Application Of Human IPSC-derived Retinal Organoids In Studying The Role Of Retinal Pigment Epithelium And NRL Gene In Photoreceptor Differentiation

The process of vision depends on coordination between photoreceptors and retinal pigment epithelium(RPE),where disruption of this homeostasis could lead to blindness.Therefore,the interaction between RPE and retina is critical during developmental stages as well as for the normal functioning of adult retina.The matured human retina is a complicated and delicate sense organ of the central nervous system that lacks the regenerative capability.Thus any damage to it may lead to impaired vision or blindness.Retina is directly exposed to intense light and is more prone to several potent damaging factors,leading to degeneration of photoreceptors.RPE has evolved multiple approaches to alleviate these notorious effects on the retina,which are the leading causes of photoreceptor degeneration and subsequent blindness.Moreover,RPE is crucial for normal outer retinal physiology,including phagocytosis of shed photoreceptor outer segments and secretions of neurotrophic and vasculotrophic growth factors.Retinal organoids(ROs)derived from human-induced pluripotent stem cells(hiPSCs)recapitulate the three-dimensional structure of the retina,mimic human retinal development,and provide cell sources for pre-clinical retinal transplantation.However,in vitro ROs-RPE co-culture can improve the differentiation of photoreceptors in ROs remains unknown.Herein,we succeeded in the sequential differentiation of human iPSCs into ROs,in vitro.Retinal markers of the neural retina were expressed in a spatiotemporally corrected manner and mimic in vivo retinogenesis.Then,primary mouse RPE cells were contacted-co-cultured with ROs at different time points.Our results revealed that the RPE cells accelerated photoreceptor differentiation in ROs,as the cross-talk between the RPE and ROs promoted the stage-specific expression of photoreceptor markers at different differentiation stages.Our results suggested that RPE are vital for retinal preservation,and any dysfunction of RPE appears to influence the differentiation of ROs in the embryonic eye greatly.Further,we demonstrated that direct contact of RPE and ROs is mandatory for improved differentiation of photo-markers during retinogenesis(in-vitro).Thus,we established an improved co-culture system based on modeling of human retina-RPE dynamics during retinogenesis for the evaluation of ocular therapies.Our work opens new avenues for deep portfolio of sight-physiology and stem cell based modeling of the retina.However,the molecular mechanism of RPE,contributing to the progression and maturation of photoreceptors needs to be investigated.We suggested a combination of CRISPR/Cas9 technology and stem cell differentiation will provide a better platform to accelerate the differentiation process and deep exploration of transcriptome dynamics.In the second part of our studies,we used CRISPR/Cas9 to disrupt the NRL gene in hiPSCs(BCl-eGFP)genomic DNA to know insights of the gene functions during retinogenesis,in vitro.The differentiation of stem cells into rod photoreceptors required an extended time period,thereby providing an attractive model to investigate transcriptomic dynamics and gene regulatory networks(GRNs),which control their unique neuronal-morphological features and developmental functions.GRNs that guided differentiation and assemblies of different cell types in the central nervous system are not fully explored yet,due to complicated inherited nature and interdependency of signaling pathways.In the study of model animals,we know that the transcription factor NRL is involved in the fate determination of rod cells,but we still do not know whether NRL has exactly the same function in the development of human retina.Therefore we performed the transcriptome profiling of developing NRL-positive and NRL-negative photoreceptors derived from hiPSCs(BCl-eGFP),in vitro.Our CRISPR/Cas9 protocol relied on two sgRNAs,which were complementary to a small fraction of NRL gene in the CDS region to degrade the specific DNA fragment.We succeeded to create knockout constructs by targeting NRL gene transcript,using the CRISPR/Cas9 system,which efficiently abolished the expression NRL in photoreceptor cells leading to loss of NR2E3 during in vitro differentiation.We found that knockout of NRL in hiPSC derived ROs led to complete absence of Rod cells and down regulation of cone cells markers such as R/G OPSIN and OPSIN1SW.Furthermore,the use of ATAC-seq and RNA-seq,for exploring the chromatin accessibility and transcriptomic changes in NRL-knockout versus wild type photoreceptors is under progress to determine the deep insight into mechanistic underlying the fate determination of rod in human retinogenesis,in vitro(under progress).Therefore,our research provides an in vitro research platform for the systematic analysis of human retinal development.