Generation Of Functional Lentiod Bodies From Human Induced Pluripotent Stem Cells Derived From Urinary Cells And The Investigation Of Mechanisms During Lens Development

PurposeCataract is the cause of more than half of all cases of blindness worldwide.Surgical removal of cataractous lenses is thought to be the only effective treatment.With the increasing aging population,the demand for cataract surgery increased significantly every year,which brought a heavy burden to society and the family.Interestingly,two recent studies have reported that lanosterol and a small chemical compound can prevent the aggregation of lens protein in both in vitro systems and animal models,this finding is the basis for a new strategy of cataract prevention and treatment.However,further investigation of the usefulness of these compounds for cataract treatment in humans is impeded by the lack of appropriate human cataract disease models.The aim of this study is to develop a stable in vitro system for human lens regeneration using pluripotent stem cells(iPSCs)to differentiate into human lens bodies(LB)with optical properties.And investigate the mechanisms in human lens development using the established LB generation system.MethodsIsolated human urinary cells were infected with four Yamanaka factors to generate urinary human iPSCs.The pluripotency of iPSCs were verified by alkaline phosphatase(AP)staining,the expression of pluripotent stem cell markers,embryoid body formation and teratoma experiments.iPSCs were induced to differentiate into lens progenitor cells and LBs using our newly established "fried egg" method.The expression of lens-specific markers including DLX3,SIX1,EYA1,PAX6,CRYAA,CRYAB,PROX1,FOXE3,SOX1,CRYB,CRYGC,MIP was examined by quantitative real-time PCR(RT-qPCR)and immunostaining.The structure and magnifying ability of LBs were investigated using transmission electron microscopy and observing the magnification of the letter "X," respectively.The transcriptome of LBs versus iPSCs was examined through microarray analysis.The expressions of selected mRNAs and IncRNAs were verified by RT-qPCR.SiRNA was used to investigate the function of lncRNA p10540 during the autophagy process.The expression of IncRNA p10540 and LC3B in lens capsules from age-related cataract patients were verified by RT-qPCR.ResultsWe successfully obtained iPSCs which were positive for AP staining and for the ESC antigens,and were able to form embryonic bodies.Teratoma analysis revealed the capacity of the cells to develop into tissues representative of the three germ layers which indicate that the cells were indeed iPSCs.We developed a "fried egg" differentiation method to generate functional LBs from iPSCs.The iPSC-derived LBs exhibited crystalline lens-like morphology and a transparent structure and expressed lens-specific markers CRYAA,CRYAB,CRYB,CRYGC,MIPP.During LB differentiation,the placodal markers SIX1,EYA1,DLX3,PAX6,and the specific early lens markers SOX1,PROX1,FOXE3,CRYAA,CRYAB were observed at certain time points.Microscopic examination revealed the presence of lens epithelial cells adjacent to the lens capsule as well as both immature and mature fiber-like cells.Optical analysis further demonstrated the magnifying ability(1.7)of the LBs.Autophagy was confirmed to exist in both mouse embryonic lens and LB differentiation.Among the massive IncRNAs expressed with a significant difference between iPSCs and LBs,lncRNA p10540,which was predicted to have a co-relationship with the autophagy marker LC3B,was highly expressed in differentiating lens fibers in LBs.LncRNA p10540 knockdown resulted in the downregulation of LC3B?,therefore inhibiting the autophagy process.Furthermore,the expression of IncRNA p10540 was found to decrease in the lens capsules of patients older than 75 years old as well as LC3B when compared to those from patients younger than 75 years old.ConclusionOur study confirmed that urinary cells can be an ideal somatic cell source for iPSCs induction.The LBs generated from the newly established "fired egg" method showed approximately 3-mm diameter and optical functions.They were the largest and most mature LBs to our knowledge,thereby establishing an in vitro system that can be used to study human lens development and cataractogenesis and perhaps even be useful for drug screening.Our results confirmed the importance of autophagy and IncRNA during LB differentiation,and identified IncRNA p10540,a potential autophagy regulator in organelle degradation during human lens development,may affect the pathogenesis of age-related cataract.