Differentiation and Three-dimensional Organization of Retinal Ganglion Cells using Human Induced Pluripotent Stem Cells

poster abstractRetinal Ganglion Cells (RGCs) are a type of neuron which function to relay visual messages between the retina and brain, and are characterized by their long axons which form part of the optic nerve. Dysfunction in this communication pathway is highly implicated in degenerative blinding disorders such as glaucoma. Unique applications using human induced pluripotent stem cells (hiPSCs) offer the ability to model human diseases, and potentially develop novel therapeutic approaches to rescue or replace damaged cells. In order to better understand the progression of degenerative eye diseases, a remaining challenge is to precisely identify the sequence of events which contribute to the diseased state, and how their features differ from non-diseased cells. Efforts were therefore undertaken to visually document the maturation of RGCs by analyzing their morphology and three-dimension organization at varying stages of development. Induced retinal cells were harvested at six different stages of development and fixed in 4% paraformaldehyde (PFA) solution to arrest their development. Cells were then cryoprotected in combinations of sucrose and Optimal Cutting Temperature (OCT) solutions, and frozen using powered dry ice. Following cryostat sectioning, samples were subject to immunocytochemistry staining to visualize for retinal-like organization of cells. Preliminary results have indicated the presence of the RGC marker Brn3, as well as markers for other retinal cell types. Future tests intend to characterize these retinal cell types according to their morphology and three-dimensional organization

Stepwise Differentiation of Retinal Ganglion Cells from Human Pluripotent Stem Cells Enables Analysis of Glaucomatous Neurodegeneration

Human pluripotent stem cells (hPSCs), including both embryonic and induced pluripotent stem cells, possess the unique ability to readily differentiate into any cell type of the body, including cells of the retina. Although previous studies have demonstrated the ability to differentiate hPSCs to a retinal lineage, the ability to derive retinal ganglion cells (RGCs) from hPSCs has been complicated by the lack of specific markers with which to identify these cells from a pluripotent source. In the current study, the definitive identification of hPSC-derived RGCs was accomplished by their directed, stepwise differentiation through an enriched retinal progenitor intermediary, with resultant RGCs expressing a full complement of associated features and proper functional characteristics. These results served as the basis for the establishment of induced pluripotent stem cells (iPSCs) from a patient with a genetically inherited form of glaucoma, which results in damage and loss of RGCs. Patient-derived RGCs specifically exhibited a dramatic increase in apoptosis, similar to the targeted loss of RGCs in glaucoma, which was significantly rescued by the addition of candidate neuroprotective factors. Thus, the current study serves to establish a method by which to definitively acquire and identify RGCs from hPSCs and demonstrates the ability of hPSCs to serve as an effective in vitro model of disease progression. Moreover, iPSC-derived RGCs can be utilized for future drug screening approaches to identify targets for the treatment of glaucoma and other optic neuropathies

Analysis of Retinal Ganglion Cell Development: From Stem Cells to Synapses

Human pluripotent stem cells (hPSCs) have the ability to self renew inde?nitely while maintaining their pluripotency, allowing for the study of virtually any human cell type in a dish. The focus of the current study was the di?erentiation of hPSCs to retinal ganglion cells (RGCs), the primary cell type a?ected in optic neuropathies. hPSCs were induced to become retinal cells using a stepwise di?erentiation protocol that allowed for formation of optic vesicle (OV)-like structures. Enrichment of OV like structures allowed for the de?nitive identi?cation of RGCs. RGCs displayed the proper temporal, spatial, and phenotypic characteristics of RGCs developing in vivo. To test the ability of hPSC-RGCs to serve as a disease model, lines were generated from a patient with an E50K mutation in the Optineurin gene, causative for normal tension primary open angle glaucoma. E50K RGCs displayed signi?cantly higher levels of apoptosis compared to a control lines. Apoptosis was reduced with exposure to neuroprotective factors. Lastly, hPSC-derived RGCs were studied for their ability to develop functional features possessed by mature in vivo RGCs. hPSC-derived RGCs displayed a few immature functional features and as such, strategies in which to expedite synaptogenesis using hPSC-derived astrocytes were explored. Astrocyte and RGG co-cultures displayed expedited synaptic and functional maturation, more closely resembling mature in vivo RGCs. Taken together, the results of this study have important implications for the study of RGC development and by extension, the advancement of translational therapies for optic neuropathies

Robust Differentiation of mRNA-Reprogrammed Human Induced Pluripotent Stem Cells Toward a Retinal Lineage.

