2023, Vol. 27 ›› Issue (1): 34-41

Expression of autocrine macrophage migration inhibitory factor and its receptors of human embryonic stem cells

Wei Yanzhao1, Zheng Xiaohan2, Gao Shijun1, Huang Ting2, Wei Xufang2, Chen Xinxu3, Zhao Zhenqiang3   

Abstract: BACKGROUND: Embryonic stem cells have a great potential in helping to understand cell development, regenerative medicine, tissue engineering, and cell therapy due to their self-renewal and multidirectional differentiation characteristics. To have a deeper understanding and effective use of human embryonic stem cells, it is necessary to explore and discover the potential molecules that affect the proliferation and survival of human embryonic stem cells. Macrophage migration inhibitory factor is expressed in a variety of human cells. At first, it was thought to be mainly involved in inflammation, but it was later found to play an important role in cell proliferation, differentiation, angiogenesis, and tumorigenesis. However, whether human embryonic stem cells express this important factor and their functions in human embryonic stem cells remain unclear.
OBJECTIVE: To determine whether human embryonic stem cells express macrophage migration inhibitory factor and its related receptors, determine which receptor interacts with macrophage migration inhibitory factor, and initially explore the effect of macrophage migration inhibitory factor on the proliferation and survival of human embryonic stem cells.
METHODS: Human embryonic stem cells were cultured. The level of macrophage migration inhibitory factor in the cell culture medium was detected by enzyme-linked immunosorbent assay. The distribution of macrophage migration inhibitory factor in the cell was observed by immunofluorescence confocal microscope. Cell immunofluorescence and western blot assay were utilized to detect the expression of embryonic stem cell related factors. Immunofluorescence confocal microscopy and immunoprecipitation were applied to detect the binding of macrophage migration inhibitory factor and its receptor. Group intervention: Human embryonic stem cells were intervened with ISO-1 (0, 12, 24, 48, 96, 192 μmol/L) for 24 hours to determine the best inhibitory concentration. Different mass concentrations of macrophage migration inhibitory factor (30, 100, 300 μg/L) were incubated with the optimal inhibitory concentration of ISO-1 for 24 hours. CCK-8 assay was used to detect cell proliferation activity. TUNEL staining was used to detect cell apoptosis level.
RESULTS AND CONCLUSION: (1) Macrophage migration inhibitory factor was expressed in the cytoplasm, membrane and culture medium of human embryonic stem cells. (2) Human embryonic stem cells expressed macrophage migration inhibitory factor, mainly expressing its receptors CXCR2 and CXCR7. (3) Macrophage migration inhibitory factor mainly bound to the receptor CXCR7. (4) The effect of macrophage migration inhibitory factor inhibitor ISO-1 on cell proliferation and survival: Compared with the blank group, as the ISO-1 concentration increased, the cell gap increased significantly and the cell viability gradually decreased (P < 0.0001). TUNEL assay showed that cell apoptosis rate was significantly increased (P < 0.000 1). (5) After adding different mass concentrations of migration inhibitory factor (30, 100, 300 μg/L), it could weaken ISO-1 (192 μmol/L) induced negative effects on cells; cell viability was significantly increased (P < 0.05); and apoptosis rate was significantly reduced (P < 0.01). (6) The results have shown that human embryonic stem cells express and secrete macrophage migration inhibitory factor, which is an important factor and can be detected in the cytoplasm, cell membrane, and outside the cell. Human embryonic stem cells mainly express the receptors CXCR2 and CXCR7 of macrophage migration inhibitors, instead of the classic receptor CD74. The protein receptor that interacts with macrophage migration inhibitory factor on human embryonic stem cells is CXCR7. No evidence of interaction with CXCR2 has been found, and its role needs further study. In addition, studies have found that macrophage migration inhibitory factors play an important role in maintaining the proliferation and survival of human embryonic stem cells.
Key words: embryonic stem cell, stem cell, factor, macrophage migration inhibitory factor, cell proliferation, proliferation