Nutrient Microenvironments Reprogram Pigment Epithelium Metabolism and Phenotype

Reviewer #1 (Public review):

Summary:

The authors utilize both human iPSC-derived RPE and human fetal RPE cultures to interrogate the effect of various types of commonly used cell culture media on several key biological- and disease-relevant RPE properties. These include a comparison of RPE morphology, polarity, transepithelial electrical potential, lipid metabolism, autophagy, and targeted metabolomic profiles across 6 different media compositions.

Strengths:

This manuscript is very well written, and data are presented in a well-organized manner. The authors address media composition as a fundamental variable that will influence the interpretation of assays performed in RPE cell cultures, particularly metabolic studies. Figure 6 provides a useful summary of the study's findings across commonly used media types, and the manuscript's discussion offers insight into which media may be best suited to address specific experimental questions. Overall, this manuscript will not only serve as an important resource for vision scientists utilizing RPE culture models, but it also serves to remind the broader cell biology community of the importance of considering the potential (confounding) experimental effect(s) of various culture media and to consider tailoring the selection of culture media types to the specific experimental question.

Weaknesses:

(1) While the authors report that iPSCs were obtained from several healthy patients and that at least two clones were generated from each individual, it is not clear to this reviewer whether the experiments with each culture media type were performed on the same set of iPSC-RPE in each case. The authors mentioned iPSC differentiation variability as a limitation, but it would be helpful to understand (and quantify) the experimental variability that may exist with the same culture media using iPSCs from different patients and/or separate iPSC clones from the same patient.

(2) Since a major purpose of the manuscript is to highlight how cell culture conditions influence RPE biology and metabolism, it would be helpful to also report whether Mycoplasma testing was performed and confirmed to be negative across all cell lines.

(3) The effect of culture media on mean RPE area and hexagonality was compared in this study. Interestingly, Figure 1E demonstrates higher mean RPE cell area but also substantial variability in cell area for media 2 (MEM-alpha and B27) and media 4 (HPLM and B27). It would be helpful to include a discussion of the potential biological implications of variable cell area across these 2 media types.

(4) The authors speculate that FBS-containing media may encourage a more mesenchymal or de-differentiated state. This could be experimentally determined by interrogating mesenchymal markers (alpha-SMA, fibronectin, etc) by immunoblot and/or immunofluorescence microscopy, similar to how the RPE markers were evaluated in Figure 1.

(5) It would be helpful for the discussion section to include a comparison of key differences (where they exist) between iPSC-RPE and fetal RPE across culture media types.

Reviewer #2 (Public review):

Summary:

In this study, Lim et al. provide a comprehensive analysis of the metabolic and physiologic effects of different media compositions on iPSC-RPE. This analysis includes commonly used iPSC-RPE media bases (MEMα, DMEM-HG/F12 basal media) as well as human plasma-like medium (HPLM) in attempts to establish a more physiologically relevant culture environment.

Strengths:

The analyses in this study provide a very thorough survey of metabolic function as well as an RPE-relevant physiologic characterization. This will be a great resource for optimizing assay conditions for disease-based studies using iPSC-RPE.

Weaknesses:

In the Seahorse studies provided in Figure 3. basal readings for OCR are abnormally low compared to Oligomycin treatment and background, suggesting difficulties with the assay. Findings should be taken with caution.

Reviewer #3 (Public review):

Summary:

The authors systematically compare six culture-media formulations using induced pluripotent stem cell-derived retinal pigment epithelium and fetal retinal pigment epithelium. They examine cell morphology, marker expression, barrier function, polarized secretion, lipid accumulation, ultrastructure, mitochondrial respiration, glycolytic function, and intracellular and extracellular metabolites. The results demonstrate that culture-medium composition and the choice of serum or B27 supplementation substantially influence retinal pigment epithelium phenotype and metabolism. Rather than identifying a single optimal medium, the study provides a comparative framework to guide medium selection according to the biological question being investigated.

