Stimulating neurogenesis of distinct retinal lineages in human retinal pigmented epithelium (RPE) with proneural transcription factors

Reviewer #1 (Public review):

Summary:

In this study, the authors identified transcription factor combinations capable of inducing retinal neuronal programs in cultured fetal human retinal pigment epithelial (RPE) cells. Using a pooled screening strategy, single-cell RNA sequencing, lineage barcoding, and immunohistochemical analyses, they identified ASCL1 and NEUROD1 as an effective combination for inducing retinal neuron-associated transcriptional states. This work aims to advance the development of therapeutic approaches for retinal regeneration by exploring the plasticity of RPE cells.

Strengths:

A major strength of the study is the comprehensive experimental design. The combination of transcription factor screening, lineage tracing, single-cell transcriptomics, and molecular validation provides a detailed characterization of the cellular responses to reprogramming factor expression.

Weaknesses:

All experiments were performed using fetal human RPE cells. Because fetal RPE remains relatively immature and retains proliferative capacity, it remains unclear to what extent the observed responses reflect true reprogramming of differentiated RPE cells versus activation of developmental plasticity already present in fetal tissue. The absence of adult human RPE controls limits assessment of the generality and translational relevance of the findings.

Reviewer #2 (Public review):

Summary:

This is an interesting study that explores how human RPE could be used as a source for new retinal neurons. This is a welcome addition to the field of retinal regeneration, which is currently focused almost exclusively on the regenerative capacity of Müller glia cells. The line of inquiry is firmly rooted in findings from amphibian and embryonic chick model systems and advances a fetal human retina RPE-based screening system as a rich resource for insights into human RPE biology, including as a potential stem cell source.

The authors investigate the potential of fetal human RPE cells to be reprogrammed into retinal neurons using overexpression of pro-neural factors. While this is a critical knowledge gap in the field of retinal regeneration with significant promise for developing regenerative therapies, several methodological concerns impact the interpretation of results. Firstly, while the authors sought to evaluate factors that enhance RPE reprogramming when co-expressed with ASCL1, nearly all co-expression constructs tested failed to achieve appreciable expression of ASCL1, leaving a central hypothesis of this study largely untested (Major concern 1). Second, although the authors were able to detect a cluster of photoreceptor-like cells in their screen, they were unable to identify which reprogramming construct generated this cluster (Major concern 2). Finally, an essential control that definitively demonstrates the value of combinatorial transcription factor reprogramming is missing (Major concern 3).

In summary, the authors establish a valuable new paradigm for culturing and reprogramming fetal human RPE, and even more importantly, demonstrate successful reprogramming to neural fates. However, the discussion and interpretation of results needs to be modified significantly to make it clear that (i) the outcome of many co-expression paradigms remains effectively unknown/untested due to failed over-expression of ASCL1, and that (ii) the reprogramming construct giving rise to photoreceptor-like cells could not be conclusively identified from their initial screen.

Strengths:

(1) Powerful new screening system advanced for exploring the regenerative potential of human fetal RPE cells.

(2) Co-expression vector system for testing additive effects of proneural transcription factors.

Weaknesses:

Major concerns:

(1) The authors executed a screen for combinations of factors that can enhance ASCL1-mediated reprogramming of RPE into retinal neurons. However, the expression level of ASCL1 was remarkably low in virtually all co-expression paradigms (see Figure 3C). Notably, the reprogramming combination with the highest potency (ASCL1 + NEUROD1) was also the one exhibiting the highest level of ASCL1 expression. The "failed" reprogramming of most of the co-expression constructs (ASCL1+LMO1, ASCL1+EZH2, and ASCL1+RAX2) is potentially a false negative resulting from low transgenic expression of ASCL1.

(2) The authors' interpretation is that the overexpression of NeuroD1 and Ascl1 generated a new cluster that expressed markers of photoreceptors such as RXRG and RCVRN (see Figure 3D). However, there does not actually appear to be any overlap between the ASCL1+NEUROD1 cluster (orange dots, left panel) and the cells expressing markers of photoreceptors (yellow/green/purple?/black? dots, right panel; yellow being ASCL1-EZH2, green being ASCL1, purple being ASCL1-and black being control - though color coding here is admittedly somewhat confusing). Thus, the photoreceptor-like cluster of interest actually seems to correspond to gray cells that were unmapped/exposed to an unknown programming cocktail. So, it remains completely unknown which reprogramming construct generated this cluster.

(3) To conclusively establish the additive role of NEUROD1 in reprogramming, it would be prudent to compare ASCL1 + FA directly to ASCL1+NeuroD1+FA. This control was not included but is needed for a more complete interpretation of results.