环境细胞内因子Lhx2促进多能干细胞造血分化的机制研究

Till now, no ideal in vitro hematopoietic differentiation method has been developed to obtain transplantable hematopoietic stem cells （HSCs）, which is in part due to lack of knowledge on hematopoietic microenvironment. Knockout of transcription factor Lhx2 leads to fetal hematopoietic defect. Of note, Lhx2 is expressed in fetal liver stromal cells but not in fetal blood cells. HSCs from Lhx2-/- fetal liver can reconstitute normal hematopoietic system in recipients. Our preliminary results show that enforced expression of Lhx2 in OP9 can accelerate hematopoietic differentiation of co-cultured pluripotent stem cells (PSC)，but the mechanism remains elusive. We hypothesize that Lhx2 accelerate PSC differentiation into hematopoietic lineage through altering the expression pattern of membrane and secreted proteins in OP9 niche cell. Based on our previous observations, we plan to further investigate the downstream effectors of Lhx2 that regulate the PSC fate decision to hematopoietic lineage. We are going to dissect the role and mechanism of Lhx2 in hematopoietic differentiation of PSC through RNA-seq approaches. Further functional evaluation of the candidate effectors will be carried out based on in vitro OP9-PSC co-culture model and in vivo teratoma model. Our research will provide insights into derivation of functional hematopoietic cells through in vitro lineage differentiation approach.

目前没有理想的体外定向分化方法获得可供移植的造血干细胞，部分原因是对胚胎造血微环境认识不足。转录因子Lhx2敲除会导致胎肝造血异常，有趣的是胎肝基质细胞表达Lhx2，但造血细胞本身不表达，敲除Lhx2的胚胎造血干细胞能重构造血系统，表明环境细胞内的Lhx2通过改变造血微环境影响胚胎造血。前期研究表明过表达Lhx2的 OP9细胞与多能干细胞（PSC）共培养能够促进PSC细胞向血液细胞定向分化，但其机制未明。我们假设，Lhx2通过调控环境细胞膜蛋白及分泌蛋白表达模式，促进PSC向造血细胞定向分化。本研究在已有研究基础上，借助OP9与ES体外共培养及体内形成畸胎瘤两种造血分化模型，利用组学分析技术，解析Lhx2促进PSC向造血定向分化的效应机制，为开发体外定向分化新技术，获得功能性血液细胞提供理论依据。

研究表明环境细胞内的Lhx2 通过改变造血微环境影响造血。本项目通过前期体外实验发现造血发育环境细胞系OP9 过表达Lhx2能够促进多能干细胞向血液细胞定向分化，但机制并不清楚。在前期工作基础上，本项目中我们在免疫缺陷小鼠胎瘤造血模式中发现OP9-Lhx2也显著增加了造血祖细胞的比例，且畸胎瘤中分离的表型造血干细胞能支持短期造血重构。进一步的研究发现Lhx2通过上调mApln促进造血分化。在基本完成原有研究的基础上，本项目还新增小分子化合物和造血发育调控转录因子促进多能干细胞定向造血分化的研究内容。本项目建立了一种高效、无血清、无饲养层从人多能干细胞获得造血祖细胞的技术体系，在这个体系中小分子化合物UM171可以提高造血分化效率。另外，本项目构建了诱导性表达Runx1的多能干细胞系，结果表明在内皮造血转变阶段诱导表达Runx1能够促进造血分化效率，进一步的PCR array分析还发现胚胎来源pre-HSC与体外分化获得的pre-HSC差异表达Hoxa9。本项目从微环境细胞系、小分子化合物等外因角度和造血发育调控转录因子的内因角度探索，为开发体外定向分化新技术获得功能性血液细胞提供理论基础和实验依据。