Kumar : Differentiation of Retinal Ganglion Cell Subtypes from Human Induced Pluripotent Stem Cells
Differentiation of Retinal Ganglion Cell Subtypes from Human Induced Pluripotent Stem Cells
Indiana University School of Medicine
Chaman Kumar1, Melody Hernandez1,2,3, Kang-Chieh Huang1, Jason S. Meyer1,2,3,4
1 Indiana University School of Medicine, 2 Department of Medical and Molecular Genetics, 3 Stark Neurosciences Research Institute, 4 Department of Ophthalmology
Intrinsically photosensitive retinal ganglion cells (ipRGCs) are a subset of retinal ganglion cells that respond to light independently from rod and cone photoreceptor input. ipRGCs are non-image forming but are integral for regulating metabolic functions, such as circadian rhythms and pupillary reflexes, through the use of the photopigment melanopsin. However, the developmental processes that lead to the generation of ipRGCs over other subtypes of RGCs are unclear. Additionally, previous studies have suggested that some RGC subtypes, including ipRGCs, are more resilient to disease-associated stressors, yet the mechanisms that confer this increased resilience remain incompletely understood.
To address these questions, particularly in a human-relevant system, we have focused upon the use of human induced pluripotent stem cells (iPSCs) to assess their ability to give rise to RGCs, with a particular emphasis upon ipRGC differentiation. Cells were initially edited by the CRISPR/Cas9 system to express the Thy1 gene at the Brn3b locus and the tdTomato gene at the melanopsin locus. The former edit allows for the purification of RGCs from retinal organoids derived from iPSCs, while the latter edit allows for the identification of ipRGCs among the broader RGC population. Cells were grown in a directed, stepwise manner following established protocols.
By immunostaining for cell type specific markers, I have documented the different stages of retinal differentiation and provided insight into the mechanisms of ipRGC differentiation.
We found that iPSCs can be effectively differentiated into retinal cells, including the formation of 3D retinal organoids. Retinal ganglion cells can be purified and matured in vitro to generate models for optic neuropathies. Within the RGC population, evidence demonstrates the presence of distinct RGC subtypes.
Translational/Human Health Impact:
My project will highlight how differentiating iPSCs into functional ipRGCs will allow for more accurate ophthalmological disease modeling in vitro. This process is also essential to study the spatial and temporal characteristics of retinal development that can inform models of retinogenesis and may be integral for future RGC transplants and disease modeling.