A) Phase contrast image of human fetal RPE harvested from a donor eye of gestation day 137 (D137) and cultured for 22 days in vitro (DIV). B) MKI67 staining of cultured RPE that was fixed at different timepoints revealed peak proliferation around 8 days in vitro (DIV8). Proliferation ceased and cell density reached its maximum at around DIV13. n =3, ages: D113, D115, D126. Horizontal bars indicate mean. C) D130 RPE was cultured until DIV20 and stained for RPE markers ZO1, OTX2 and PMEL. D) ZO1 staining from confluent RPE or native fetal RPE wholemounts was used for segmentation of outlines of individual cells via REShAPE [16]. Cultured RPE showed a slight increase in cell area but no difference in the number of neighbors or hexagonality. cultured: n=3, ages: D78, D130, D137. native: n = 4, ages D72, D78, D85, D145. Black bars indicate the mean and dashed red line indicates 6 neighbors.

A) Overview of scRNAseq experimental timeline. Cultured human RPE of a D137 donor was infected at DIV12 with a lentivirus carrying either a CMV>ASCL1-TuGFP, pHES1>ASCL1-eGFP or pHES1>eGFP cassette; one sample was left uninfected. Cells were harvested at DIV20 and 8 days post infection (DPI). B) CMV and Hes1 promoter were both capable of driving the expression of transgenes producing GFP/ASCL1 double positive cells. Only CMV>ASCL1-TuGFP produced cells with increased TUBB3 (TUJ) expression and axon-like morphology (white arrow). C) ScRNAseq analysis revealed a big cluster of cells with equal contributions from all samples (RPE). Two clusters contained exclusively cells that were transduced with ASCL1 carrying virus and were dominated by CMV>ASCL1-TuGFP expressing cells (NPre, neur). Three clusters showed expression patterns characteristic for proliferating cells (cycling), fibroblasts (fibro) and subconfluent RPE (imRPE). D) NPre cells showed a downregulation in RPE markers and an upregulation of general neuronal genes, as well as ASCL1 targets. Neur exhibited mature neuronal expression pattern. E) From a previously published dataset in which fetal human Müller glia were reprogrammed with ASCL1, we identified genes characteristic for NPre and neur. We compared their combined expression in the form of module scores between Müller glia and RPE derived reprogrammed cells. Genes that were induced upon ASCL1 overexpression in Müller cells and yielded NPre (precursor module) or neur (neuronal module), were also upregulated in the respective clusters in ASCL1-transduced RPE.

A) Example structure of constructs present in the screen library. CMV drives the expression of fluorescent reporter mScarlet3. Adjacent but in antisense direction, EF1a promotor regulates the expression of the transgene cassette. It contains the sequences of the candidate genes followed by a construct-specific barcode (BC) and a polyadenylation signal (polyA). B) Human fetal RPE was derived from two donors aged D91 (ctrl/lib 1) and D147 (lib 2) and infected during peak proliferation (DIV8/DIV9). Cells were harvested at DPI11 (ctrl, lib 1) or DPI18 (lib 2) for scRNAseq. B) UMAP embedding revealed one big cluster with equal contributions from all samples and was determined to be RPE. Cycling cells and fibroblastic (fibro) cells were also present in all samples. Only cells transduced with the library formed new clusters NPre and neur. These showed increased expression of ASCL1, its downstream targets, and several neuronal markers concomitant with a downregulation of RPE markers. C) Cells were assigned to the construct they were transduced with by the detection of its specific barcode. Transgene expression matched the assigned construct. Cells with unknown barcode (uk) showed no specific upregulation of transgenes or the fluorescent protein while those that were assigned to the control vector (ctrl), exhibited strong expression of mScarlet. Most of the cells of NPre expressed the ASCL1 construct, while neur seemed to arise from the combination of ASCL1 and NEUROD1 overexpression. D) left: Zoomed in UMAP focusing on the cluster containing reprogrammed cells. Righ, black arrowt: Cells in the neur cluster expressed photoreceptor genes RXRG and RCVRN.

