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

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.

Cone photoreceptors in human stem cell-derived retinal organoids demonstrate intrinsic light responses that mimic those of primate fovea.

High-definition vision in humans and nonhuman primates is initiated by cone photoreceptors located within a specialized region of the retina called the fovea. Foveal cone death is the ultimate cause of central blindness in numerous retinal dystrophies, including macular degenerative diseases. 3D retinal organoids (ROs) derived from human pluripotent stem cells (hPSCs) hold tremendous promise to model and treat such diseases. To achieve this goal, RO cones should elicit robust and intrinsic light-evoked electrical responses (i.e., phototransduction) akin to adult foveal cones, which has not yet been demonstrated. Here, we show strong, graded, repetitive, and wavelength-specific light-evoked responses from RO cones. The photoresponses and membrane physiology of a significant fraction of these lab-generated cones are comparable with those of intact ex vivo primate fovea. These results greatly increase confidence in ROs as potential sources of functional human cones for cell replacement therapies, drug testing, and in vitro models of retinal dystrophies.