Michael L. Risner, 1 Silvia Pasini, 1 Xitiz Chamling, 2 Nolan R. McGrady, 1 Jeffrey L. Goldberg, 3 Donald J. Zack, 2 and David J. Calkinscorresponding author 1
1Vanderbilt Eye Institute, Department of Ophthalmology & Visual Sciences, Vanderbilt University Medical Center, Nashville, TN, USA
2Wilmer Eye Institute, Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
3Byers Eye Institute, Department of Ophthalmology, Stanford University School of Medicine, Palo Alto, CA, USA
corresponding authorCorresponding author.
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.
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.
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.
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.
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.
The whole cell configuration was determined by a change in access resistance and verified by transmission of Lucifer Yellow into the cell by exciting the dye with 405 nm (pE-4000, CoolLED, Andover, UK).
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Transl Vis Sci Technol
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