The ability and efficiency of mRNA-reprogrammed human induced pluripotent stem cells (hiPSCs) to yield retinal cell types in a directed, stepwise manner was tested. hiPSCs derived through mRNA-based reprogramming strategies offer numerous advantages owing to the lack of genomic integration or constitutive expression of pluripotency genes. Such methods represent a promising new approach for retinal stem cell research, especially translational applications

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

Summary: Retinal 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. : In this article, Langer and colleagues present extensive characterization of RGC subtypes derived from human pluripotent stem cells, with multiple subtypes identified by subtype-specific molecular markers. Their results present a more detailed analysis of RGC diversity in human cells and yield the use of different markers to identify RGC subtypes. Keywords: iPSC, retina, retinal ganglion cell, RGC subtype, stem cell, ipRGC, alpha RGC, direction selective RGC, RNA-se

Three-Dimensional Retinal Organoids Facilitate the Investigation of Retinal Ganglion Cell Development, Organization and Neurite Outgrowth from Human Pluripotent Stem Cells

Abstract Retinal organoids are three-dimensional structures derived from human pluripotent stem cells (hPSCs) which recapitulate the spatial and temporal differentiation of the retina, serving as effective in vitro models of retinal development. However, a lack of emphasis has been placed upon the development and organization of retinal ganglion cells (RGCs) within retinal organoids. Thus, initial efforts were made to characterize RGC differentiation throughout early stages of organoid development, with a clearly defined RGC layer developing in a temporally-appropriate manner expressing a complement of RGC-associated markers. Beyond studies of RGC development, retinal organoids may also prove useful for cellular replacement in which extensive axonal outgrowth is necessary to reach post-synaptic targets. Organoid-derived RGCs could help to elucidate factors promoting axonal outgrowth, thereby identifying approaches to circumvent a formidable obstacle to RGC replacement. As such, additional efforts demonstrated significant enhancement of neurite outgrowth through modulation of both substrate composition and growth factor signaling. Additionally, organoid-derived RGCs exhibited diverse phenotypes, extending elaborate growth cones and expressing numerous guidance receptors. Collectively, these results establish retinal organoids as a valuable tool for studies of RGC development, and demonstrate the utility of organoid-derived RGCs as an effective platform to study factors influencing neurite outgrowth from organoid-derived RGCs

Astrocytes Regulate the Development and Maturation of Retinal Ganglion Cells Derived from Human Pluripotent Stem Cells

Summary: Retinal ganglion cells (RGCs) form the connection between the eye and the brain, with this connectivity disrupted in numerous blinding disorders. Previous studies have demonstrated the ability to derive RGCs from human pluripotent stem cells (hPSCs); however, these cells exhibited some characteristics that indicated a limited state of maturation. Among the many factors known to influence RGC development in the retina, astrocytes are known to play a significant role in their functional maturation. Thus, efforts of the current study examined the functional maturation of hPSC-derived RGCs, including the ability of astrocytes to modulate this developmental timeline. Morphological and functional properties of RGCs were found to increase over time, with astrocytes significantly accelerating the functional maturation of hPSC-derived RGCs. The results of this study clearly demonstrate the functional and morphological maturation of RGCs in vitro, including the effects of astrocytes on the maturation of hPSC-derived RGCs. : In this article, VanderWall and colleagues demonstrate the morphological and functional maturation of hPSC-derived RGCs over time, including the role of astrocytes in their functional maturation. Results indicated that hPSC-derived RGCs were capable of exhibiting robust neurite outgrowth and functional properties, with the direct contact with astrocytes significantly enhancing this maturation. As astrocytes are a major component of the nerve fiber layer and optic nerve, the results of these studies constitute a model for studying these cellular interactions in vitro. Keywords: stem cell, retina, retinal ganglion cell, astrocyte, development, differentiation, pluripotent stem cel