Strengths:

The head-to-head comparison of six media under otherwise similar culture conditions addresses an important source of variability in retinal pigment epithelium research. The study uses a broad range of complementary approaches, including imaging, transepithelial resistance, electron microscopy, extracellular flux analysis, and targeted metabolomics. The inclusion of both induced pluripotent stem cell-derived and fetal retinal pigment epithelium increases the potential relevance of the findings across different cell sources. The matched comparisons of serum and B27 supplementation within MEMα and human plasma-like medium are particularly informative because they help distinguish supplement-associated effects from those caused by the basal medium. Overall, the dataset has the potential to serve as a valuable resource for selecting culture conditions and interpreting findings across retinal pigment epithelium studies.

Weaknesses:

The most important limitation is that the experimental unit and degree of biological replication are not clearly defined. It is unclear whether individual observations represent independent donors, clones, differentiated lines, culture preparations, wells, images, or sections. This makes it difficult to determine the independence, robustness, and generalizability of several comparisons.

The metabolic analyses also require additional methodological clarification. For intracellular metabolomics, the culture format, cellular biomass, extraction volume, pooling strategy, and normalization method are not reported sufficiently. Normalization of extracellular measurements to unspent medium accounts for differences in starting metabolite abundance but not for differences in cell number or biomass. Similarly, normalization of intracellular signals to medium 1 does not correct for differences in the amount of cellular material extracted.

For the Seahorse experiments, the main figures present unnormalized values even though the media produce differences in cell number, size, and protein content. These raw measurements represent total metabolic activity per well and may not reflect activity per cell. It is also unclear how normalization was performed because the Methods describe cell-count and protein measurements from two wells, whereas the stress tests included five to six wells per condition. In addition, measurements obtained after transfer into a common assay medium reflect metabolic adaptations retained from the preceding culture conditions rather than real-time metabolism within the original media.

Other limitations include insufficient information about the biological replication underlying the sub-RPE deposit analysis and the inability to fully interpret the effects of X-VIVO 10 because its composition is proprietary. Finally, public availability of the underlying metabolomics data would be important for a study intended to serve as a community resource.

Author response:

We thank the editors and reviewers for their thoughtful feedback on our manuscript. We are encouraged that they recognized the importance accounting for media consideration when modeling in vitro disease phenotypes, as well as the value of this dataset as a resource for the field. We appreciate the points raised regarding biological replicates and data normalization methods, and we plan to address these fully in our formal response and in revisions to the manuscript. Below, we provide preliminary responses to several comments and indicate how we anticipate addressing them in the revised manuscript.

(1) Reviewer 1 & 3: Unclear definition of iPSC lines/clones used in data generation.

We thank the reviewers for pointing out this ambiguity in the Methods. The project was completed with multiple donor iPSC lines, with at least 2 clones generated from each line, and each experiment was performed using at least three independent iPSC RPE lines. To minimize confounding variability, we took several precautions where possible: each experiment was performed within the same culture plate (coated with Matrigel from the same lot number), using RPE seeded at the same time to ensure comparable maturity, and RPE of the same passage number were used across multiple experiments to reduce de-differentiation or senescence effects. We agree that RPE derived from different iPSC differentiation batches can vary. For this reason, all iPSC RPE used in this study were generated from a single differentiation attempt. Because the goal of this project was to isolate the impact of nutrient composition on RPE phenotype, rather than to characterize variability arising from clonal or donor differences, we did not stratify our analysis by clone or donor. For imaging-based assays, multiple fields were selected at random from each well to ensure representative sampling. TEM analysis of sub-RPE deposits was performed with n=3 independent filters per medium condition, with three panoramic sections imaged per filter. We will add these details, including the number of clones used per iPSC line, to the revised Methods to clarify experimental unit and level of replication for each assay and will include sample number in legends.

(2) Reviewer 2: In the Seahorse studies provided in Figure 3. basal readings for OCR are abnormally low compared to Oligomycin treatment and background, suggesting difficulties with the assay. Findings should be taken with caution.

