Retinal Ganglion Cell Diversity and Subtype Specification from Human Pluripotent Stem Cells

Abstract

SummaryRetinal ganglion cells (RGCs) are the projection neurons of the retina and transmit visual information to postsynaptic targets in the brain. While this function is shared among nearly all RGCs, this class of cell is remarkably diverse, comprised of multiple subtypes. Previous efforts have identified numerous RGC subtypes in animal models, but less attention has been paid to human RGCs. Thus, efforts of this study examined the diversity of RGCs differentiated from human pluripotent stem cells (hPSCs) and characterized defined subtypes through the expression of subtype-specific markers. Further investigation of these subtypes was achieved using single-cell transcriptomics, confirming the combinatorial expression of molecular markers associated with these subtypes, and also provided insight into more subtype-specific markers. Thus, the results of this study describe the derivation of RGC subtypes from hPSCs and will support the future exploration of phenotypic and functional diversity within human RGCs.

Dual SMAD inhibition and Wnt inhibition enable efficient and reproducible differentiations of induced pluripotent stem cells into retinal ganglion cells

“…In our study, the expression of CARTPT is seen in RGCs matured only in LSB, CHIR and LSBX conditions at DIV35 indicating that these conditions develop specific subtypes of mature RGCs that are known to be markers for ON-OFF direction-selective RGCs. [55] FSTL4 is a gene expressed in ON DS-RGCs and is colocalized with BRN3B in few RGCs [56]. We have seen increased expression of FSTL4 gene transcript in CHIR induced RGCs when compared with other conditions at DIV35 indicating that development of various subtypes of mature RGCs in our culture conditions.…”

“…In the current study, we employed a two-step/stage differentiation to induce RGC differentiation from iPSCs by modification of existing methodologies. [28,29,56] The first stage involves differentiation of iPSCs to RPCs. The RPCs were matured in a stage specific manner using small molecules to modulate sonic hedgehog pathway, Wnt pathway and Notch signaling to produce abundant RGCs reliability, that stain positive for RGC markers and emulate action potential.…”

“…Several published methods to generate RGCs from iPSC and ESCs in 2D cultures used stepwise differentiation protocols generated different types of RGCs based on morphological and functional characteristics [56]. The RGC subtypes were characterized by their distinct single-cell RNA sequencing profiles, morphological features and distinct expression profiles.…”

“…The RGC subtypes were characterized by their distinct single-cell RNA sequencing profiles, morphological features and distinct expression profiles. [56] Based on the nature and type of physiological responses, several RGC subtypes like ON-, OFF-and alpha-RGCs were observed. Evaluation of the gene expression profile of transcripts relevant several RGC markers at DIV18 and DIV28 time point of RGC differentiation confirming the validity of our method.…”

Retinal Ganglion Cell Replacement: Current Status and Challenges Ahead

“…These different protocols have been the basis for many others that followed, and they have been replicated using mESCs, hESCs, and iPSCs (mouse and human) ( Table 1) (Osakada et al, 2008;Parameswaran et al, 2010;Tucker et al, 2011a;Tucker et al, 2011b;La Torre et al, 2012;Nakano et al, 2012;Boucherie et al, 2013;Tucker et al, 2013;Decembrini et al, 2014;Riazifar et al, 2014;Zhong et al, 2014;La Torre et al, 2015;Sluch et al, 2015;Tanaka et al, 2015;Volkner et al, 2016;Aparicio et al, 2017;Chao et al, 2017;Teotia et al, 2017;Vergara et al, 2017;Yokoi et al, 2017;Daniszewski et al, 2018;DiStefano et al, 2018;Kobayashi et al, 2018;Langer et al, 2018). Retinal organoids have also proven useful as platforms to study different aspects of development using not only mouse cells, but also human cells Phillips et al, 2014;Ohlemacher et al, 2016;Takata et al, 2017;Eldred, 2018).…”