Stimulating neurogenesis of distinct retinal lineages in human retinal pigmented epithelium (RPE) with proneural transcription factors
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eLife Assessment
This important study provides a contribution to retinal regeneration research by using overexpression of pro-neural factors to reprogram fetal human RPE cells into retinal neurons. The authors provide solid evidence of fetal RPE reprogramming into neural and photoreceptor-like states using scRNA-seq and imaging validation. There remains concerns regarding comparisons between the effectiveness of different combinatorial transcription factor codes.
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Abstract
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.
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eLife Assessment
This important study provides a contribution to retinal regeneration research by using overexpression of pro-neural factors to reprogram fetal human RPE cells into retinal neurons. The authors provide solid evidence of fetal RPE reprogramming into neural and photoreceptor-like states using scRNA-seq and imaging validation. There remains concerns regarding comparisons between the effectiveness of different combinatorial transcription factor codes.
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Reviewer #2 (Public review):
Summary:
This is an interesting study that explores how human RPE could be used as 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 Muller 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 …
Reviewer #2 (Public review):
Summary:
This is an interesting study that explores how human RPE could be used as 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 Muller 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 feta 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 Fig. 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.
Comments on revised version.
We thank the authors for their thoughtful responses to our concerns and for modifying a figure to improve clarity. However, we were disappointed to see that the responses to our major concerns were not incorporated into the revised manuscript, as we believe they provide context that is essential to proper interpretation of the data. Minimal revisions of the current manuscript would address our first two major concerns. This could be achieved by adding two additional sentences, for example incorporating the sentences below which are excerpted from the authors response to our concerns:
The following excerpt from the authors response would address major concern 1 if incorporated into the relevant section in the main text of the manuscript:
"Insufficient ASCL1 expression in the combinations might have led to lack of reprogramming, representing a false-negative result."
And this excerpt would address major concern 2:
"The farthest subcluster with highest RCVRN/RXRG expression was not assigned ASCL1-NEUROD1 identity."
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Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
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 …
Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
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.
We agree with the assessment that the reprogramming of fetal RPE due to its potential fate plasticity poses a different challenge than the adult. In fact, embryonic chick RPE will only undergo neural trans differentiation when treated with FGF before embryonic day 4. Therefore, we specifically focused on RPE at early developmental stages for our experiments. It is interesting in that light that the ages of fetal human retina are far more advanced in their development than the E4 chick, yet still have the capacity for neural reprogramming; while it will be interesting to explore in the future how long during fetal development this plasticity exists, we were limited by the fetal ages that were available to us. Moreover, the facility that supplied our tissues (Birth Defects Research Lab) are no longer able to use NIH funds to distribute fetal tissues as they have for the last >40 years, making any future studies with primary fetal tissues much more difficult.
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.
We agree with the reviewer that insufficient ASCL1 expression in the combinations might have led to lack of reprogramming, representing a false-negative result. We do not rule out that individual, rather than pooled, expression vectors of the “failed” varieties might prove effective. A future version of such a screen might need a control to test proper vector functionality of each member in the pool. However, the expression of ASCL1 alone from a vector in the same pool worked well. Additionally, the expression of ASCL1 from the ASCL1-NEUROD1 vector was not nearly as high as from ASCL1 alone and still led not only to reprogramming but also to a shift in the gene expression landscape as seen in 4D. This shift is consistent across experiments and vector constructs.
(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.
We apologize for the confusion in Figure 3D, which stemmed from lack of proper labeling.
The right panel shows the expression of photoreceptor genes in a feature plot, not the vector assignment. The color code is different between the left most panel (which shows vector assignment) and the other two panels (which show gene expression). To make this more clear, we have now separated this into a panel D and a panel D’, and added a caption as well as a scale bar for the D’ panel.
The reviewer is correct that the farthest subcluster with highest RCVRN/RXRG expression was not assigned ASCL1-NEUROD1 identity. The inherent sparsity of scRNAseq experiments and the small size of that particular cluster made definite identification challenging. Furthermore, vector assignment is tied to vector and thus barcode expression. It is conceivable that as cells matured towards a photoreceptor-like cells, they silenced the vector insertion locus but initiated marker expression. Additionally, these photoreceptor-like cells were assigned to the same cluster (neur, Fig. 3B) as the positively identified ASCL1-NEUROD1 cells by unsupervised clustering, express NEUROD1 (Suppl. Fig. 2D) and seem to follow the same trajectory. This suggests that they stem from ASCL1-NEUROD1 overexpression. Moreover, the follow-up experiments showing the expression of the relevant genes in the reprog4 cluster (Fig. 4) and the corresponding stainings (Fig. 5) provide evidence that ASCL1-NEUROD1 overexpression causes expression of RCVRN.
