Abstract
Purpose :
Metabolic dysfunction of the retinal pigment epithelium (RPE) plays a critical role in the pathogenesis and progression of age-related macular degeneration (AMD). RPE derived from induced pluripotent stem cells (iPSC) of patients with AMD and other retinal diseases are used to characterize disease-relevant metabolic and morphological phenotypes. However, phenotypic variability among different iPSC-derived RPE culture systems is a major challenge in the field. To examine whether one source of variability may be nutrient availability in the culture system, this study seeks to characterize the morphology and metabolic profile of RPE cultured in commonly used media types.
Methods :
We conducted a literature review of iPSC RPE studies and noted the most commonly used RPE culture media. Three widely used media formulations and a novel physiologically relevant media developed based on concentrations of metabolites found in human plasma, human plasma-like media (HPLM), were studied. The media groups are as follows: 1) MEMα + NEAA, N1, THT and 1% FBS, 2) MEMα + NEAA, THT and 2% B27, 3) DMEM-high glucose/Ham’s F-12 + NEAA and B27, 4) X-VIVO 10, 5) HPLM + NEAA, N1, THT and 1% FBS, 6) HPLM + NEAA, THT and 2% B27. iPSC RPE derived from healthy subjects were seeded on Matrigel-coated surfaces and cultured under various media and maintained for up to 8 weeks. RPE were characterized using morphological assessment, TEM, TER, cytokine and RPE marker expression, and targeted metabolomics.
Results :
RPE maintained in all commonly used iPSC RPE culture media retained expression of RPE markers including RPE65, BEST1, CRALBP and Ezrin. However, variations in RPE size, density, and orthogonality across the media groups were noted. Similarly, differences in TER and cytokine secretion were observed. Nutrient consumption and metabolite transport in the RPE were different depending on the culture medium composition, which was also found to be highly diverse in nutrient and metabolite content. Notably, the presence of B27 in the media increased RPE orthogonality, TER, as well as fatty acid oxidation.
Conclusions :
Differing nutrient microenvironments influence iPSC RPE phenotype and metabolism, likely impacting the in vitro presentation of RPE disease phenotypes.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.