Authors

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

Affiliations

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.

Topic

Neuroscience

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.

DOI: https://dx.doi.org/10.1167%2Ftvst.10.10.1

Extract

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).

Product Associated Features

The pE-4000 Universal Illumination System offers 16 selectable wavelengths from 365 - 770 nm, making it a highly flexible illuminator covering a wide variety of fluorophores

Product Type

pE-4000

Journal

Transl Vis Sci Technol

Year of Publication

2021

Country of Publication

USA