中国组织工程研究

中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (33): 5348-5356.doi: 10.12307/2023.714

• 胚胎干细胞 embryonic stem cells • 上一篇    下一篇

巨噬细胞迁移抑制因子促进人胚胎干细胞分化为腹侧中脑多巴胺能神经祖细胞

郑晓晗1，2，3，冯晓丽1，胡  兰4，高仕君1，2，3，魏艳召1，2，3，黄  婷1，2，3，孙圣童2，魏绪芳2，王  埮1，2，3，赵振强1，2，3   

  1. 海南医学院第一附属医院，1神经内科，4检验科，海南省海口市   570100；2 海南医学院，海南省海口市   570100；3 海南省热带脑科学研究与转化重点实验室， 海南省海口市   570100
  • 收稿日期:2022-09-24 接受日期:2022-11-16 出版日期:2023-11-28 发布日期:2023-03-30
  • 通讯作者: 赵振强，博士，主任医师，海南医学院第一附属医院神经内科，海南省海口市 570100；海南医学院，海南省海口市 570100； 海南省热带脑科学研究与转化重点实验室， 海南省海口市 570100 王埮，博士，主任医师，海南医学院第一附属医院神经内科，海南省海口市 570100；海南医学院，海南省海口市 570100；海南省热带脑科学研究与转化重点实验室， 海南省海口市 570100
  • 作者简介:郑晓晗，女，1995 年生，福建省福州市人，汉族，海南医学院在读硕士，主要从事干细胞移植与神经系统疾病的研究。
  • 基金资助:
    国家自然科学基金(81860238)，项目负责人：赵振强；海南省重点研发计划项目(ZDYF2018233)，项目负责人：赵振强；海南省自然科学基金项目(821RC694)，项目负责人：赵振强；海南省临床医学中心建设项目(琼卫医函[2021]276号)；海南省卫生健康行业科研项目(21A200354)，项目负责人：冯晓丽；海南省研究生创新科研课题(琼教高[2021]116号)，项目负责人：郑晓晗

Macrophage migration inhibitory factor promotes the differentiation of human embryonic stem cells into ventral midbrain dopaminergic neural progenitor cells

Zheng Xiaohan1, 2,  3, Feng Xiaoli1, Hu Lan4, Gao Shijun1, 2,  3, Wei Yanzhao1, 2,  3, Huang Ting1, 2,  3, Sun Shengtong2, Wei Xufang2, Wang Tan1, 2,  3, Zhao Zhenqiang1, 2,  3   

  1. 1Department of Neurology, 4Department of Clinical Laboratory, First Affiliated Hospital of Hainan Medical University, Haikou 570100, Hainan Province, China; 2Hainan Medical University, Haikou 570100, Hainan Province, China; 3Hainan Provincial Key Laboratory of Tropical Brain Research and Translation, Haikou 570100, Hainan Province, China
  • Received:2022-09-24 Accepted:2022-11-16 Online:2023-11-28 Published:2023-03-30
  • Contact: Zhao Zhenqiang, MD, Chief physician, Department of Neurology, First Affiliated Hospital of Hainan Medical University, Haikou 570100, Hainan Province, China; Hainan Medical University, Haikou 570100, Hainan Province, China; Hainan Provincial Key Laboratory of Tropical Brain Research and Translation, Haikou 570100, Hainan Province, China Wang Tan, MD, Chief physician, Department of Neurology, First Affiliated Hospital of Hainan Medical University, Haikou 570100, Hainan Province, China; Hainan Medical University, Haikou 570100, Hainan Province, China; Hainan Provincial Key Laboratory of Tropical Brain Research and Translation, Haikou 570100, Hainan Province, China
  • About author:Zheng Xiaohan, Master candidate, Department of Neurology, First Affiliated Hospital of Hainan Medical University, Haikou 570100, Hainan Province, China; Hainan Medical University, Haikou 570100, Hainan Province, China; Hainan Provincial Key Laboratory of Tropical Brain Research and Translation, Haikou 570100, Hainan Province, China
  • Supported by:
    the National Natural Science Foundation of China, No. 81860238 (to ZZQ); Hainan Key Research & Development Program, No. ZDYF2018233 (to ZZQ); Hainan Natural Science Foundation Project, No. 821RC694 (to ZZQ); Hainan Clinical Medical Center Construction Project, No. QWYH[2021]276; Hainan Health Industry Scientific Research Project, No. 21A200354 (to FXL); Hainan Postgraduate Innovation Research Project, No. QJG[2021]116 (to ZXH)

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摘要：


文题释义：

巨噬细胞迁移抑制因子：是一类具有独特结构的多效免疫调节细胞因子，具有类似趋化因子的功能。自1966年被发现以来，巨噬细胞迁移抑制因子在人体内的功能，尤其是它在固有免疫、免疫细胞招募和炎症反应等方面有了很多的研究进展。巨噬细胞迁移抑制因子广泛表达于多种器官和细胞中，影响多种生物学过程，如增殖、分化、迁移、凋亡和细胞周期进程。
腹侧中脑多巴胺能神经祖细胞：是由神经祖细胞定向中脑腹侧区域分化而来。通常情况下，相对于人多能干细胞和神经祖细胞而言，移植已经定向的多巴胺能祖细胞对于治疗帕金森病更具可行性。高纯度的腹侧中脑多巴胺能神经祖细胞可用于直接移植于体内和体外分化为多巴胺能神经元，有利于降低分化的异质性。

