内皮微环境NOTCH信号通路促进人多能干细胞造血分化  

NOTCH signaling of the vascular niche prompts differentiation of functional HSCs from hPSCs

作　　者：赵月 王文秀 张博文[2,3] 何丽娟 曾泉[2,3] 范增 岳文[2,3] 裴雪涛 习佳飞[2,3] 

机构地区：[1]广西医科大学,南宁530000 [2]军事医学科学院野战输血研究所,全军干细胞与再生医学重点实验室,血液安全保障技术研究北京市重点实验室,北京100850 [3]华南干细胞与再生医学研究中心,广州510005

出　　处：《军事医学》2017年第9期767-774,785,共9页Military Medical Sciences

基　　金：国家重点研发计划资助项目(2017YFA0103100,2017YFA0103103,2017YFA0103104);广州市健康医疗协同创新重大专项资助项目(201704020224)

摘　　要：目的体外诱导人诱导性多能干细胞(human induced pluripotent stem cells,hiPSC)向造血分化,富集生血内皮细胞,将其与高表达NOTCH信号通路配体DLL4的内皮细胞共培养,体外建立内皮微环境促进多能干细胞造血分化的研究模型,进一步研究明确内皮微环境影响造血干/祖细胞产生的作用及机制。方法将携带runx1c报告基因的hiPSC通过spin EB方法诱导分化形成拟胚体(embryonic body,EB),在诱导分化第10天通过免疫磁珠分选技术(magnetic-activated cell sorting,MACS)分选富集出CD34^+的生血内皮细胞,将生血内皮细胞与高表达DLL4的内皮微环境细胞Vera Vec共培养,进一步诱导其向造血干/祖细胞分化,最终建立体外研究NOTCH信号通路促进多能干细胞向造血干/祖细胞分化的研究模型。结果建立了单细胞培养hiPSC的技术方法,将单细胞培养的携带runx1c报告基因的hiPSC通过spin EB方法诱导其向造血细胞分化,在诱导分化第10天利用MACS技术分离富集了CD34^+的生血内皮细胞。同时获得了转染E4ORF1的人脐静脉内皮细胞(human umbilical vein endothelial cell,UVEC)Vera Vec细胞,并对其NOTCH信号通路相关基因的表达进行了鉴定。结果表明,其在m RNA水平和蛋白水平均高表达NOTCH信号通路的配体DLL4。将分离富集的生血内皮细胞与高表达DLL4的内皮细胞共培养。结果显示,其能明显促进携带runx1c报告基因的hiPSC诱导分化时红色荧光td Tomato的表达和造血细胞的形成,以此作为模型可进一步研究内皮微环境促进多能干细胞向造血干/祖细胞分化的模型。结论成功建立了spin EB方法诱导多能干细胞分化的技术方法,能高效在体外富集生血内皮细胞,并将生血内皮细胞与高表达NOTCH信号通路配体DLL4的内皮细胞共培养,在体外构建了内皮微环境促进内皮生血转化的研究模型,供进一步研究内皮微环境促进造血干/祖细胞分化的作用及其机制,为体外利Objective To generate hemogenic endothelial cells（HECs） from human induced pluripotent stem cells（hiPSCs） in vitro in order to learn more about the mechanism by which the vascular niche affects HECs production and selfrenewal.Methods hiPSCs with reporter gene runx1c were differentiated to hematopoietic cells by spin EB method.The CD34 positive cells were sorted by magnetic-activated cell sorting（MACS） at day 10 after hematopoietic differentiation.Afterwards,these CD34 positive cells were co-cultured with DLL4 overexpressed vascular niche cells Vera Vec to further differentiate to HECs.The HECs derived from the hiPSCs were characterized by FACS.Results We first established an hiPSCs single cell culture method for spin EB differentiation.Single cell cultured hiPSCs with reporter gene runx1c were differentiated to form embryonic bodies（EBs） by spin EB method.The HECs were enriched from the day 10.Meanwhile,we cultured the E4ORF1 transfected human umbilical vein endothelial cell（HUVEC） line（Vera Vec） and examined the expression of NOTCH signaling pathway related genes.According to the results,Vera Vec had a high expression level of NOTCH ligand DLL4 at both m RNA and protein levels.And the CD34 positive HECs were co-cultured with DLL4 overexpressed Vera Vec cells,which promoted the expression of td Tomato during hematopoitic differentiation and increased HSCs production.Conclusion A method of inducing hiPSCs differentiation by spin EB has been established,which can enrich HECs.This model can be applied to study the mechanism by which the vascular niche promotes hematopoietic differentiation from hPSCs.The generated functional HSCs are of great social and military values for HSCs transplantation and battlefield radiation injury treatment.

关 键 词：人诱导性多能干细胞 造血干细胞 内皮微环境 NOTCH信号通路 

分 类 号：R82[医药卫生—临床医学]