Thomas Andrew Reh

There are currently few promising approaches for treatment of photoreceptor pathologies: for example, gene therapy to augment or replace mutated genes, has proven successful in preclinical studies, and some of these therapies are moving towards the clinic. Another approach aims to unlock the inherent stem-cell potential of non-neuronal retinal cells to regenerate neurons in situ. This line of research is based on the discovery that some vertebrates can restore even severely damaged retina from RPE with all the necessary cell types to regain full functionality. To determine whether this approach can be applied to humans, we established a robust in vitro culture system using fetal human RPE, and employed a barcode-multiplexed, single cell RNAseq based screen to find factors that would reprogram human RPE into photoreceptors. With this approach we were able to identify NEUROD1 as a complimentary factor to ASCL1. Their combined overexpression together with a treatment with bFGF and Activin A inhibitor produced RPE-derived neuronal cells with expression patterns characteristic of photoreceptors and other lineages.

There are currently few promising approaches for treatment of photoreceptor pathologies: for example, gene therapy to augment or replace mutated genes, has proven successful in preclinical studies, and some of these therapies are moving towards the clinic. Another approach aims to unlock the inherent stem-cell potential of non-neuronal retinal cells to regenerate neurons in situ. This line of research is based on the discovery that some vertebrates can restore even severely damaged retina from RPE with all the necessary cell types to regain full functionality. To determine whether this approach can be applied to humans, we established a robust in vitro culture system using fetal human RPE, and employed a barcode-multiplexed, single cell RNAseq based screen to find factors that would reprogram human RPE into photoreceptors. With this approach we were able to identify NEUROD1 as a complimentary factor to ASCL1. Their combined overexpression together with a treatment with bFGF and Activin A inhibitor produced RPE-derived neuronal cells with expression patterns characteristic of photoreceptors and other lineages.

Retinal development has been extensively studied and key transcriptional regulators that drive fate decisions have been identified for major cell classes. These findings were confirmed and deepened in recent years with the advance of single cell RNA sequencing (Scrase). However, many processes that guide progenitor to postmitotic cell differentiation remain elusive, especially since some genes seem to yield different cell populations without apparent correlation with expression level or timing. Here, differential transcript isoform usage might play a role in diversifying the function of developmental genes. In short-read based scRNAseq, isoforms can only be identified if a read maps to a unique sequence or exon junction. However, due to the sparsity and very short reads, these events are extremely rare. We combined a commercial scRNAseq kit, that produces barcoded, full-length cDNA with Oxford Nanopore Technologies based long-read sequencing to generate the first single cell long-read sequencing dataset of fetal human retina. It can help elucidate the role of alternative splicing in retinal development and guide the design of transcript-specific gene therapies for retinal regeneration.

There are currently few promising approaches for treatment of photoreceptor pathologies: for example, gene therapy to augment or replace mutated genes, has proven successful in preclinical studies, and some of these therapies are moving towards the clinic. Another approach aims to unlock the inherent stem-cell potential of non-neuronal retinal cells to regenerate neurons in situ. This line of research is based on the discovery that some vertebrates can restore even severely damaged retina from RPE with all the necessary cell types to regain full functionality. To determine whether this approach can be applied to humans, we established a robust in vitro culture system using fetal human RPE, and employed a barcode-multiplexed, single cell RNAseq based screen to find factors that would reprogram human RPE into photoreceptors. With this approach we were able to identify NEUROD1 as a complimentary factor to ASCL1. Their combined overexpression together with a treatment with bFGF and Activin A inhibitor produced RPE-derived neuronal cells with expression patterns characteristic of photoreceptors and other lineages.

In the central region of the human retina, the high-acuity foveola is notable for its dense packing of green (M) and red (L) cones and absence of blue (S) cones. To identify mechanisms that pattern cones in the foveola, we examined human fetal retinas and differentiated retinal organoids. During development, sparse S-opsin-expressing cones are initially observed in the foveola. Later in fetal development, the foveola contains a mix of cones that either coexpress S- and M/L-opsins or exclusively express M/L-opsin. In adults, only M/L cones are present. Two signaling pathway regulators are highly and continuously expressed in the central retina: Cytochrome P450 26 subfamily A member 1 (CYP26A1), which degrades retinoic acid (RA) and Deiodinase 2 (DIO2), which promotes thyroid hormone (TH) signaling. Both CYP26A1 mutant organoids and high RA conditions increased the number of S cones and reduced the number of M/L cones in retinal organoids. In contrast, sustained TH signaling promoted the generation of M/L-opsin-expressing cones and induced M/L-opsin expression in S-opsin-expressing cones, showing that cone fate is plastic. Our data suggest that CYP26A1 degrades RA to specify M/L cones and limit S cones and that continuous DIO2 expression sustains high levels of TH to transition S-opsin-expressing cones into M/L cone fate, resulting in the foveola containing only M/L cones. Given the vulnerability of the foveola in macular degeneration and other retinal disorders, these findings provide a mechanistic framework for engineering organoids for therapeutic applications.

Chromatin structure plays a central role in defining cell identity by regulating gene expression. During development, shifts in chromatin structure facilitate changes in gene expression needed to specify distinct cell types. To understand how changes in chromatin structure influence the developmental trajectory of neural progenitor cells, we developed CUT&TIME, a technique that uses a hyperactive 6-methyl adenosine (6mA) methyltransferase pulsed in living cells to map historical chromatin accessibility genome-wide in single cells. We show that CUT&TIME produces a record of the chromatin landscape during neurogenesis in the developing retina, specifically as neural progenitors produce the major projection neuron type, retinal ganglion cells (RGCs). We further show that this method is compatible with single cell profiling technologies, which allows us to visualize and capture the diversity of chromatin states that produce RGCs. Additionally, we identify changes in promoter accessibility associated with the transition from progenitor to RGC. Together, these data demonstrate that CUT&TIME captures a historical record of chromatin structure, which can be used to identify early changes in accessibility associated with cell-fate commitment.

Strategies to stimulate the regeneration of neurons in the adult central nervous system can offer universal solutions for neurodegenerative diseases. Taking lessons from naturally regenerating species, such as the zebrafish, we have previously shown that vector-mediated expression of proneural transcription factors can stimulate neurogenesis from the resident Müller glia (MG) population in the adult mouse retina, both in vitro and in vivo . To bring this closer to translation, we now show that vector-mediated expression of the proneural transcription factor ASCL1 can reprogram adult macaque MG into functional neurons. To this end, we established purified MG cultures and show they retain a mature transcriptomic profile that correlates with foveal and peripheral MG. Importantly, MG-derived neurons express retinal ganglion cell markers, can fire action potentials and have a transcriptome that overlaps with developing human and adult macaque retinal ganglion cells. To refine this approach for clinical application, we incorporated microRNA-124 target sites in the reprogramming cassette and show that this restricts expression to MG in mixed primary cultures and intact explant cultures of adult macaque retina. Regulating ASCL1 expression with microRNA-124 target sites maintained the reprogramming efficiency from adult MG cultures and improved the yield of RGC-like neurons from infant MG cultures. Most importantly, with this vector cassette we successfully reprogrammed macaque MG from both adult and infant retina into HuC/D+ neurons. Our findings demonstrate that ASCL1 can induce neurogenesis from macaque MG across ages and provide a targeted, effective strategy for potential clinical translation in retinal repair.