Intrinsic Morphologic and Physiologic Development of Human Derived Retinal Ganglion Cells In Vitro

Abstract

Purpose Human retinal ganglion cells (hRGC) derived from human pluripotent stem cells are promising candidates to model, protect, and replace degenerating RGCs. Here, we examined intrinsic morphologic and physiologic development of hRGCs. Methods We used CRISPR-Cas9 to selectively express tdTomato under the RGC-specific promoter, BRN3B. Human pluripotent stem cells were chemically differentiated into hRGCs and cultured up to 7 weeks. We measured soma area, neurite complexity, synaptic protein, axon-related messenger RNA and protein, and voltage-dependent responses. Results Soma area, neurite complexity, and postsynaptic density protein 95 increased over time. Soma area and neurite complexity increased proportionally week to week, and this relationship was dynamic, strengthening between 2 and 3 weeks and diminishing by 4 weeks. Postsynaptic density 95 localization was dependent on culture duration. After 1 to 2 weeks, postsynaptic density 95 localized within somas but redistributed along neurites after 3 to 4 weeks. Axon initial segment scaffolding protein, Ankyrin G, expression also increased over time, and by 7 weeks, Ankyrin G often localized within putative axons. Voltage-gated inward currents progressively developed, but outward currents matured by 4 weeks. Current-induced spike generation increased over time but limited by depolarization block. Conclusions Human RGCs develop up to 7 weeks after culture. Thus, the state of hRGC maturation should be accounted for in designing models and treatments for optic neuropathies. Translational Relevance We characterized hRGC morphologic and physiologic development towards identifying key time points when hRGCs express mechanisms that may be harnessed to enhance the efficacy of neuroprotective and cell replacement therapies.

Ex Vivo Integration of Human Stem Retinal Ganglion Cells into the Mouse Retina

“…In the rat retina, immunostaining for the postsynaptic marker PSD-95 in the IPL can be seen as early as P0 and reaches an adult staining pattern at P10 [ 40 ]. Our quantitative assessment of PSD-95 distribution demonstrates that in the presence of adult explants at 7 DIV, PSD-95 is enriched in neurites, a phenomenon that has also been previously observed in hRGCs over time in culture ( Figure 7 A–C) [ 20 ]. However, our quantificatin of PSD-95 may not represent functional post-synaptic densities.…”

“…Previously, we found immunofluorescence for the postsynaptic marker, PSD-95, decreases in hRGC somas after 4 weeks and increases in neurites after 3 and 4 weeks compared to 1-week cultures, suggesting maturation of hRGCs over time in culture [ 20 ]. Notably, our previous experiments were performed in cultures without BDNF + forskolin supplementation.…”

“…At the molecular level, human embryonic stem RGCs (hRGC) appear similar to native RGCs, expressing transcripts for BRN3B, ATOH7, ISL1, and SOX4 [ 17 , 18 , 19 ]. Moreover, these cells express the RGC-specific protein, RBPMS, and neuron-associated protein, TUJ1 [ 18 , 20 ]. hRGCs produce excitatory inward currents in response to glutamate, and generate action potentials in response to depolarizing stimuli [ 17 , 20 ].…”

“…Moreover, these cells express the RGC-specific protein, RBPMS, and neuron-associated protein, TUJ1 [ 18 , 20 ]. hRGCs produce excitatory inward currents in response to glutamate, and generate action potentials in response to depolarizing stimuli [ 17 , 20 ]. Furthermore, hRGC neurites accumulate excitatory postsynaptic proteins, suggesting the potential to form contacts with other cells [ 20 , 21 ].…”

Neutral sphingomyelinase inhibition promotes local and network degeneration in vitro and in vivo

“…H9 BRN3B-P2A-tdTomato-P2A-THY1.2 hESCs differentiated toward a RGC lineage (hRGC) were prepared for cultured as described in an earlier report [ 30 ]. At the time of use, hRGCs were thawed by gently swirling the vial in a 37 °C water bath for 1 min, and the vial was disinfected with 70% ETOH.…”