. Therapeutic potential of umbilical cord blood cells for type 1 diabetes mellitus. J Diabetes. 2015;7(6): 762-773.

[10] Liu S, Yuan M, Hou K, et al. Immune characterization of mesenchymal stem cells in human umbilical cord Wharton's jelly and derived cartilage cells. Cell Immunol. 2012;278(1-2):35-44.

[11] Yang H, Sun J, Li Y, et al. Human umbilical cord-derived mesenchymal stem cells suppress proliferation of PHA-activated lymphocytes in vitro by inducing CD4(+)CD25(high)CD45RA(+) regulatory T cell production and modulating cytokine secretion. Cell Immunol. 2016;302:26-31.

[12] Vellasamy S, Sandrasaigaran P, Vidyadaran S, et al. Mesenchymal stem cells of human placenta and umbilical cord suppress T-cell proliferation at G0 phase of cell cycle. Cell Biol Int. 2013;37(3):250-256.

[13] Braghirolli DI, Zamboni F, Acasigua GA, et al. Association of electrospinning with electrospraying: a strategy to produce 3D scaffolds with incorporated stem cells for use in tissue engineering. Int J Nanomedicine. 2015;10:5159-5169.

[14] González S, Mei H, Nakatsu MN, et al. A 3D culture system enhances the ability of human bone marrow stromal cells to support the growth of limbal stem/progenitor cells. Stem Cell Res. 2016;16(2):358-364.

[15] Rath SN, Nooeaid P, Arkudas A, et al. Adipose- and bone marrow-derived mesenchymal stem cells display different osteogenic differentiation patterns in 3D bioactive glass-based scaffolds. J Tissue Eng Regen Med. 2016;10(10):E497-E509.

[16] Griffith LG, Swartz MA. Capturing complex 3D tissue physiology in vitro. Nat Rev Mol Cell Biol. 2006;7(3):211-224.

[17] Ferlin KM, Prendergast ME, Miller ML, et al. Influence of 3D printed porous architecture on mesenchymal stem cell enrichment and differentiation. Acta Biomater. 2016;32:161-169.

[18] Li SL, Liu Y, Hui L. Construction of engineering adipose-like tissue in vivo utilizing human insulin gene-modified umbilical cord mesenchymal stromal cells with silk fibroin 3D scaffolds. J Tissue Eng Regen Med. 2015;9(12):E267-275.

[19] Qin Y, He YH, Hou N, et al. Sonic hedgehog improves ischemia-induced neovascularization by enhancing endothelial progenitor cell function in type 1 diabetes. Mol Cell Endocrinol. 2016;423:30-39.

[20] Colomeu TC, Figueiredo D, Cazarin CB, et al. Antioxidant and anti-diabetic potential of Passiflora alata Curtis aqueous leaves extract in type 1 diabetes mellitus (NOD-mice). Int Immunopharmacol. 2014;18(1):106-115.

[21] Grisé KN, Olver TD, McDonald MW, et al. High Intensity Aerobic Exercise Training Improves Deficits of Cardiovascular Autonomic Function in a Rat Model of Type 1 Diabetes Mellitus with Moderate Hyperglycemia. J Diabetes Res. 2016;2016: 8164518.

[22] 付清松, 夏玉军, 张明, 等. 三维球体间充质干细胞移植对大鼠缺血再灌注损伤脑组织TNF-α及凋亡相关蛋白表达的影响[J].第二军医大学学报, 2015,36(8):845-850.

[23] Antebi B, Zhang Z, Wang Y, et al. Stromal-cell-derived extracellular matrix promotes the proliferation and retains the osteogenic differentiation capacity of mesenchymal stem cells on three-dimensional scaffolds. Tissue Eng Part C Methods. 2015;21(2):171-181.

[24] Occhetta P, Centola M, Tonnarelli B, et al. High-Throughput Microfluidic Platform for 3D Cultures of Mesenchymal Stem Cells, Towards Engineering Developmental Processes. Sci Rep. 2015;5:10288.

[25] Papadimitropoulos A, Piccinini E, Brachat S, et al. Expansion of human mesenchymal stromal cells from fresh bone marrow in a 3D scaffold-based system under direct perfusion. PLoS One. 2014;9(7):e102359.

[26] Caron I, Rossi F, Papa S, et al. A new three dimensional biomimetic hydrogel to deliver factors secreted by human mesenchymal stem cells in spinal cord injury. Biomaterials. 2016;75:135-147.

[27] Li Y, Guo G, Li L, et al. Three-dimensional spheroid culture of human umbilical cord mesenchymal stem cells promotes cell yield and stemness maintenance. Cell Tissue Res. 2015; 360(2):297-307.

[28] Rodina AV, Tenchurin TK, Saprykin VP, et al. Migration and Proliferative Activity of Mesenchymal Stem Cells in 3D Polylactide Scaffolds Depends on Cell Seeding Technique and Collagen Modification. Bull Exp Biol Med. 2016;162(1):120-126.

[29] Zheng Z, Shen W, Le H, et al. Three-dimensional parallel collagen scaffold promotes tendon extracellular matrix formation. Zhejiang Da Xue Xue Bao Yi Xue Ban. 2016;45(2):120-125.

[30] Shami A, Gonçalves I, Hultgårdh-Nilsson A. Collagen and related extracellular matrix proteins in atherosclerotic plaque development. Curr Opin Lipidol. 2014;25(5):394-399.

[31] 刘锐,刘畅,殷甜甜. 三维培养下化学方法诱导肌源性干细胞向胰岛素分泌细胞分化的研究[J]. 辽宁医学学报, 2016,37(1):4-7.

[32] Zhang Y, Zhang Y, Gu W, et al. TH1/TH2 cell differentiation and molecular signals. Adv Exp Med Biol. 2014;841:15-44.

[33] Perez-Mazliah D, Langhorne J. CD4 T-cell subsets in malaria: TH1/TH2 revisited. Front Immunol. 2015;5:671.

[34] Matsuzaki J, Tsuji T, Imazeki I, et al. Immunosteroid as a regulator for Th1/Th2 balance: its possible role in autoimmune diseases.Autoimmunity. 2005;38(5):369-375.

[35] Raz I, Eldor R, Naparstek Y. et al. Immune modulation for prevention of type 1 diabetes mellitus.Trends Biotechnol. 2005;23(3):128-134.

[36] Heurtier AH, Boitard C.T-cell regulation in murine and human autoimmune diabetes: the role of TH1 and TH2 cells. Diabetes Metab. 1997;23(5):377-385.

[37] Haritha C, Shankar V, Trivedi HL, et al. Mesenchymal Stem Cells Transplantation with Non-myeloablative Conditioning for Type I Diabetes Mellitus : Lab to Clinic. International Journal of Radiation Oncology Biology Physics. 2009; 75(3) :S486-S487.

[38] Masteller EL, Tang Q, Bluestone JA. Antigen-specific regulatory T cells--ex vivo expansion and therapeutic potential. Semin Immunol. 2006;18(2):103-110.

[39] Tan T, Xiang Y, Chang C, et al. Alteration of regulatory T cells in type 1 diabetes mellitus: a comprehensive review. Clin Rev Allergy Immunol. 2014;47(2):234-243.

[40] Lee RH, Seo MJ, Reger RL, et al. Multipotent stromal cells from human marrow home to and promote repair of pancreatic islets and renal glomeruli in diabetic NOD/scid mice.Proc Natl Acad Sci U S A. 2006;103(46):17438-17443.