胚胎干细胞来源的拟血岛巨噬细胞对红细胞分化的促进作用  

Erythroblastic island-like macrophages from human embryonic stem cells promotes erythroid differentiation

作　　者：李璇 王晓玲 崔甜甜 范增 赵玲萍 颜颢 徐振钊 何丽娟 周军年 王海洋 张彪 曾泉 习佳飞 岳文 裴雪涛 Li Xuan;Wang Xiao-Ling;Cui Tian-Tian;Fan Zeng;Zhao Ling-Ping;Yan Hao;Xu Zhen-Zhao;He Li-Juan;Zhou Jun-Nian;Wang Hai-Yang;Zhang Biao;Zeng Quan;Xi Jia-Fei;Yue Wen;Pei Xue-Tao(Stem Cell and Regenerative Medicine Laboratory,Institute of Health Service and Transfusion Medicine,Academy of Military Medical Sciences,Beijing 100850,China;South China Research Center for Stem Cell and Regenerative Medicine,South China Institute of Biomedicine,Guangzhou,Guangdong 510005,China;Experimental Hematology and Biochemistry Laboratory,Institute of Health Service and Transfusion Medicine,Academy of Military Medical Sciences,Beijing 100850,China)

机构地区：[1]军事科学院军事医学研究院卫生勤务与血液研究所干细胞与再生医学实验室,北京100850 [2]华南生物医药研究院华南干细胞与再生医学研究中心,广东广州510005 [3]军事科学院军事医学研究院辐射医学研究所血液与生化实验室,北京100850

出　　处：《解放军医学杂志》2022年第12期1180-1189,共10页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金青年基金(82101969)。

摘　　要：目的探索胚胎干细胞体外向红细胞诱导分化过程中出现的拟血岛结构中巨噬细胞的表型,以及拟血岛巨噬细胞对红细胞分化的作用。方法利用单细胞聚团拟胚体(Spin-EB)方法诱导人胚胎干细胞向红细胞分化,流式细胞术和成像流式细胞术分析诱导体系中红系细胞和巨噬细胞特征标志物的表达及细胞形态,实时荧光定量聚合酶链反应(qRT-PCR)检测红系及巨噬细胞特征标记基因在转录水平的表达情况,利用免疫荧光染色鉴定拟血岛巨噬细胞的表型,并分析拟血岛巨噬细胞对红系细胞分化的作用。结果吉姆萨染色结果显示,胚胎干细胞体外诱导过程中出现了类似于体内红细胞发育中的血岛结构;免疫荧光染色显示,此拟血岛结构为红系细胞(CD235a)围绕在巨噬细胞(CD68)的周围。该拟血岛巨噬细胞表型为CD45+CD235a+CD163+CD169+CD106+(比例为0.092%±0.013%),与体内血岛巨噬细胞相似,流式细胞术结果亦表明CD45+CD235a+CD163+CD106+CD169+的血岛巨噬细胞被CD235a+红系细胞包围。去除拟血岛结构后诱导所得CD71+CD235a+细胞占比为37.37%±1.68%,明显低于未处理组(46.97%±4.16%,P<0.05)。免疫荧光染色结果显示,CD169+巨噬细胞周围的红系细胞表达CD43。结论胚胎干细胞体外红系诱导体系中可形成与体内血岛类似的拟血岛样结构,拟血岛巨噬细胞可能通过CD169与CD43的相互作用促进体外红细胞分化。Objective To explore the phenotypes of erythroblastic island-like(EBI-like)macrophages during erythroid differentiation from human embryonic stem cells in vitro and the function of EBI-like macrophages in the differentiation of erythroid.Methods To study the function of EBI-like macrophages in the differentiation of erythroid,we used the Spin-EB method to induce human embryonic stem cells(hESCs)to differentiate into red blood cells.The expression of specific surface markers of erythrocytes and macrophages and the cell morphology were tested by flow cytometry and imaging flow cytometry.Multiple gene expressions were detected by qRT-PCR.An immunofluorescence assay was used to identify the phenotype of EBI-like macrophages.Results Erythroid cells were induced by embryonic stem cells in vitro.Giemsa staining revealed that EBI-like structures derived from the hESCs were similar to natural EBI.Immunofluorescence staining confirmed that the erythroid cells(CD235a)were surrounded by macrophages(CD68);CD45+CD235a+CD163+CD169+CD106+EBI-like macrophage(0.092%±0.013%)resembles in vivo EBI macrophage.Imaging flow cytometry observed that CD45+CD235a+CD163+CD106+CD169+EBI-like macrophages were surrounded by CD235a+erythroid cells.The proportion of induced CD71+CD235a+cells was 37.37%±1.68%after removing the EBI structure,which was significantly lower than the untreated group(46.97%±4.16%).We found that central macrophages of EBI-like may play a role in promoting erythroid differentiation through the interaction of CD169 and CD43.Conclusions EBI-like structure in the erythroid induction system of hESCs is similar to the natural EBI.The central EBI-like macrophages maybe promote the differentiation of erythrocytes in vitro through the interaction of CD169 and CD43.We provided a theoretical and practical reference for optimizing the erythroid induction system in vitro.

关 键 词：胚胎干细胞 巨噬细胞 体外诱导 红细胞 拟血岛 

分 类 号：R34[医药卫生—基础医学]