PONE-D-22-26643Denuded Descemet's membrane supports human embryonic stem cell-derived retinal pigment epithelial cell culturePLOS ONE

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Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

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Reviewer #1: The purpose of this study was to investigate whether decellularized Descemet’s membrane (DM) supports the maturation of hESC-derived RPE cells. DMs were isolated from human corneas, corneal endothelium was peeled off and additional Thermolysin treatment was applied. Decellularization was confirmed by immunohistochemistry and Atomic Force Microscopy (AFM). dDM was dried out on tissue culture plates or PET tissue culture inserts and sterilized by UV. hESC-derived RPE cells attached faster and showed higher initial proliferation when plated on the corneal endothelial side of acellular DM (dDM). RPE on dDM produced significantly more VEGF at 4 weeks than RPE cultured on tissue culture inserts. The transepithelial resistance was lower in RPE cultures on dDM, but reached an equal value on d56. hESC-RPE cultures on dDM were polarized and expressed significantly more RPE65. As a control, the authors also cultured adult eyebank-derived RPE cells which similarly showed a higher dree of maturation and significantly more RPE65 expression on dDM. Thus, the authors conclude that dDM is a suitable substrate for RPE cells.

General comments: The paper is well written, and the studies are well designed. The methods are well described, with listing experimental numbers. The figures are excellent. The results are important because dDM is a natural substrate for RPE cells, could serve as a xeno-free substrate for hESC-RPE, and could help to improve transplantation outcomes.

The authors also discuss the limitations of their study as they have not yet tried to transplant RPE sheets on dDM. It is unclear whether dDM has the same diffusion properties as Bruch’s membrane. This is an important question that needs to be answered because of the RPE role in transporting nutrients for photoreceptors from the choroid.

Specific comments:

A data availability statement is missing from the manuscript and only entered in the manuscript submission form. The authors state that some restrictions will apply, but do not specify which restrictions. The authors state that all relevant data are withing the manuscript and its supporting information files.

Author contributions: The authors should list the specific contributions for each author.

(Minor):

p.14, line 340: “showed” should be “shown”

Reviewer #2: Daniele et al., in the manuscript “Denuded Descemet's membrane supports human embryonic stem cell-derived retinal pigment epithelial cell culture” have shown that denuded DM could be used as a scaffold for potential transplantation of the RPE cells for cell transplantation therapy. However, there few concerns that needs to addressed for strengthening the manuscript.

1. The transplantation of RPE is believed to be one of the primary way to restore the RPE functionality in diseases such as AMD. However, it is also well known in the RPE related diseases the local RPE milieu harbor oxidative stress conditions driven by the dying cells and changes in the molecular components of the milieu. The scaffold that carries the RPE cells should be able to withstand the stress of the oxidative stress so that the cells can hopefully restore the normal functionality. It would be worthwhile if the authors provide some evidence of the role of DM in dealing with oxidative stress environment by experimental findings. The authors may show only the expression of soluble VEGF levels and few qPCR markers to show stability of cells cultured on DM under oxidative stress conditions. Or else the authors may add these points in the discussion section.

2. In Figure 4, the staining for BEST protein is not clear, please replace a better image. Since the IF images are taken at lower magnification, the readers would appreciate an inset with higher magnification.

3. It is unclear the figure of 4(B) and Supplementary figure 3. They seem to be same conditions by different images. In case something is missed please make the changes accordingly.

4. The authors have access to adult primary RPE cells. That is an ideal control to compare and show the physiological property of differentiated cells. Please include the results of the adult RPE cells along with the differentiated RPE cells for transepithelial resistance, VEGF, PEDF production.

5. The authors have missed showing the phagocytic property of the RPE cells which is vital component that gets affected in the RPE disease conditions. Please show the results in comparison with adult primary RPE cells.

Reviewer #3: The purpose of this paper is to use the denuded Descemet's membrane (DM) as a reliable substrate for establishing a polarized RPE monolayer for subsequent retinal implantation. Thus, this system might serve as a substitute for Bruch’s membrane (BM).

The denuded DM (dDM) has been used in the past as a substrate mostly for endothelial cells, but there are also have been prior reports of using the DM as a substrate for RPE cells, similar to the main tenet of this paper. In that sense, the paper is not entirely innovative.

The authors characterized their DM-RPE system using a large body of careful experimental approaches, (including consideration of statistics, i.e. replications and sample sizes) and demonstrated that they indeed can decellularize (denude) the DM, repopulate its endothelial side with different kinds of RPE cells derived from hESC-, adult human-, and human iPSC (no justification was provided for the use of these three kinds of RPE cells) and demonstrated that, by comparison to their controls, the dDM has beneficial effects on some, but not all of the RPE cells tissue-specific features.

Criticisms:

1. Confounding the authors' claim of superiority of the dDM is the lack of effect of some of the RPE-specific markers of cell maturation (such as CRALBP and MERTK) as well as the results of their TEER measurements, in which control cells have essentially > 25% higher TEER values than the cells growing on the dDM.

2. The characterization of the dDM by AFM tells something about the surface roughness but has little information as to the surface structure, e.g. morphology (e.g. fibrous)

3. The main problem I have with this paper is that in my opinion, the authors used the wrong controls. For the most part, their controls were RPE cells seeded onto either LN-521+CIV- precoated plastic wells or pre-coated TC inserts. None of these controls properly mimics the 3D structure/microtopography of the dDM. Instead, the authors should have maintained the 3D configuration, e.g. by using micro-/nanofibrous mats electrospun of collagen I (and then applying their LN-521+CIV-coating) or using a combination of ECM proteins as found in the natural subendothelial space in the DM. Furthermore, given that it might be impossible to obtain DMs in sufficient quantities to carry out large-scale clinical studies, the use of a more complex “man-made” substrate might reveal whether the EDM is indeed superior, and/or whether this “synthetic” substrate might suffice to obtain a functional RPE layer ready for implantation.

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Reviewer #1: No

Reviewer #2: Yes: Debashish Das

Reviewer #3: Yes: Peter I. Lelkes, Ph.D.

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Denuded Descemet's membrane supports human embryonic stem cell-derived retinal pigment epithelial cell culture

PONE-D-22-26643R1

Dear Dr. Daniele,

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Kind regards,

Panayiotis Maghsoudlou

Academic Editor

PLOS ONE