Immune properties of human CD200+ sub-population from human placenta-derived mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.1528

Abstract

Abstract:

BACKGROUND: In different studies, mesenchymal stem cells exhibit contradictory immune characteristics. This may be because mesenchymal stem cells are a group of complex cells and different subgroups exhibit different immune characteristics.
OBJECTIVE: To isolate and identify a sub-population of placenta-derived mesenchymal stem cells with high positive surface antigen of CD200, and to investigate the immune characteristics of this sub-population.
METHODS: Flow cytometry was utilized to sort CD200⁺ placenta-derived mesenchymal stem cells. Phenotypic characterization and multilineage differentiation of the sorted cells were determined. Cytokine secretion, involving interleukin-6 and interleukin-8, was measured by ELISA. Mixed lymphocyte proliferation assay was used to determine the immune characteristics of CD200⁺ placenta-derived mesenchymal stem cells.
RESULTS AND CONCLUSION: The CD200⁺ placenta-derived mesenchymal stem cells exhibited homogeneous spindle shape, and highly expressed CD44, CD73, CD90 and CD105, negatively expressed CD34, CD45, CD80 and CD86. After induced differentiation, the cells were positively stained by oil red O staining and alizarlin red staining, which were the markers of adipocytes and osteoblasts. The results of ELISA indicated that the expression level of interleukin-6 and interleukin-8 in the CD200⁺ placenta-derived mesenchymal stem cells was strikingly higher than placenta-derived mesenchymal stem cells and CD200^- placenta-derived mesenchymal stem cells. Compared with normal and CD200^- placenta-derived mesenchymal stem cells, CD200⁺ placenta-derived mesenchymal stem cells inhibited T cell proliferation significantly, lowered the levels of interferon-γ and interleukin-2, and upregulated the level of interleukin-10. We found that CD200⁺ placenta-derived mesenchymal stem cells have better immunoregulatory capability compared with normal and CD200^-placenta-derived mesenchymal stem cells, which may provide an ideal theoretical basis for clinical use.

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: Placenta, Mesenchymal Stem Cells, Immunomodulation, Tissue Engineering

CLC Number:

R459.9

Liu Ting, Ma Xiaona, Ma Haibin, Yang Tingting, Jin Yiran, Liang Xueyun. Immune properties of human CD200⁺sub-population from human placenta-derived mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(1): 79-84.

Figures/Tables 1

2.1 CD200+-PMSCs的分选结果经流式细胞术检测，分选前PMSCs的CD200表达水平在60%-70%，分选后CD200+-PMSCs的CD200表达水平在90%以上，见图1。 2.2 CD200+-PMSCs的形态特征分选获得的CD200+- PMSCs保持了间充质干细胞的形态特征，绝大多数细胞形态均一，细胞核较大，胞质透明，呈涡旋状生长，见图2A，而CD200--PMSCs没有表现出形态上的差异，见图2B。 2.3 CD200+-PMSCs表达与间充质干细胞一致的表面抗原分选后CD200+-PMSCs高表达CD73、CD90和CD105，不表达CD34、CD45、CD80和CD86，见图3，具有典型的间充质干细胞表面标志。 2.4 CD200+-PMSCs亚群细胞具有多向分化潜能成脂诱导分化3 d左右，细胞增殖停止，细胞开始逐渐变大，诱导分化12 d左右，镜下可见细胞形态呈不规则多边形，胞质内出现多个圆泡，油红O染色后可见圆泡被染成红色，即为脂滴，证明分选获得的CD200+-PMSCs成功诱导分化为脂肪细胞，见图4A，B。成骨诱导分化5-7 d，细胞生长已经连成片，继续诱导至14 d左右，镜下可见细胞层层叠叠紧密生长，挤成一团，茜素红染色后可以观察到有多处成片细胞被染成红色，即为诱导产生的钙结节，证明分选获得的CD200+-PMSCs成功诱导分化为成骨细胞，见图4C，D。 2.5 CD200+-PMSCs的白细胞介素6、白细胞介素8因子分泌量 CD200+-PMSCs的白细胞介素6分泌量明显高于PMSCs(P < 0.01)和CD200--PMSCs(P < 0.001)。白细胞介素8的分泌量也高于PMSCs(P < 0.05)和CD200-- PMSCs(P < 0.05)，差异均有显著性意义，见图5。 2.6 CD200+-PMSCs对PHA刺激的T淋巴细胞增殖有明显的抑制作用当活化的T淋巴细胞分别与PMSCs、CD200+-PMSCs、CD200--PMSCs以10∶1的比例共培养时，这3种细胞均能明显抑制T淋巴细胞的增殖，其中CD200+-PMSCs的抑制作用明显强于CD200--PMSCs(P < 0.01)，差异有显著性意义，而与PMSCs没有显著差异。当共培养细胞比例为50∶1时，T淋巴细胞的增殖仍能被抑制(P < 0.05)，但是抑制作用有所减弱，并且3种细胞的抑制作用没有显著差异，见图6。 2.7 CD200+-PMSCs对PHA刺激的T淋巴细胞分泌因子的调节作用见图7。活化的T淋巴细胞与间充质细胞以10∶1的比率共培养48 h后，CD200+-PMSCs能够明显下调干扰素γ水平，与PMSCs、CD200--PMSCs比较差异有显著性意义(P < 0.05)。 CD200+-PMSCs同样能够明显下调活化的T淋巴细胞分泌白细胞介素2的水平，与PMSCs(P < 0.05)、CD200--PMSCs(P < 0.001)比较差异有显著性意义。活化的T淋巴细胞分泌白细胞介素10的水平均能够被3种细胞显著上调，但是3种细胞的上调作用没有显著差异。"

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