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。

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.