While characterizing in vitro differentiation

While characterizing in vitro differentiation of cone precursors we made several observations regarding this process. The proportion of cone precursors was high using our protocol, which might be a consequence of poor generation/survival of other retinal neurons including rods. Alternatively, a soluble factor acting as a negative feedback signal for cone genesis, analogous to the action of GDF11 in retinal ganglion cell development (Kim et al., 2005), may fail to reach biologically relevant concentrations. Timely inhibition of Notch suggested that the temporal competence confining the peak of cone genesis to early stages of retinogenesis (Carter-Dawson and LaVail, 1979) is preserved in mESC-derived retinas differentiated in vitro. Notch inhibition may increase commitment to cone fate or accelerate their differentiation. While the proportion of hbv virus was increased at the time point used for transplantation (equivalent to early postnatal stage), there was no significant gain in total yield. Furthermore, we found that RA signaling plays an evolutionarily conserved role in regulating photoreceptor maturation. The addition of exogenous all-trans RA to late stages of photoreceptor differentiation in ESC-derived retinas led to the suppression of cone maturation. This is similar to observations that exogenous RA impairs cone maturation in the fish retina (Hyatt et al., 1996). Expression of enzymes responsible for RA synthesis declined significantly at late differentiation stages, in accordance with previously reported biochemical analyses showing RA to be abundant at early retinogenesis in vivo (when cones are born) but declining neonatally (when rods are produced [McCaffrery et al., 1993]). High levels of RA in embryonic retina might therefore act to prevent precocious cone maturation during embryonic development, while acting to stimulate rod differentiation (as also observed in mESC-derived retinas) in the neonatal retina. This could provide a mechanism to synchronize maturation of the two photoreceptor types despite the lag between their birth peaks. When isolated from their differentiation niche and transplanted into an adult environment, mESC-derived cone precursors at equivalents of both embryonic and early postnatal stages are able to undergo further differentiation in vivo, similar to precursor cells derived from donor mice (Lakowski et al., 2010). Strikingly, many of these cells go on to express M OPSIN, even in the severely degenerate Aipl1 retina. The signals present in the mature host retina appear to trigger M OPSIN expression, not observed in vitro despite extended culture periods. This suggests that the developmental capacity for subsequent M cone differentiation is retained in the retinal organoid system, but certain inductive signals, such as thyroid hormone provision from the circulation (Lu et al., 2009), are likely missing. Using a mESC retinal organoid system as a source, we could reproducibly transplant cones in numbers on a par with the total number of cones in an adult mouse retina (Jeon et al., 1998). We achieved extensive cone cell replacement in the Aipl1 mouse, a model of Leber congenital amaurosis end-stage retinal disease in which the vast majority of host cones are lost due to degeneration (Ramamurthy et al., 2004). Unsurprisingly, given the advanced degenerative state of the recipient retina, the morphology of transplanted cones was compromised. This contrasts with apparently mature features of GFP-labeled cells observed in cone transplants into non-degenerative retina, which are now understood to arise from cytoplasmic material transfer from the subretinal graft to host rods (Decembrini et al., 2017; Ortin-Martinez et al., 2016). Nonetheless, cones in the Aipl1 recipients appeared to make physical contact with inner retinal neurons and expressed components associated with synaptic transmission, alongside phototransduction-related proteins, suggesting advanced differentiation and maturation. Although beyond the scope of the present study, future studies will be required to investigate further the potential functionality of these transplanted cells.