Immunogenicity of insulin producing cells differentiating from human umbilical cord mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.0488

Abstract

Abstract:

BACKGROUND: Human umbilical cord mesenchymal stem cells (hUC-MSCs) have low immunogenicity and it is unclear whether insulin producing cells (IPCs) that differentiate from hUC-MSCs have low immunogenicity.
OBJECTIVE: To investigate the immunogenicity of IPCs differentiating from hUC-MSCs in vitro and after IPCs transplantation into the host.
METHODS: (1) The hUC-MSCs were induced to differentiate into IPCs according to the modified scheme. Flow cytometry assay was used to detect the immunophenotype and apoptotic rate of IPCs in a cytotoxicity test. (2) Cell counting kit-8 was used to detect the proliferative capacity of human peripheral blood mononuclear cells in the one-way mixed lymphocyte assay. (3) The IPCs were then transplanted into the abdominal cavity and left renal capsule of mice, and then the infiltration of immune cells was detected by flow cytometry and immunohistochemistry.
RESULTS AND CONCLUSION: The IPCs highly expressed HLA-ABC and lowly expressed HLA-DR, CD40 and CD80. The apoptosis rate of IPCs increased with the increase of pre-sensitized splenocytes in the cytotoxicity test. In the one-way mixed lymphocyte assay, IPCs inhibited the proliferation of human peripheral blood mononuclear cells when the target ratio was 10:1 and 50:1. After IPCs transplantation, the number of lymphocytes was increased in the transplanted site. In summary, our results show that IPCs that differentiate from hUC-MSCs maintain low immunogenicity in vitro, but have some immunogenicity after transplantation into the host due to microenvironment changes.

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 Cells, Cell Differentiation, Insulin-Secreting Cells, Tissue Engineering

CLC Number:

R394.2

Li Lan-lan, Li Ning, Yang Xiao-fei, Chen Tao, Ou Gang-wei, Li Fu-rong. Immunogenicity of insulin producing cells differentiating from human umbilical cord mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(13): 2045-2050.

Figures/Tables 1

2.1 hUCMSCs和IPCs分离和鉴定从孕妇脐带分离、纯化的hUCMSCs，经流式细胞分析表达CD29(99.86%)、CD73(99.8%)、CD105(99.94%)；几乎不表达CD34 (1.45%)、CD45(1.23%)和HLA-DR(1.02%)，与文献[16-17]报道的间充质干细胞表型相符。成脂成骨诱导分化实验呈阳性。对分化的IPCs鉴定，胰岛素分泌阳性率23%，纯化的胰岛素分泌细胞阳性率87%。 2.2 分化的IPCs表面免疫分子的表达如图1所示，流式细胞术分析hUCMSCs分化的IPCs，HLA-ABC表达率为(97.93±1.71)%，HLA-DR表达率为(1.13±0.31)%，CD40表达率为(2.41±0.53)%，CD80表达率为(2.33±0.96)%，与hUCMSCs组HLA-ABC(90.17±3.34)%、HLA-DR (1.06± 0.33)%、CD40(1.83±0.85)%、CD80(2.01±0.90)的表达相似(P > 0.05)。 2.3 hUCMSCs或IPCs对外周血单个核细胞增殖能力的影响如图2所示，在单向混合淋巴细胞实验中，外周血单个核细胞与hUCMSCs共培养后，外周血单个核细胞的刺激指数是0.24±0.04，与阳性对照组和阴性对照组比较差异有显著性意义(P < 0.05)。外周血单个核细胞与IPCs共培养后，外周血单个核细胞的刺激指数是0.79±0.06，低于阳性对照组，与阴性对照组比较差异无显著性意义(P > 0.05)，明显高于hUCMSCs组(P < 0.05)。结果表明，hUCMSCs分化的IPCs免疫原性增强，但免疫抑制能力减弱。 2.4 预致敏小鼠淋巴细胞对hUCMSCs或IPCs的细胞毒性及培养上清中细胞因子的表达如图3A所示，预致敏小鼠淋巴细胞与hUCMSCs或IPCs在4个效靶比下共培养，发现在不同效靶比之间预致敏小鼠淋巴细胞对hUCMSCs或IPCs有细胞毒性差异(P < 0.05)，随着效靶比的升高预致敏小鼠淋巴细胞对hUCMSCs或IPCs的细胞毒性越大，即凋亡率越高。当效靶比为100∶1时，hUCMSCs和IPCs的凋亡率分别为(34.01±0.44)%和(39.1±0.66)% (P > 0.05)。结果表明，IPCs具有低免疫原性而不能激活体内记忆T细胞和细胞毒性T细胞。 IFN-γ，IL-2和IL-4是Th1/Th2的细胞因子，在共培养细胞的上清液中检测这些细胞因子，如图3B-D所示，随着效靶比的升高，预致敏小鼠淋巴细胞与hUCMSCs或IPCs共培养组IFN-γ和IL-2水平升高，而IL-4水平降低(P < 0.05)。在不同效靶比下，hUCMSCs共培养组与IPCs共培养组比较差异无显著性意义(P > 0.05)。结果表明IPCs与hUCMSCs呈低免疫原性未能激活免疫细胞。 2.5 hUCMSCs或IPCs腹腔移植后炎性细胞分析结果流式细胞术分析显示hUCMSCs组腹腔灌洗液细胞总数为(7.10±0.55)×105，约55%的细胞表达CD45，即白细胞；约7%的细胞表达 CD3e，即T淋巴细胞。IPCs组腹腔灌洗液细胞总数为(7.92±0.09)×105，约61%的细胞表达CD45，约12%的细胞表达CD3e；与hUCMSCs组相比，IPCs组细胞数明显增加(P < 0.05)，见图4。结果表明IPCs对宿主具有免疫刺激作用，可引起炎性细胞浸润至细胞移植部位。 2.6 hUCMSCs或IPCs左肾移植后病理学变化如图5所示，hUCMSCs或IPCs左肾包膜移植15 d后，可见移植部位存在大量的移植细胞，并伴有少量的淋巴细胞及单核细胞浸润，PBS组无炎症细胞浸润。与移植15 d相比，移植30 d后IPCs组免疫细胞渗出明显增多，而hUCMSCs组免疫细胞明显减少，IPCs组免疫细胞渗出明显高于hUCMSCs组。结果表明随时间推移，IPCs移植到体内免疫原性增强并能引起淋巴细胞浸润到移植位点。"

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