(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.
We thank the reviewer for this suggestion. Such a control would indeed clarify the role of FA treatment on reprogramming with ASCL1 alone. However, to address the additive role of NEUROD1, we presented in this study the comparison of ASCL1-NEUROD1 with ASCL1 alone. Even without the addition of FA, we noted a shift in expression pattern (Fig. 4D) and the formation of neurogenic clusters (Suppl.Fig. 4B) with the addition of NEUROD1. Moreover, as noted above, it is no longer possible for us to obtain tissue from the BDRL to carry out additional experiments at this time.
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
(1) The antibody section should mention the dilution used and how primary antibodies were validated (in the current study, or cited from previous literature).
We have added the dilutions of the used primary antibodies. They are targeted against well-known markers in the field and have been validated by our lab and others.
(2) A supplementary table summarizing and comparing pro-photoreceptor genes detected vs functional photoreceptor genes would be helpful to readers.
We think that the heatmap in 4C provides a good overview of detected genes of interest.
(3) The manuscript would benefit if figures have scale bars and marker labels above or below the images, but not superimposed (so as not to block the gene signal in some cells). (Figure 1C).
We changed the labels in 1C and added a scale bar to be more consistent with the other figures in the manuscript.
(4) Figure 2B and 5B (neurogenic dome) are missing scale bars.
We added scale bars in 2B and 5B as well as the supplemental figures.
(5) Plasmid design: dual promoters without insulators should be addressed (MCV and Ef1a). If possible, an RPE65-specific promoter to drive ASCL1-NEUROD1, to show that short temporal expression is not capable of inducing the desired reprogramming.
We agree that an RPE-specific promoter would not only gauge the strength and duration that is necessary for reprogramming, but would also support translational efforts. While we have addressed that question somewhat with the HES1-ASCL1 construct in our initial experiments, more in-depth investigation to choose an optimal promoter will be necessary in the future.
Reviewer #2 (Recommendations for the authors):
(1) The first sentence of the abstract is identical to the third sentence of the introduction. Rephrase to avoid redundancy. There are currently a few promising approaches for treatment: 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 [2], [3].
We rephrased the sentence to:
Current developments in gene therapy, which aims to deliver healthy copies of mutated genes, show promising results and are increasingly becoming available in the clinic.
(2) Panel 1C would be more informative if images were enlarged/zoomed in ~4x or higher.
We have enlarged the panels as requested by the reviewer.
(3) Panel 1D reports size/area differences for cultures at a variety of time points. Data should thus be represented as a line chart to reflect whether cell size is changing over time, rather than pooling data points from disparate time points into a single group.
We agree that such a chart would provide a measure of RPE maturation over time; however, our focus is the relationship between cultured and native RPE. As there seems to be little spread in the measurements between ages, and the range of ages between cultured and native RPE is comparable, we believe that the current chart represents a valid visualization.
(4) Panel 2D would be clearer if cluster labels were moved to the top of the figure (such that they sit above the columns corresponding to those clusters). It could be further clarified by adding category labels to the marker genes (i.e., RPE markers, ascl1 targets, Neural markers, etc.).
We thank the reviewer for this suggestion. We shifted the cluster labels and created new labels for the gene groups.
(5) No comma necessary here: Using transcription factor binding site analysis on these peaks, revealed an enrichment of members of the ETS family like ELK1/4 and ETV1/4.
Thank you for your attention to detail. We have removed that comma.
(6) Add dashed box to Panel 3D to indicate region of detail displayed in panels to the right (as was done in Panel 3C).
We have added a dashed box in panel 3D.
(7) Distracting typos in the legend of Figure 3.
We have corrected the typos.
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eLife Assessment
This study provides an important contribution to retinal regeneration research by using overexpression of pro-neural factors to reprogram fetal human RPE cells into retinal neurons. The authors provide solid evidence of fetal RPE reprogramming into neural and photoreceptor-like states using scRNA-seq and imaging validation; however, there are concerns regarding comparisons between the effectiveness of different combinatorial transcription factor codes.
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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 …
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 …
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.
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