Re-formation of synaptic connectivity in dissociated human stem cell-derived retinal organoid cultures

Abstract

Human pluripotent stem cell (hPSC)-derived retinal organoids (ROs) can efficiently and reproducibly generate retinal neurons that have potential for use in cell replacement strategies [Capowski et al. , Development 146, dev171686 (2019)]. The ability of these lab-grown retinal neurons to form new synaptic connections after dissociation from ROs is key to building confidence in their capacity to restore visual function. However, direct evidence of reestablishment of retinal neuron connectivity via synaptic tracing has not been reported to date. The present study employs an in vitro, rabies virus-based, monosynaptic retrograde tracing assay [Wickersham et al., Neuron 53, 639–647 (2007); Sun et al., Mol. Neurodegener. 14, 8 (2019)] to identify de novo synaptic connections among early retinal cell types following RO dissociation. A reproducible, high-throughput approach for labeling and quantifying traced retinal cell types was developed. Photoreceptors and retinal ganglion cells—the primary neurons of interest for retinal cell replacement—were the two major contributing populations among the traced presynaptic cells. This system provides a platform for assessing synaptic connections in cultured retinal neurons and sets the stage for future cell replacement studies aimed at characterizing or enhancing synaptogenesis. Used in this manner, in vitro synaptic tracing is envisioned to complement traditional preclinical animal model testing, which is limited by evolutionary incompatibilities in synaptic machinery inherent to human xenografts.

Human iPSC-derived photoreceptor transplantation in the cone dominant 13-lined ground squirrel

“…We also observed some migration of SOX2 + /HuNu + cells, indicating the limited migration ability of either stem cells or glia ( Figure S4 C). Finally, we assessed synaptic development by staining for a human-specific synaptic marker synaptophysin ( Ludwig et al., 2023 ). We observed coexpression of the synaptic marker in GFP + cells in both sets of transplant conditions ( Figures 3 E and 4 E).…”

Reliability of human retina organoid generation from hiPSC-derived neuroepithelial cysts

“…This might include other regions of the brain, like the diencephalon, that are part of the natural lineage of retinal development. On the other hand, combined development of cerebral organoids with eye-like structures or with retina organoids as assembloids also might be beneficial to model their connections (Fligor et al, 2021;Gabriel et al, 2021;Ludwig et al, 2023). While some studies in HROs noted the presence of non-retinal or extra-retinal domains of variable size, e.g., other parts of the eye or nervous system, a systematic quantitative analysis has not yet been performed (Browne et al, 2017;Deng et al, 2018;Phillips et al, 2018), and these domains are not always evident in sections that focus on an isolated region of a single organoid.…”

Identification of Evolutionarily Conserved VSX2 Enhancers in Retinal Development

“…Retinal organoids serve as a powerful tool for modeling human retinal development and disease (Foltz and Clegg, 2019, (Kruczek and Swaroop, 2020, (Wahle et al, 2023, (Ludwig et al, 2023, (Saha et al, 2022, (Phillips et al, 2014). To understand the role of the Vsx2 SE modules in human retinal organoids, we individually deleted Region 0 or Region 3 and assessed their growth and development over three established developmental stages (Capowski et al, 2019).…”