先天性白内障病人特异性iPSCs及其晶状体小体模型的构建和发病机制研究

We have reported and studied a large number of new congenital cataract (CC)-linked mutations in many genes over the past decade. In order to further investigate the pathological mechanism of these mutations during cataract formation, we’d like to first establish an in vitro system in which lens development and cataract process could be mimicked using human induced pluripotent stem cells (hiPSCs). Human exfoliated renal epithelial cells which present in urine were collected, cultured and infected with four Yamanaka factors to generated urinary human iPSCs (hiPSCs). After adapting the 3-stage system of lentoid bodies (LBs) formation from human embryonic stem cells (hESCs), hiPSCs were differentiated into LBs which show the similar structure and functions as human lens in chemically defined conditions. Molecular mechanisms were then explored by comparing the gene expression profile, structure and biological functions between LBs differentiated from patient iPSCs and LBs differentiated from patient iPSCs after the mutation has been corrected through CRISPR-Cas9. Finally, zebrafish and mice cataract disease models carrying the chosen mutations were created and used for mechanism investigation. Using the patient’s specific cataract disease models (LBs) which were established in this proposal not only could the vivo process of lens development and cataract be mimicked, but also could the species differences which are faced by researchers be avoided. Exploration may lead to clinical opportunities for stem cells therapy and medicine targets in CC.

申请人前期已深入研究了大量基因突变对先天性白内障（先白）发生发展的影响。为了进一步探讨已知突变基因引起先白的病理机制，申请人利用iPSCs技术，采用无创手段，收集培养人尿液细胞诱导成iPSCs，并优化“三步法诱导人胚胎干细胞分化成晶状体小体（lentoid bodies, LBs）”，将尿液来源iPSCs诱导分化成具有初步人晶状体结构的LBs，体外模拟人晶状体发育过程和先白发病进程即构建病人特异性先白模型，并通过CRISPER-cas9基因修复、microarray基因表达谱分析、实时定量PCR、免疫荧光等实验手段，探索已知突变基因引起先白的病理机制，并在斑马鱼及小鼠动物模型上加以验证。本课题构建的LBs模型即模拟了晶状体及先白在体内的发展状态又可避免先白研究中长期面临的动物模型造成的种属差异性问题。研究结果也将为先白的干细胞治疗及临床药物靶点的寻找提供新的思路和理论依据。

先天性白内障是儿童首位致盲性眼病，其中30%和遗传相关。本课题基于前期研究创立了“荷包蛋”法，通过人尿液细胞来源iPSCs成功诱导获得体外再生晶状体（LBs），并从形态学、细胞生物学、分子生物学、结构、光学特性等各方面对获得的LBs进行鉴定分析；并收集了不同基因突变的先天性白内障家系，利用患者及家系中正常对照尿液细胞来源iPSCs，通过 “荷包蛋”法诱导分化获得患者特异性及正常个体来源LBs，对比分析不同突变与正常LBs的细胞生物学、分子生物学、结构及光学特性；进一步利用CRISPR-Cas9技术在患者特异性iPSCs中修复了突变基因，并研究由此诱导分化所得LBs的相关表型。结果表明：通过“荷包蛋法”诱导iPSCs可获得成熟LBs，其分化过程包括基板期、早期晶状体期及成熟晶状体期，与晶状体胚胎发育一一对应。成熟LBs表达晶状体纤维细胞标志物（CRYAA、CRYAB、CRYB、CRYGC、MIP），具有晶状体上皮细胞、晶状体纤维细胞以及囊膜等结构，且具有良好的透明性和1.7倍的放大倍率，是目前已知的最接近人晶状体的理想体外晶状体模型。在此基础上利用基因突变的先天性白内障患者尿液细胞来源iPSCs诱导分化获得患者特异性LBs，与正常个体来源的LBs相比，CRYGD和CRYBB2突变LBs均表现出明显混浊，且其混浊程度特征与相应患者白内障的表型存在很高的对应性。本研究通过“荷包蛋”法利用人源性iPSCs在体外诱导分化获得了目前世界上最大最成熟且具有光学功能的体外再生晶状体，并首次构建了患者特异性先天性白内障体外疾病模型，为今后的先天性白内障病理机制研究与白内障治疗候选药物筛选提供了新的平台。