A) RPE from a D115 human donor was cultured for 8 days before infection. At DIV10 bFGF and Activin A inhibitor (FA) were added to the media and on DIV13, when proliferation declined, EdU application was started. Cells treated with control virus, with CMV>ASCL1-NEUROD1 or CMV>ASCL1-NEUROD1 plus FA were harvested on DPI 20 and processed for FACS enrichment and scRNAseq. Only samples that received ASCL1-NEUROD1 overexpression generated new clusters and FA treatment increased their proportion. B) The RPE cluster was equally represented by all samples, while there was an enrichment in FA treated cells in the cycling cluster. The other clusters were dominated by cells originating from samples infected with the transgene virus. C) Residual but markedly downregulated markers of RPE were found in all reprog clusters. ASCL1 and NEUROD1 overexpression activated downstream targets like ID1/3 and DLL3 as well as general neuronal markers like DCX and MAP2. Reprogrammed cluster 4 (Reprog 4, black arrow) was enriched in mature neuronal markers of different lineages like VSX1/VSX2 (bipolar cells), ELAVL3 (ganglion cells), NRL (rods), RCVRN (photoreceptors). D) ScRNAseq data from CMV>ASCL1 (CMV-A), EF1a>ACSL1 (scr-A), CMV>ASCL-NEUROD1 (CMV-AN) and EF1a>ASCL1-NEUROD1 (scr-AN) from previous experiments were integrated and coembedded in a UMAP. While there is significant overlap between single and dual transcription factor overexpression, the addition of NEUROD1 clearly shifts the resulting cell fate. Top plot represents the scaled density of cells along the first UMAP axis. E) Projection of reprogrammed RPE treated either with CMV>ASCL1 (left) or CMV>ASCL1-NEUROD1 (right) onto a combined reference UMAP from D59 and D76 human fetal retina. ASCL1 overexpression alone produces cells that align with retinal progenitors (RPC), bipolar cells (BP) and photoreceptors (PR). The addition of NEUROD1 produces a higher proportion of bipolar cells and photoreceptors (mostly from reprog 2 and 4) but also shows prominent subclusters mapping to native retinal ganglion cells (RGC) and the transition point between RPC to mature neurons.

Cultured RPE from the same donor and experimental run that was used for scRNAseq (Fig. 4) were processed for immunostaining.

All images that are shown here are from the CMV>ASCL1-NEUROD1+FA paradigm. A) Transduced cells, represented by mScarlet signal, were accumulated in clusters and exhibited distinct DCX positive axon-like arborization and were surrounded by EdU positive, proliferating RPE. Reprogrammed cells were not positive for EdU (dashed yellow ellipse). B) Orthogonal section view of a neurogenic dome produced by CMV>ASCL1-NEUROD1 and FA treatment (DPI20) of RPE derived from a D110 donor. Dashed, yellow outlines depict the location of the hollow inner space. Red lines indicate the location of the glass coverslip that the cells were grown on. The dome appears flattened due to being compressed between the coverslip and the microscope slide. Right: DCX transcript expression zoomed on reprogrammed clusters. C) Some clusters contained cells that were positive for either HuC/D or VSX1 in a mutually exclusive manner confirming the transcriptomic pattern of reprog 4 (Fig. 4). D) VSX2 and RCVRN expression was heterogeneous, sometimes present in the same cluster (center row) or one being expressed without the other (top row: only VSX2; bottom row: only RCVRN). Scale bar: 50 µm.

Primary antibodies used in this study.

Summary of lentiviral vectors.

A) Heatmap of RPE genes, markers for fibroblasts and epithelial to mesenchymal transition. Reprogrammed cells retain residual RPE expression pattern and are closer related to RPE than to fibroblasts. B) Differentially expressed genes between RPE, NPre and neur clusters.

A) From human fetal single cell transcriptomic data we found pseudotime trajectories leading from retinal progenitor cells to photoreceptors. We identified genes whose expression change along that axis. RPC: retinal progenitor cells, AC: Amacrine cells, BP: Bipolar cells, PR: Photoreceptors. B) Genes from A) were additionally evaluated by looking whether they are upregulated during FGF-mediated RPE transdifferentiation in chicken. C) From single cell ATAC sequencing of human fetal retina and bulk ATAC sequencing of RPE we found peaks of open chromatin that contained OTX2 binding motifs. We then identified peaks that are common to RPE and photoreceptors (PR) but not bipolar cells (BP). These peaks were further analyzed for enriched binding motifs of other transcription factors. D) ASCL1 expression is highest in the clusters originating from cells that received the library with reprogramming vectors Npre, neur. NEUROD1 expression causes a shift in expression thus creating a smaller, distinct cluster of reprogrammed cells. MKI67 marks proliferating cells and NES is highly expressed in clusters of reprogrammed cells. E). Heatmap with RPE, fibroblast and EMT markers indicating the RPE origin of the reprogrammed cells.

A) Overexpression of ASCL1 and NEUROD1 in human fetal RPE (D103) results in mScarlet positive clusters 10 days post infection even without FA treatment. These cells are also positive for early neuronal markers DCX and/or MAP2 albeit without prominent arborization. B) UMAP of integrated D59/76 fetal human retina used for reference mapping.

A) Overexpression of ASCL1 and NEUROD1 and treatment with FGF and AAI in human fetal RPE results in mScarlet positive clusters with vast DCX-positive arborization. B) Top: Transduction of cultured RPE with the CMV>ASCL1-NEUROD1 lentivirus produces neurogenic clusters even in the absence of FA treatment. Bottom: Transduction with the control virus does not produce DCX positive cells nor mScarlet positive clusters even with the addition of FA. C/D) Control virus infection and FA treatment does not produce cells positive for cell type specific markers VSX1, HuC/D (C) or RCVRN, VSX2 (D).