Induced pluripotent stem cell transplantation for systemic lupus erythematosus in a mouse model

doi:10.3969/j.issn.2095-4344.1826

Abstract

Abstract:

BACKGROUND: The biological characteristics of autologous mesenchymal stem cells for transplantation of systemic lupus erythematosus (SLE) are susceptible to the disease. Cell therapy with low immunogenic stem cells that are not affected by the disease has become a research hotspot. To date, treatment of systemic lupus erythematosus with autologous induced pluripotent stem cell transplantation has not been reported.
OBJECTIVE: To evaluate the therapeutic effect of transplantation of induced pluripotent stem cells derived from autologous fibroblasts in mice with systemic lupus erythematosus.
METHODS: Fifty-four 8-week-old MRL/lpr mice were randomly divided into three groups. Model control group was not treated, reagent control group was injected with PBS solution through tail vein, and stem cell treatment group was injected with autologous induced pluripotent stem cells through tail vein. Serum creatinine, urea nitrogen, anti-double-stranded DNA antibodies and anti-nuclear antibodies were detected by ELISA at 4 weeks of cell treatment. Kidney tissue morphology was observed by hematoxylin-eosin staining on paraffin sections. Urinary protein concentrations were detected by Coomassie brilliant blue method. Flow cytometry was used to detect the percentage of regulatory T cells in peripheral blood, percentage of splenic dendritic cells, and ratio of splenic helper T1 cells to helper T2 cells.
RESULTS AND CONCLUSION: The levels of serum creatinine, urea nitrogen, anti-double-stranded DNA antibody and anti-nuclear antibody in the stem cell treatment group were lower than those in the model control and reagent control groups, while decreased urine protein concentration, increased percentages of regulatory T cells in peripheral blood, increased percentage of splenic dendritic cells, and increased splenic Th1/Th2 ratio were detected in the stem cell treatment group as compared with the other two groups. Compared with the model control and reagent control groups, there was decreased glomerular volume, reduced number of mesangial cells, degeneration of renal tubular epithelial cells and decreased tubular type in the stem cell treatment group. In summary, induced pluripotent stem cell transplantation from autologous skin fibroblasts for treat systemic lupus erythematosus can alleviate renal tissue damage, improve renal function and play an immunoregulatory role in mice.

Key words: induced pluripotent stem cells, systemic lupus erythematosus, stem cell transplantation, somatic cell reprogramming, kidney, immune function, regulatory T cells

CLC Number:

Ma Linjie, Xue Wentao, Tan Jupeng. Induced pluripotent stem cell transplantation for systemic lupus erythematosus in a mouse model[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(33): 5286-5292.

