Interferon-gamma combined with lipopolysaccharide polarizes
human umbilical cord-derived mesenchymal stem cells to a MSC2 phenotype

doi:10.3969/j.issn.2095-4344.2050

Abstract

Abstract:

BACKGROUND: Mesenchymal stem cells have been widely applied in autoimmune diseases and graft-versus-host diseases because of their immunomodulatory capabilities. However, mesenchymal stem cells have plasticity in immunomodulation, which leads to heterogeneity and instability when used in vivo.

OBJECTIVE: To investigate the polarization of human umbilical cord derived mesenchymal stem cells to an immunosuppressive phenotype (MSC2) in the inflammatory microenvironment induced by interferon-γ and lipopolysaccharide.

METHODS: Human umbilical cord-derived mesenchymal stem cells were isolated and cultured in vitro, and then were identified by morphological characteristics, surface markers, adipogenesis and osteoinduction activity. Human umbilical cord-derived mesenchymal stem cells were treated with interferon-γ (10 μg/L), lipopolysaccharide (100 μg/L), or their combination for 24 hours, respectively, and were then co-cultured with phytohemagglutinin pre-treated peripheral blood mononuclear cells for 5 days under direct or Transwell indirect contact. The percentages of regulatory T cells and T helper 1 cells were analyzed by flow cytometry at different times. The mRNA expression levels of Toll-like receptors 2, 3 and 4 were detected by real-time fluorescence quantitative PCR.

RESULTS AND CONCLUSION: (1) Human umbilical cord derived mesenchymal stem cells exhibited spindle-shaped or fibroblast-like morphology, highly expressed CD73, CD90 and CD105, and lacked expression of CD34, CD45 and HLA-DR. (2) Under direct or indirect co-culture, human umbilical cord-derived mesenchymal stem cells pre-treated with interferon-γ and lipopolysaccharide could promote the generation of regulatory T cells, which was superior to the interferon-γ, lipopolysaccharid, un-treated and control groups (P < 0.05). (3) The percentage of T helper 1 cells gradually decreased over time. (4) Under indirect co-culture, human umbilical cord derived mesenchymal stem cells pre-treated with interferon-γ and lipopolysaccharide were polarized into immunosuppressive MSC2 phenotype at an earlier period and highly expressed Toll-like receptor 3 (P < 0.05). (5) In conclusion, the combination of interferon-γ (10 μg/L) and lipopolysaccharide (100 μg/L) results in the high-efficient polarization of mesenchymal stem cells toward the MSC2 phenotype under indirect co-culture, and the immunosuppressive capability of MSC2 is independent of intercellular contact, which provides clinical evidence for the MSC2-derived exosome therapy in the future.

Key words: human umbilical cord derived mesenchymal stem cells, interferon-γ, lipopolysaccharide, regulatory T cells, Toll-like receptors

CLC Number:

Huang Tian, Huang Xin, Lai Peilong, Geng Suxia, Chen Xiaomei, Wang Yulian, Guo Liyan, Zeng Gaochun, Han Fengzhen, Li Xiaohong, Du Xin, Weng Jianyu. Interferon-gamma combined with lipopolysaccharide polarizes human umbilical cord-derived mesenchymal stem cells to a MSC2 phenotype[J]. Chinese Journal of Tissue Engineering Research, 2020, 24(13): 2020-2027.

Figures/Tables 4

(2)γ干扰素、脂多糖刺激人脐带间充质干细胞对Th1细胞比例的影响：共培养第3天，与单纯外周血单个核细胞组相比，刺激与无刺激人脐带间充质干细胞各组的Th1细胞比例均明显升高(P < 0.05)。共培养第4天，与单纯外周血单个核细胞组及无刺激人脐带间充质干细胞组相比，经γ干扰素、脂多糖或γ干扰素+脂多糖刺激人脐带间充质干细胞组Th1细胞比例均出现下降趋势(P > 0.05)。共培养第5天，与单纯外周血单个核细胞组相比，刺激人脐带间充质干细胞各组Th1细胞比例均明显下降，单纯外周血单个核细胞组为(19.8±3.4)%、γ干扰素刺激组为(13.7±1.1)%、脂多糖刺激组为(13±4)%、γ干扰素联合脂多糖刺激组为(12.3±4.0)%，差异有显著性意义(P < 0.05)。结果表明：随着培养时间延长，单纯外周血单个核细胞组的Th1细胞比例逐步上升，人脐带间充质干细胞共培养各组的Th1细胞比例逐步下降；在共培养第5天时，刺激人脐带间充质干细胞各组抑制Th1分化的作用优于无刺激人脐带间充质干细胞组，见图2C。γ干扰素和/或脂多糖刺激后可以降低Th1细胞比例，同时也表明其发挥免疫调节作用需要四五天的时间。 2.2.2 间接共培养 (1)γ干扰素、脂多糖刺激人脐带间充质干细胞对调节性T细胞比例的影响：共培养第3天，γ干扰素联合脂多糖刺激组调节性T细胞比例为(15.9±1.6)%，明显高于单纯外周血单个核细胞组(9.7±0.5)%、γ干扰素刺激组(12.6± 1.2)%、脂多糖刺激组(11.8±1.3)%和无刺激组 (10.4± 0.8)%，差异均有显著性意义(P < 0.05)；γ干扰素联合脂多糖刺激组和γ干扰素刺激组优于无刺激组(P < 0.05)，见图3A、B。共培养第4天和第5天，与单纯外周血单个核细胞组相比，刺激与无刺激的人脐带间充质干细胞各组调节性T细胞比例均明显增加(P < 0.05)。结果表明:间接共培养下，刺激后的人脐带间充质干细胞在早期(第3天)即可促进调节性T细胞比例增加，同时γ干扰素单独或联合脂多糖刺激人脐带间充质干细胞均可显著增强调节性T细胞比例。 "

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