Synapse formation and in vitro synaptic tracing in hPSC-derived retinal neurons

Abstract

Purpose : Synapses—the basic unit of neuron function—are a central challenge of retinal cell replacement therapies for blinding eye disorders. Synapse formation within human pluripotent stem cell (hPSC)-derived retinal organoids (ROs) has been observed by electron microscopy, but the capacity for de novo synaptogenesis among hPSC-derived retinal neurons (RNs) following isolation from ROs has not been definitively established. In this study, we validated a synaptic tracing assay to screen for functional synapses in vitro among dissociated hPSC-RN cultures.

Methods : A monosynaptic retrograde tracing assay using a GFP-encoding lentivirus (lenti-GFP) and mCherry-encoding pseudotyped rabies virus (RaV) was modified to assess hPSC-RN synapse formation. Stage 2 (D80) ROs were dissociated and plated onto 96 well plates. 10 days post-plating, cultures were treated with lentiviral constructs to label a subset of RNs. After 5 days, cultures were infected with RaV, which enters only lenti-GFP-transduced cells. Synaptic tracing cultures were fixed 5 days post RaV transduction (D100) to screen for the presence of potentially postsynaptic (GFP+ and mCherry+) and presynaptic RNs (GFP-, mCherry+). To control for material transfer, a glycoprotein-null, RaV transmission-incompetent lentivirus was used. Confocal and high-content images were taken for qualitative and quantitative analysis.

Results : RaV-mCherry and lenti-GFP vectors labeled pre- and potentially postsynaptic RNs in dissociated hPSC-RN cultures. RaV transmission to presynaptic RNs was significantly greater (p<0.0001) in experimental cultures (~6.3% of all cells) relative to control cultures (<1.5%), validating the assay for presynaptic RN identification. Functional synapses, as demonstrated by RaV transmission, were identified in post-dissociation cultures. Major classes of hPSC-RNs, including photoreceptors (PRs), ganglion cells (GCs), and interneurons were detected among traced presynaptic cells. PRs and GCs—neurons of particular importance for cell replacement—represented ~40% and ~28% of traced presynaptic cells, respectively.

Conclusions : This study successfully demonstrates the use of a synaptic tracing assay to identify de novo synapse formation among hPSC-RNs isolated from ROs in vitro. While overall synaptic formation rates were expectedly low, this system represents a platform for high-throughput testing of methods to improve hPSC-RN synapse formation in vitro.

This is a 2021 ARVO Annual Meeting abstract.