Figures/Tables 2

2.1 MRL/lpr小鼠诱导性多能干细胞的鉴定碱性磷酸酶染色结果显示，克隆样生长的细胞中分布着大量紫红色颗粒，即为碱性磷酸酶染色阳性，见图1A；细胞免疫荧光染色结果显示，细胞胞质内可见OCT4、SSEA-1和SOX2等多能性标记物呈阳性表达，见图1B-D；拟胚体分化荧光染色结果显示，形成拟胚体的细胞胞质内可见标记内胚层的AFP、标记中胚层的Desmin和标记外胚层的nestin呈阳性表达，见图1E。以上结果表明，从MRL/lpr小鼠皮肤成纤维细胞重编程获得的细胞具有诱导性多能干细胞的特性。 2.2 实验小鼠的数量分析动物体内实验使用54只8周龄MRL/lpr小鼠和18只8龄C57BL/6小鼠，实验过程中无小鼠死亡，72只小鼠的实验结果均计入统计分析。 2.3 各组血清尿素氮、肌酐、抗双链DNA抗体和抗核抗体水平的比较与正常对照组比较，模型对照组、试剂对照组和干细胞治疗组的血清肌酐、尿素氮、抗双链DNA抗体和抗核抗体水平升高，差异有显著性意义(P < 0.01)；与模型对照组及试剂对照组比较，干细胞治疗组4项指标降低，差异有显著性意义(P < 0.01)。以上结果说明诱导性多能干细胞治疗能降低MRL/lpr小鼠血清肌酐、尿素氮、抗双链DNA抗体和抗核抗体水平，见图2。 2.4 各组肾脏组织形态结构正常对照组小鼠肾脏组织结构正常，见图3A；模型对照组和试剂对照组小鼠肾脏间质内可见肾小球体积增大，系膜细胞增生，肾小管上皮细胞呈空泡样变性，肾小管腔内出现管型，见图3B和3C；干细胞治疗组小鼠肾脏肾小球体积缩小，系膜细胞减少，肾小管上皮细胞变性改善，肾小管腔内管型减少，见图3D。苏木精-伊红染色结果说明诱导性多能干细胞治疗能减轻MRL/lpr小鼠肾脏组织损伤。 2.5 各组尿蛋白浓度的比较与正常对照组比较，模型对照组、试剂对照组和干细胞治疗组尿蛋白浓度均升高，差异有显著性意义(P < 0.01)；与模型对照组及试剂对照组比较，干细胞治疗组的尿蛋白浓度降低，差异有显著性意义(P < 0.01)；模型对照组与试剂对照组比较，差异无显著性意义(P > 0.05)，见图4。以上结果提示诱导性多能干细胞治疗改善了小鼠肾脏功能。 2.6 各组外周血调节性T细胞百分率的比较与正常对照组比较，模型对照组、试剂对照组和干细胞治疗组的调节性T细胞百分率降低，差异有显著性意义(P < 0.05)；与模型对照组及试剂对照组比较，干细胞治疗组调节性T细胞百分率升高，差异有显著性意义(P < 0.05)；模型对照组与试剂对照组比较，差异无显著性意义(P > 0.05)，见图5。以上结果提示诱导性多能干细胞治疗提高了外周血调节性T细胞的百分率。 2.7 各组脾脏树突状细胞百分率的比较与正常对照组比较，模型对照组和试剂对照组的树突状细胞百分率降低，差异有显著性意义(P < 0.05)；与模型对照组及试剂对照组比较，干细胞治疗组树突状细胞百分率升高，差异有显著性意义(P < 0.05)；模型对照组与试剂对照组比较，差异无显著性意义(P > 0.05)，见图6。以上结果提示诱导性多能干细胞治疗提高了脾脏树突状细胞百分率。 2.8 脾脏Th1/Th2比值的比较与正常对照组比较，模型对照组和试剂对照组的Th1细胞百分率降低而Th2细胞百分率升高，干细胞治疗组的Th1细胞和Th2细胞百分率均升高，差异有显著性意义(P < 0.05)；与模型对照组及试剂对照组比较，干细胞治疗组Th1细胞百分率升高，差异有显著性意义(P < 0.05)；模型对照组Th1细胞和Th2细胞百分率与试剂对照组比较，差异无显著性意义(P > 0.05)，见图7。与正常对照组比较，模型对照组和试剂对照组Th1/Th2比值降低，差异有显著性意义(P < 0.05)；与模型对照组和试剂对照组比较，干细胞治疗组Th1/Th2比值升高，差异有显著性意义(P < 0.05)。以上结果提示诱导性多能干细胞治疗提高了小鼠脾脏Th1细胞百分率和Th1/Th2比值。"

References

[1]Finzel S, Schaffer S, Rizzi M, et al. Pathogenesis of systemic lupus erythematosus. Z Rheumatol. 2018;77(9):789-798.

[2]Di Battista M, Marcucci E, Elefante E, et al. One year in review 2018: systemic lupus erythematosus. Clin Exp Rheumatol. 2018;36(5):763-777.

[3]Linge P, Fortin PR, Lood C, et al. The non-haemostatic role of platelets in systemic lupus erythematosus. Nat Rev Rheumatol. 2018;14(4):195-213.

[4]Oon S, Huq M, Godfrey T, et al. Systematic review, and meta-analysis of steroid-sparing effect, of biologic agents in randomized, placebo-controlled phase 3 trials for systemic lupus erythematosus. Semin Arthritis Rheum. 2018;48(2): 221-239.

[5]Zhu Y, Feng X. Genetic contribution to mesenchymal stem cell dysfunction in systemic lupus erythematosus. Stem Cell Res Ther. 2018;9(1):149.

[6]Wang D, Sun L. Stem cell therapies for systemic lupus erythematosus: current progress and established evidence. Expert Rev Clin Immunol. 2015;11(6):763-769.

[7]Chalayer É, Ffrench M, Cathébras P. Aplastic anemia as a feature of systemic lupus erythematosus: a case report and literature review. Rheumatol Int. 2015;35(6):1073-1082.

[8]Liang J, Sun L. Mesenchymal stem cells transplantation for systemic lupus erythematosus. Int J Rheum Dis. 2015;18(2): 164-171.

[9]王治国,刘学明,佟胜全,等.骨髓间充质干细胞移植治疗系统性红斑狼疮[J].中国组织工程研究,2016,20(10):1461-1467.

[10]阮光萍,王金祥,杨建勇,等.系统性红斑狼疮小鼠骨髓间充质干细胞的成骨、成脂分化能力下降[J].中国组织工程研究,2014,18(1): 1-6.

[11]Liao J, Chang C, Wu H, et al. Cell-based therapies for systemic lupus erythematosus. Autoimmun Rev. 2015;14(1): 43-48.

[12]Martin PE, O'Shaughnessy EM, Wright CS, et al. The potential of human induced pluripotent stem cells for modelling diabetic wound healing in vitro. Clin Sci (Lond). 2018;132(15):1629-1643.

