VEGF、FGF2和BMP4联合通过ERK通路高效诱导hiPSCs向内皮细胞分化  

The combination of VEGF,FGF2 and BMP4 can efficiently induce the differentiation of hiPSCs into endothelial cells through ERK pathway

作　　者：刘莲 汪萍萍 肖晓 尹姮 戴子怡 张慧慧 李涛 Liu Lian;Wang Pingping;Xiao Xiao;Yin Heng;Dai Ziyi;Zhang Huihui;Li Tao(School of Medicine,Hunan Normal University,Changsha 410013,China;Grade 20 Medical laboratory technology class of medical school,Hunan Normal University,Changsha,Hunan,China,410013;Grade 19 Medical laboratory technology class of medical school,Hunan Normal University,Changsha,Hunan,China,410013)

机构地区：[1]湖南师范大学医学院,长沙410013

出　　处：《中国组织化学与细胞化学杂志》2022年第1期1-10,共10页Chinese Journal of Histochemistry and Cytochemistry

基　　金：国家自然科学基金(81870201);湖南师范大学学位与研究生教育改革研究项目(20JG19);湖南师范大学医学院开放基金(KF2021021)。

摘　　要：目的分析影响诱导人诱导多能干细胞(human induced pluripotent stem cells,hiPSCs)向内皮细胞分化的因素及其机制,探索提高hiPSCs向内皮细胞分化效率的诱导方法。方法利用3阶段法诱导hiPSCs向内皮细胞定向分化,首先通过激活Wnt信号通路以启动中胚层分化,而后由VEGF、FGF2、BMP4诱导细胞分化为血管前体细胞,最后在VEGF作用下分化为成熟的内皮细胞。期间利用qRT-PCR检测一系列分化相关基因的表达,通过qRT-PCR、细胞免疫荧光、流式细胞术、成管实验验证细胞分化效果。通过VEGF、FGF2、BMP4两两配伍,比较细胞因子对诱导分化的影响;再进一步验证ERK和PI3K通路抑制剂对内皮诱导分化的影响;同时利用细胞免疫荧光分析不同起始细胞密度对诱导分化效果的影响。结果hiPSCs可诱导分化为内皮细胞;VEGF、FGF2、BMP4三者联合具有高效内皮诱导分化效果;相较于PI3K,抑制ERK信号通路可显著抑制内皮分化效率,促进hiPSCs向平滑肌样细胞分化;低起始细胞密度更有利于提高内皮分化效率。结论VEGF、FGF2、BMP4联合诱导可取得最佳诱导效果,其中ERK是介导hiPSCs向内皮分化的重要信号通路。Objective To explore the induction methods to improve the differentiation efficiency of human induced pluripotent stem cells(hiPSCs)into endothelial cells through analyzing the factors and mechanism affecting the differentiation of hiPSCs into endothelial cells.Methods hi PSCs were induced to differentiate into endothelial cells by a three-stage protocol.Firstly,the Wnt signaling pathway was activated to initiate mesodermal differentiation,then VEGF,FGF2 and BMP4 were used to induce the differentiation into vascular progenitor cells,and finally,VEGF was used to favor the generation of mature endothelial cells.The efficiency of cell differentiation was verified by qRT-PCR,immunofluorescence staining,flow cytometry and tube formation assay.The effects of cytokines on induced differentiation were compared by pairing VEGF,FGF2 and BMP4.The effect of ERK and PI3K pathway inhibitors on endothelial induced differentiation was further verified.Immunofluorescence staining was used to investigate the effect of different initial cell density on induced differentiation.Results hi PSCs can be induced to differentiate into endothelial cells;the combination of VEGF,FGF2 and BMP4 has an efficient effect on endothelial differentiation;compared with PI3K,inhibition of ERK signaling pathway can significantly inhibit the efficiency of endothelial differentiation,promoting the differentiation of hiPSCs into smooth muscle-like cells;low initial cell density is more conducive to improve the efficiency of endothelial differentiation.It was found that the combination of VEGF,FGF2 and BMP4 could achieve better endothelial induction effect.Compared with PI3K,ERK pathway is more important for endothelial differentiation.Low initiation cell density is more likely to increase endothelial differentiation efficiency.Conclusion The combination of VEGF,FGF2 and BMP4 can achieve an optimal induction efficiency,and ERK is an important signal pathway mediating differentiation of hiPSCs into endothelial cells.

关 键 词：人诱导多能干细胞 内皮细胞 分化诱导 细胞因子 细胞密度 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]