背景：细胞替代疗法是一种可以治疗帕金森病的非常有前景的方法。目前将人多能干细胞进行神经分化的主要方案是“双 SMAD”抑制方案，利用阻断糖原合酶激酶 3β来激活WNT通路信号，随后利用音猬因子信号与骨形态发生蛋白信号之间的调控相结合，可以精确地控制人多能干细胞从底板区域到顶板区域特异性的细胞命运。巨噬细胞迁移抑制因子(macrophage migration inhibitory factor，MIF)是一类具有独特结构的多效免疫调节细胞因子，在多项研究中证明MIF对神经发育和分化非常重要。然而，MIF是否参与诱导干细胞的分化还是未知的。
目的：在“双SMAD”抑制分化方案的基础上，通过在培养基中添加不同浓度MIF及其抑制剂(S，R)-3-(4-羟基苯基)-4，5-二氢-5-异噻唑乙酸甲酯(ISO-1)，观察其对人胚胎干细胞分化为中脑多巴胺能祖细胞的影响，以期寻找解决人胚胎干细胞分化为中脑多巴胺能祖细胞过程异质性问题的新途径。
方法：CCK8检测添加不同浓度MIF和ISO-1培养人胚胎干细胞的存活情况。免疫荧光法检测人胚胎干细胞上MIF受体CD74、CD44、CXCR7的表达。在胚胎干细胞分化16 d后，通过RT-qPCR和Western blot分别检测腹侧中脑标志物LMX1A、FOXA2、EN1、前脑标记物FOXG1、后脑标记物HOXA2、中脑基板标记物PAX6和Wnt1/β-catenin信号通路转录因子SOX6的表达水平。同时，通过免疫荧光法检测LMX1A、FOXA2、EN1、MAP2的表达。将胚胎干细胞继续分化到42 d后用免疫荧光法检测LMX1A、FOXA2、TH和GIRK2在多巴胺能神经元中的表达。

结果与结论：①MIF在人胚胎干细胞内表达，其相关受体CD44和CXCR7也表达，但CD74不发生表达；②添加MIF后，促进了LMX1、FOXA2、EN1的蛋白表达，促进了LMX1、FOXA2的基因表达，同时明显抑制了FOXG1和HOXA2以及PAX6的基因与蛋白表达，有效抑制了分化的异质性；③添加MIF后，多巴胺能神经祖细胞中SOX6并未如预期那样高表达，反而在基因层面表达下降，而其抑制剂ISO-1则使SOX6表达在基因水平上轻度升高；④该研究证明了MIF在人胚胎干细胞的多巴胺能分化潜能与腹侧中脑命运决定中起着重要作用，MIF的干预进一步优化了神经诱导的区域定位。

https://orcid.org/0000-0003-0249-2734(郑晓晗) 

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

关键词: 人胚胎干细胞, 巨噬细胞迁移抑制因子, 帕金森病, 多巴胺能神经祖细胞, 分化

Abstract: BACKGROUND: Cell replacement therapy is a very promising approach to treating Parkinson’s disease. The current main regimen for neural differentiation of human pluripotent stem cells is the “dual SMAD” inhibition regimen, which uses blockade of glycogen synthase kinase 3β to activate WNT pathway signaling, followed by a combination of hedgehog signaling and bone morphogenetic protein signaling regulation to precisely control the specific fate of human pluripotent stem cells from the basal to the parietal regions. Macrophage migration inhibitory factor (MIF), a class of pleiotropic immunomodulatory cytokines with a unique structure, has been shown to be important for neural development and differentiation in several studies. However, whether MIF is involved in the induction of stem cell differentiation is unknown.
OBJECTIVE: On the basis of the “dual SMAD” inhibition regimen, different concentrations of MIF and its inhibitor (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isothiazoleacetic acid methyl ester (ISO-1) were added to the culture medium to observe its effect on the differentiation of human embryonic stem cells into midbrain dopaminergic progenitor cells, to find a new way to solve the problem of heterogeneity during differentiation of human embryonic stem cells into midbrain dopaminergic progenitor cells.
METHODS: CCK8 assay was performed to detect the survival of human embryonic stem cells cultured with the addition of different concentrations of MIF and ISO-1. The expression of MIF receptors CD74, CD44 and CXCR7 on human embryonic stem cells was detected by immunofluorescence. The expression levels of ventral midbrain markers LMX1A, FOXA2, EN1, forebrain marker FOXG1, hindbrain marker HOXA2, midbrain substrate marker PAX6 and transcription factor SOX6 of Wnt1/β-catenin signaling pathway were determined by RT-qPCR and western blotting, respectively, 16 days after differentiation of embryonic stem cells levels. Meanwhile, the expression levels of LMX1A, FOXA2, EN1 and MAP2 were measured by immunofluorescence assay. The expression of LMX1A, FOXA2, TH and GIRK2 in dopaminergic neurons was determined by immunofluorescence assay after continuing the differentiation of embryonic stem cells until day 42. 
RESULTS AND CONCLUSION: (1) MIF was expressed in human embryonic stem cells, and its associated receptors CD44 and CXCR7 were also expressed, but CD74 expression did not occur. (2) The addition of MIF promoted the protein expression of LMX1, FOXA2, and EN1, and promoted the gene expression of LMX1 and FOXA2. It also significantly inhibited the gene and protein expression of FOXG1 and HOXA2 and PAX6, effectively suppressing the heterogeneity of differentiation. (3) The addition of MIF did not result in high expression of SOX6 in dopaminergic neural progenitors as expected, but rather decreased expression at the gene level, while its inhibitor ISO-1 caused a mild increase in SOX6 expression at the gene level. (4) The present study demonstrates that MIF plays an important role in the dopaminergic differentiation potential and ventral midbrain fate determination of human embryonic stem cells and that MIF intervention further optimizes the regional localization of neural induction.

Key words: human embryonic stem cell, macrophage migration inhibitory factor, Parkinson’s disease, dopaminergic neural progenitor cell, differentiation

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