We thank the reviewer for this careful reading. The apparent discrepancy likely arises from comparing the raw OCR trace (left) versus the background-subtracted “Basal respiration” bar graph (right) in Figure 3A. In the raw trace, basal OCR (~45-65 pmol/min) is appropriately higher than both the oligomycin-treated (~35-45 pmol/min) and background (~30-45 pmol/min) rates, as expected. The bar-graph value is smaller (~7-25 pmol/min) only because the non-mitochondrial rate has been subtracted out, whereas the oligomycin plateau in the raw trace has not. These raw basal values fall within Agilent's recommended starting range for the XFe96 platform (~20-160 pmol/min), consistent with the modest basal energy demand of quiescent, differentiated RPE rather than an assay problem. A recent survey of 530 published Cell Mito Stress Tests [1] found that 17% report the implausible result of maximal OCR below basal OCR, and higher basal rate can compromise the FCCP-stimulated maximal rate. We titrated our cell numbers before this assay to ensure a clear FCCP response. The substantially increased maximal rates in all six media conditions indicate a technically sound assay. We will clarify this calculation in the revised Methods.

(3) Reviewer 3: The metabolic analyses also require additional methodological clarification. For intracellular metabolomics, the culture format, cellular biomass, extraction volume, pooling strategy, and normalization method are not reported sufficiently. Normalization of extracellular measurements to unspent medium accounts for differences in starting metabolite abundance but not for differences in cell number or biomass. Similarly, normalization of intracellular signals to medium 1 does not correct for differences in the amount of cellular material extracted.

We agree that these methodological details require clarification. Intracellular metabolomics was performed on RPE lysates collected from 12-well plates (n=3 independent wells/RPE lines per medium), each extracted separately without pooling. RPE were scraped directly into a fixed volume of 300 µL chilled 80% methanol per well, regardless of the medium condition; 10 µL of the resulting lysate was dried together with an internal standard (nicotinamide-D4), reconstituted in 100 µL of mobile phase, and 5 µL was injected for LC-MS/MS analysis. Media were processed in parallel using an identical workflow: 50 µL of conditioned media was collected at 24 and 48 hours, of which 10 µL was mixed with 40 µL cold methanol, and 10 µL of the resulting supernatant was dried with internal standard, reconstituted in 100 µL mobile phase, and 5 µL injected for analysis.

Biomass data, including average nuclei count and total protein content reported in Supp. Fig. 2D, were obtained from RPE cultured in parallel under identical conditions. Because nuclei count and protein content did not consistently agree with one another across the six media, metabolite intensities were not normalized to either protein content or cell number. Instead, the intensity of each metabolite was instead normalized to Medium 1 to allow relative comparison across conditions, avoiding an additional, potentially skewed layer of correction from an imperfect biomass metric. We acknowledge this as a limitation of the method: since RPE size and biomass differ across media, our fold-changes reflect metabolite pool per well rather than per cell, which could over- or under-represent true per-cell differences in media that yield especially large or small RPE. We will clarify these details in the Methods and Discussion.

(4) Reviewer 1: Since a major purpose of the manuscript is to highlight how cell culture conditions influence RPE biology and metabolism, it would be helpful to also report whether Mycoplasma testing was performed and confirmed to be negative across all cell lines.

We thank the reviewer for the suggestion to include this information. To confirm, Mycoplasma testing was performed on all cell lines used in this study with negative results. We will add this information in the revised Methods.

(5) Reviewer 3: public availability of the underlying metabolomics data would be important for a study intended to serve as a community resource.

The metabolomics data has been deposited to UCSD Center for Computational Mass Spectrometry (CCMS) repository (Dataset: MSV000095024) and will be made publicly accessible upon publication.

References:

(1) Ransy C, Boissan M, Hammad N, Bouaboud A, Issad T, De Dieuleveult M, Miotto B, Ye M, Pasmant E, Bouillaud F. Extracellular flux analyses indicate low ATP yield and require refinement for accurate determination of maximal oxygen consumption rate. Sci Rep. 2026 Jun 10;16(1):18344.