SUMMARY To study the development of the human retina, we use single-cell RNA sequencing (RNA-seq) at key fetal stages and follow the development of the major cell types as well as populations of transitional cells. We also analyze stem cell (hPSC)-derived retinal organoids; although organoids have a very similar cellular composition at equivalent ages as the fetal retina, there are some differences in gene expression of particular cell types. Moreover, the inner retinal lamination is disrupted at more advanced stages of organoids compared with fetal retina. To determine whether the disorganization in the inner retina is due to the culture conditions, we analyze retinal development in fetal retina maintained under similar conditions. These retinospheres develop for at least 6 months, displaying better inner retinal lamination than retinal organoids. Our single-cell RNA sequencing (scRNA-seq) comparisons of fetal retina, retinal organoids, and retinospheres provide a resource for developing better in vitro models for retinal disease.

Graphical AbstractHighlights d STAT activation hampers Ascl1's ability to reprogram M€ uller glia into retinal neurons.d Progenitor-like cells from Ascl1-expressing M€ uller glia have high STAT signaling.d Ascl1 ChIP-seq shows that STAT potentially directs Ascl1 to inappropriate targets.d STAT inhibitors, along with Ascl1 and TSA, cause an increase in neuron regeneration. SUMMARYM€ uller glia (MG) serve as sources for retinal regeneration in non-mammalian vertebrates. We find that this process can be induced in mouse MG, after injury, by transgenic expression of the proneural transcription factor Ascl1 and the HDAC inhibitor TSA. However, new neurons are generated only from a subset of MG. Identifying factors that limit Ascl1-mediated MG reprogramming could make this process more efficient. In this study, we test whether injury-induced STAT activation hampers the ability of Ascl1 to reprogram MG into retinal neurons. Single-cell RNA-seq shows that progenitor-like cells derived from Ascl1expressing MG have a higher level of STAT signaling than do those cells that become neurons. Ascl1-ChIPseq and ATAC-seq show that STAT potentially directs Ascl1 to developmentally inappropriate targets. Using a STAT inhibitor, in combination with our previously described reprogramming paradigm, we found a large increase in the ability of MG to generate neurons.

Highlights d The transcription factor Atoh7 can reprogram MG to a neurogenic state in vitro d In vivo, the combination of Atoh1:Ascl1 significantly enhances MG neurogenesis d Atoh1:Ascl1 overexpression bypasses the need for injury to induce neurogenesis d Atoh1:Ascl1 regenerates a broader range of neuronal cell fates than Ascl1 alone

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Highlights d Chromatin accessibility of human fetal retina reveals transcription factor cascades d Accessible chromatin of hESC-derived retinal organoids shows conserved development d Retinal organoids and fetal retina show differences in Notch signaling