[13]Zhou R, Jiang G, Tian X, et al. Progress in the molecular mechanisms of genetic epilepsies using patient-induced pluripotent stem cells. Epilepsia Open. 2018;3(3):331-339.

[14]Rowland HA, Hooper NM, Kellett KAB. Modelling Sporadic Alzheimer's Disease Using Induced Pluripotent Stem Cells. Neurochem Res. 2018;43(12):2179-2198.

[15]Pocock JM, Piers TM. Modelling microglial function with induced pluripotent stem cells: an update. Nat Rev Neurosci. 2018;19(8):445-452.

[16]Hwang Y, Broxmeyer HE, Lee MR. Generating autologous hematopoietic cells from human-induced pluripotent stem cells through ectopic expression of transcription factors. Curr Opin Hematol. 2017;24(4):283-288.

[17]Nikitina TV, Menzorov AG, Kashevarova AA, et al. Induced pluripotent stem cell line, IMGTi003-A, derived from skin fibroblasts of an intellectually disabled patient with ring chromosome 13. Stem Cell Res. 2018;33:260-264.

[18]丁桃,李阳,唐睿珠,等.急性肺损伤患者诱导性多能干细胞系的建立[J].南方医科大学学报,2014,34(10):1414-1419.

[19]陈旭东,范文娟,袁科理,等.不同培养条件对小鼠诱导性多能干细胞分化为神经元样细胞的影响[J].细胞与分子免疫学杂志,2015, 31(9):1216-1219,1223.

[20]Schwarz N, Uysal B, Rosa F, et al. Generation of an induced pluripotent stem cell (iPSC) line from a patient with developmental and epileptic encephalopathy carrying a KCNA2 (p.Leu328Val) mutation. Stem Cell Res. 2018;33:6-9.

[21]Sui W, Hou X, Che W, et al. Hematopoietic and mesenchymal stem cell transplantation for severe and refractory systemic lupus erythematosus. Clin Immunol. 2013;148(2):186-197.

[22]Fliefel R, Ehrenfeld M, Otto S. Induced pluripotent stem cells (iPSCs) as a new source of bone in reconstructive surgery: A systematic review and meta-analysis of preclinical studies. J Tissue Eng Regen Med. 2018;12(7):1780-1797.

[23]Celhar T, Fairhurst AM. Modelling clinical systemic lupus erythematosus: similarities, differences and success stories. Rheumatology (Oxford). 2017;56(suppl_1):i88-i99.

[24]De Groof A, Hémon P, Mignen O, et al. Dysregulated Lymphoid Cell Populations in Mouse Models of Systemic Lupus Erythematosus. Clin Rev Allergy Immunol. 2017;53(2): 181-197.

[25]Shin MS, Lee N, Kang I. Effector T-cell subsets in systemic lupus erythematosus: update focusing on Th17 cells. Curr Opin Rheumatol. 2011;23(5):444-448.

[26]Smirnova EV, Krasnova TN, Proskurnina EV, et al. Role of neutrophil dysfunction in the pathogenesis of systemic lupus erythematosus. Ter Arkh. 2017;89(12):110-113.

[27]Dammacco R. Systemic lupus erythematosus and ocular involvement: an overview. Clin Exp Med. 2018;18(2):135-149.

[28]Álvarez K, Vasquez G. Damage-associated molecular patterns and their role as initiators of inflammatory and auto-immune signals in systemic lupus erythematosus. Int Rev Immunol. 2017;36(5):259-270.

[29]Mok CC. Calcineurin inhibitors in systemic lupus erythematosus. Best Pract Res Clin Rheumatol. 2017;31(3): 429-438.

[30]Floris A, Piga M, Cauli A, et al. Predictors of flares in Systemic Lupus Erythematosus: Preventive therapeutic intervention based on serial anti-dsDNA antibodies assessment. Analysis of a monocentric cohort and literature review. Autoimmun Rev. 2016;15(7):656-663.

[31]Bai Y, Tong Y, Liu Y, et al. Self-dsDNA in the pathogenesis of systemic lupus erythematosus. Clin Exp Immunol. 2018; 191(1):1-10.

[32]Momtazi-Borojeni AA, Haftcheshmeh SM, Esmaeili SA, et al. Curcumin: A natural modulator of immune cells in systemic lupus erythematosus. Autoimmun Rev. 2018;17(2):125-135.

[33]王颍翠,潘兴华.间充质干细胞对系统性红斑狼疮T、B细胞的免疫调节机制[J].中国组织工程研究,2017,21(29):4734-4741.

[34]Anolik JH. B cell biology: implications for treatment of systemic lupus erythematosus. Lupus. 2013;22(4):342-349.

[35]Wang D, Huang S, Yuan X, et al. The regulation of the Treg/Th17 balance by mesenchymal stem cells in human systemic lupus erythematosus. Cell Mol Immunol. 2017;14(5): 423-431.