Biology subjects

Lim, R. R.

Publications and source records attributed to Lim, R. R..

3 recordsLinked to original sources

bioRxiv2026

Nutrient microenvironments reprogram RPE metabolism

Induced pluripotent stem cell-derived retinal pigment epithelium (iPSC RPE) has become a widely used model for studying the mechanisms of age-related macular degeneration (AMD). However, the nutrient composition of currently used RPE culture media is highly variable, posing a major challenge to reproducibility in RPE metabolism and phenotype. We systematically investigate how six distinct nutrient microenvironments shape RPE phenotype, function and metabolism in both iPSC RPE and fetal RPE (fRPE). These included MEM, DMEM-HG/F12 basal media, physiological human plasma-like medium (HPLM) supplemented with FBS or B27, and X-VIVO 10. Although canonical RPE markers were expressed across all conditions, B27 supplementation and X-VIVO 10 increased RPE cell size, hexagonality, and transepithelial resistance. Culture in HPLM+FBS induced accumulation of lipid droplets and sub-RPE deposits, whereas X-VIVO 10 resulted in the formation of large intracellular vacuoles. B27 supplementation enhanced basal respiration, while X-VIVO 10 increased glycolytic capacity. Amino acid consumption was broadly conserved across media types, including complete depletion of proline in all conditions by 48 hours; however, lipid and nucleotide metabolism varied substantially between conditions. Notably, B27 supplementation in specific media types reversed the net direction of several metabolites, with creatine, serine and taurine shifting from consumption to production, while riboflavin and guanine shifted from production to consumption. These findings establish the nutrient environment as a key determinant of RPE phenotype, function and metabolism. Our work provides a valuable resource for media selection and interpretation of cellular and metabolic phenotypes relevant to RPE disease modeling.

cell biology↗

bioRxiv2024

Retinal microglia-derived S100A9 incite NLRP3 inflammasome in a Western diet fed Ossabaw pig retina

PurposeWe established S100A9 as a myeloid-derived damage-associated molecular pattern (DAMPs) protein associated with increasing severity of diabetic retinopathy (DR) in type 2 diabetic subjects. The present study investigates the retinal localization, expression, and mechanisms of action for S100A9 in the young obese Ossabaw pig retina. MethodsRetinae from Ossabaw pigs fed a Western diet for 10 weeks were evaluated for S100 and inflammatory mediator expression using quantitative PCR and Western blot. Double immunohistochemistry was performed to identify the cellular sources of S100A9 in the pig retina. Primary pig retinal microglial cells (pMicroglia) were examined for S100A9 production. S100A9-induced responses were also investigated, and inhibitor studies elucidated the mechanism of action via the NLRP3 inflammasome. A specific inhibitor, Paquinimod (ABR-215757), was administered in vitro to assess the rescue of S100A9-induced NLRP3 inflammasome activation in pMicroglia. ResultsThe expression of the S100 family in the obese Ossabaw pig retina showed a significant elevation of S100A9, consistent with increased levels of circulating S100A9. Moreover, the retina had elevated levels of inflammatory mediators IL-6, IL-8, MCP-1, IL-1{beta} and NLRP3. Retinal microglia in obese Ossabaw were activated and accompanied by an increased expression of intracellular S100A9. pMicroglia isolated from pig retina transformed from ramified to amoeboid state when activated with LPS and produced high S100A9 transcript and protein levels. The S100A9 protein, in turn, further activated pMicroglia by heightened production of S100A9 transcripts and secretion of pro-inflammatory IL-1{beta} protein. Inhibition of TLR4 with TAK242 and NLRP3 with MCC950 attenuated the production of IL-1{beta} during S100A9 stimulus. Finally, pre-treatment with Paquinimod successfully reduced S100A9-driven increases of glycosylated-TLR4, NLRP3, ASC, Caspase-1, and IL-1{beta} production. ConclusionWe demonstrated that microglial-derived S100A9 perpetuates pro-inflammatory responses via the NLRP3 inflammasome in the retina of young Western-diet-fed Ossabaw pigs exhibiting diabetic retinopathy.

neuroscience↗

bioRxiv2024

Metabolic phenotyping of healthy and diseased human RPE cells

PurposeMetabolic defects in the retinal pigment epithelium (RPE) underlie many retinal degenerative diseases. This study aims to identify the nutrient requirements of healthy and diseased human RPE cells. MethodsWe profiled nutrient utilization of various human RPE cells, including differentiated and dedifferentiated fetal RPE (fRPE), induced pluripotent stem cell derived-RPE (iPSC RPE), Sorsby fundus dystrophy (SFD) patient-derived iPSC RPE, CRISPR-corrected isogenic SFD (cSFD) iPSC RPE, and ARPE-19 cell lines using Biolog Phenotype MicroArray Assays. ResultsDifferentiated fRPE cells and healthy iPSC RPE cells can utilize 51 and 48 nutrients respectively, including sugars, intermediates from glycolysis and tricarboxylic acid (TCA) cycle, fatty acids, ketone bodies, amino acids, and dipeptides. However, when fRPE cells lose their epithelial phenotype through dedifferentiation, nutrient utilization becomes restricted to 17 nutrients, primarily sugar and glutamine-related amino acids. SFD RPE cells can utilize 37 nutrients; however, compared to cSFD RPE and healthy iPSC RPE, they are unable to utilize lactate, some TCA cycle intermediates, and short-chain fatty acids. Nonetheless, they show increased utilization of branch-chain amino acids (BCAAs) and BCAA-containing dipeptides. Dedifferentiated ARPE-19 cells grown in traditional culture media cannot utilize lactate and ketone bodies. In contrast, nicotinamide supplementation promotes differentiation towards an epithelial phenotype, restoring the ability to use these nutrients. ConclusionsEpithelial phenotype confers metabolic flexibility to healthy RPE for utilizing various nutrients. SFD RPE cells have reduced metabolic flexibility, relying on the oxidation of BCAAs. Our findings highlight the potentially important roles of nutrient availability and utilization in RPE differentiation and diseases.

biochemistry↗