TY - JOUR
T1 - Intrinsic morphologic and physiologic development of human derived retinal ganglion cells in vitro
AU - Risner, Michael L.
AU - Pasini, Silvia
AU - Chamling, Xitiz
AU - McGrady, Nolan R.
AU - Goldberg, Jeffrey L.
AU - Zack, Donald J.
AU - Calkins, David J.
N1 - Funding Information:
Supported by a departmental unrestricted award by the Research to Prevent Blindness Inc., Research to Prevent Blindness Inc. Stein Innovation Award, the Stanley Cohen Innovation Fund, and National Institutes of Health grants EY017427 (DJC), EY024997 (DJC), EY008126 (DJC), and EY029903 (JLG, DJC, DJZ). Imaging supported through the Vanderbilt University Medical Center Cell Imaging Shared Resource core facility and NIH grants CA68485, DK20593, DK58404, and DK59637.
Publisher Copyright:
© 2021 The Authors.
PY - 2021/8
Y1 - 2021/8
N2 - 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.
AB - 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.
KW - Clustered regularly interspaced short palindromic repeats
KW - Development
KW - Glaucoma
KW - Neuroregeneration
KW - Retinal ganglion cells
KW - Stem cells
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U2 - 10.1167/tvst.10.10.1
DO - 10.1167/tvst.10.10.1
M3 - Article
C2 - 34383881
AN - SCOPUS:85112743496
VL - 10
JO - Translational Vision Science and Technology
JF - Translational Vision Science and Technology
SN - 2164-2591
IS - 10
M1 - 1
ER -