Improved Retinal Organoid Differentiation by Modulating Signaling Pathways Revealed by Comparative Transcriptome Analyses with Development In Vivo

Abstract

Stem cell-derived retinal organoids recapitulate many landmarks of in vivo differentiation but lack functional maturation of distinct cell types, especially photoreceptors. Using comprehensive temporal transcriptome analyses, we show that transcriptome shift from postnatal day 6 (P6) to P10, associated with morphogenesis and synapse formation during mouse retina development, was not evident in organoids, and co-expression clusters with similar patterns included different sets of genes. Furthermore, network analysis identified divergent regulatory dynamics between developing retina in vivo and in organoids, with temporal dysregulation of specific signaling pathways and delayed or reduced expression of genes involved in photoreceptor function(s) and survival. Accordingly, addition of docosahexaenoic acid and fibroblast growth factor 1 to organoid cultures specifically promoted the maturation of photoreceptors, including cones. Our study thus identifies regulatory signals deficient in developing retinal organoids and provides experimental validation by producing a more mature retina in vitro, thereby facilitating investigations in disease modeling and therapies.

Transcriptomic Analysis of the Developmental Similarities and Differences Between the Native Retina and Retinal Organoids

“…The mouse retinal development dataset GSE101986 provided by Hoshino et al 4 included 12-stage samples comprising four embryonic time points (E11, E12, E14, and E16) and eight postnatal time points (P0, P2, P4, P6, P10, P14, P21, and P28). The mouse retinal organoid development dataset GSE102794 was provided by Brooks et al 13 Mouse retinal organoids derived from miPSCs were collected for RNA-seq analysis in triplicate at 10 time points during differentiation [day (D) 0, 4, 7, 10, 12, 15, 18, 22, 25, and 32]. The human native retinal development dataset GSE104827 provided by Hoshino et al 4 contained 13 samples at different development time points (D52/54, 53, 57, 67, 80, 94 [2 samples], 105, 107, 115, 125, 132, and 136).…”

“…For example, Hoshino et al 4 extracted RNA from 17 human fetal retinal samples (fetal days [D]52-136) and performed a transcriptome analysis by RNA sequencing (RNA-seq). Similarly, Aldiri et al 5 extracted RNA from human fetal retinal samples (fetal weeks [13][14][15][16][17][18][19][20][21][22][23][24] for transcriptome analysis. The discovered course of retinal development of the two studies is consistent.…”

Retinogenesis of the Human Fetal Retina: An Apical Polarity Perspective

“…In the absence of vascularized stem cell-derived retinal organoids, bioreactors help facilitate this mechanism. A number of teams have also very recently come up with methods that lead to better cone and rod specification in human retinal organoids [57,[210][211][212][213].…”

Limitations and Promise of Retinal Tissue From Human Pluripotent Stem Cells for Developing Therapies of Blindness

“…A layer of photoreceptors with NRL[+] rods ( Swaroop et al, 1992 ), OPN1SW[+] (S-cones) and TRß2[+] ( Ng et al, 2001 ) M-cones robustly forms in organoids derived by multiple techniques, which highlights retinal organoids as a good model of human photoreceptor genesis and maturation in a 3D tissue in a dish. This enables to dissect the important of multiple small molecules, morphogens and canonical signaling pathways such as basic fibroblast growth factor (bFGF), docosahexaenoic acid, bone morphogenic protein (BMP), taurine, Retinoic Acid (RA), WNT (Wingless) ( Murali et al, 2005 ; Pandit et al, 2015 ; Zhong et al, 2014 ; Capowski et al, 2016 , 2019 ; Brooks et al, 2019 ; Gamm et al, 2019 ) important for photoreceptor development. Methods outlining mostly cone photoreceptor development from CRX[+] photoreceptor progenitors will be instrumental for modeling human macula in a dish as well as for designing transplantable 3D retinal grafts for treating patients with advanced AMD ( Zhou et al, 2015 ).…”

“…Nevertheless, the neuroanatomical structure and connectivity of young retinal organoids growing in a dish is very similar to the developing human fetal retina, which is being explored as new way to study early stages of human retinal development ( Meyer et al, 2009 ). However, all studies, where retinal organoids were cultured for prolonged period of time (6 months or longer), note the gradual changes in retinal organoids (specifically, gradual loss of RGCs and thinning of INL) ( Wahlin et al, 2017 ; DiStefano et al, 2018 ; Brooks et al, 2019 ; Capowski et al, 2019 ; Nasonkin et al, 2019 ), thus substantially reducing the ability to model diseases and derive therapeutically meaningful results from drug screening efforts. Human retinal tissue in a dish has a real potential to be a great tool for drug screening, as well as disease modeling and source of transplantable 3D retina for RP and AMD after these critical shortcomings of retinal organoid technology are addressed.…”