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Steven Mayerl, Allison Ludwig, Yu Gao, Xinyu Zhao, David M Gamm; Assessing hPSC-derived retinal neuron synapse formation in vitro via monosynaptic retrograde tracing. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2506.
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© ARVO (1962-2015); The Authors (2016-present)
Synapse formation in human pluripotent stem cell (hPSC)-derived retinal neurons (RN) has been supported by immunohistochemical and ultrastructural evidence in organoid cultures, and indirectly through electrical recordings and visual function testing following transplantation. However, direct evidence of functional hPSC-RN synaptic connections has not been definitively established. In this study, we validated a monosynaptic retrograde viral synaptic tracing assay to screen for the presence of functional synapses in vitro using dissociated cultures of human pluripotent stem cell (hPSC)-derived retinal organoid.
A monosynaptic retrograde tracing assay utilizing a lentivirus (encoding GFP) and attenuated rabies virus (RaV) (encoding mCherry) was modified for assessment of hPSC-RN synapse formation. Stage 2 (D80) retinal organoids were dissociated and plated on a 96-well plate at 200k cells/well. 20 days post-plating, cultures were treated with lentiviral constructs to infect retinal neurons. 5 days post-lentiviral infection, cultures were infected with RaV-mCherry, which only infects lentivirus-transduced cells. Cultures were fixed 5 days post RaV transduction and screened for the presence of GFP+/mCherry+ postsynaptic neurons and GFP-/mCherry+ presynaptic neurons. As a control, cells were also transduced with a lentiviral construct lacking the glycoprotein critical for synaptic transmission of RaV. Operetta high-content images were obtained for quantification. Quantification was achieved using Operetta high content imaging analysis.
RaV-mCherry and lentiviral-GFP vectors successfully labeled pre- and/or post-synaptic retinal neurons, in dissociated retinal organoid cultures. Functional synapses, as demonstrated by the transmission of RaV-mCherry between neurons, were identified in synaptic tracing experiments. Synaptic transmission was significantly greater (p=0.009) in experimental cultures than in control cultures. Functional hPSC-PR synapses represented roughly 17% of synapses identified.
This study demonstrates the utility of a RaV synaptic tracing assay to identify functional synaptic connections between of hPSC-derived retinal neurons in vitro. While overall synaptic formation rates involving hPSC-PRs were expectedly low, this system represents a promising platform for high-throughput testing of methods to improve hPSC-PR synapse formation in vitro.
This is a 2020 ARVO Annual Meeting abstract.
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