Chinese Journal of Tissue Engineering Research ›› 2023, Vol. 27 ›› Issue (33): 5348-5356.doi: 10.12307/2023.714

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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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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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