Abstract
Purpose :
Metabolic reprogramming is a critical determinant of tissue fate during development and disease. It influences tissue maturation, functionality, and disease. We used the iPSC-derived RPE model to discover metabolic reprogramming from stem cell to committed RPE and from immature to mature RPE fates and extended these findings to the corresponding transcriptomic analysis.
Methods :
iPSC lines from healthy individuals were subjected to directed RPE differentiation using the published protocol. Three technical and two biological replicates were collected for bulk RNA sequencing and untargeted metabolome. Samples were collected at the iPSC stage, neuroectoderm, RPE differentiation, committed RPE, RPE progenitors, and mature RPE stages and subjected to bulk RNA sequencing (NIH Sequencing Core), metabolomics (NIEHS core), and ultra-high-performance liquid chromatography-mass spectrometry (Thermo Vanquish).
Results :
The transcriptome PCA plot revealed three separate clusters: iPSC and neuroectoderm; RPE differentiation, committed and immature RPE, suggesting the most significant gene expression changes from neuroectoderm to RPE progenitors, and from progenitors to committed RPE fate. Interestingly, the metabolome PCA plot showed a similarity between iPSCs and committed RPE and neuroectoderm and RPE progenitors. Immature RPE clustered separately, suggesting differentiation stage-specific metabolome. Notably, metabolites of the tri cyclic carboxylic acid (TCA) cycle (e.g., succinate), critical for RPE function, were relatively higher during RPE commitment. Glycolysis was the highest at the committed RPE stage and lower at the immature and mature RPE stages. Changes in the gene expression of rate-limiting enzymes corroborated changes in TCA and glycolysis metabolites. Phospho-pentose pathway metabolites were higher at the early differentiation stage, while purine and their mono and di-phosphates derivatives were high at the immature RPE stage. Immature and mature RPE stages depend on amino acid metabolism, compared to the pluripotent and early stages of differentiation.
Conclusions :
Our comprehensive metabolic and transcriptomic maps of RPE development and maturation reveal dynamic metabolic reprogramming during RPE development. These reference maps will help better understand metabolic changes in various retinal diseases and help discover biomarkers and potential metabolic switches for treating specific retinal degenerations.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.