Morphological and metabolic analysis of Stargardt disease enucleated eye with stem cell transplantation

Abstract

Purpose : Fluorescence lifetime imaging microscopy (FLIM) is a simple method to study the metabolic activity of tissues. Metabolic activity of the retina using FLIM and histological changes are compared in a Stargardt patient’s eye transplanted with human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) with the contralateral untransplanted eye.

Methods : Formalin-fixed postmortem eyes were obtained from an 80-year-old patient with Stargardt disease who underwent hESC-RPE transplantation into the left eye derived from hESC line MA09 cells. The eyes were embedded in paraffin and sectioned; several sections were stained with H&E or PAS for light microscopy. Before imaging with FLIM, the unstained slides were deparaffinized. FLIM images were acquired using the Leica SP8 DIVE FALCON microscope with a 40x water objective. Excitation wavelengths of 740 nm for autofluorescence and metabolic imaging and 860 nm for lipofuscin were acquired at a power of ~500 µW. The emission wavelength for NAD(P)H was 425-475, 600-650 for melanin, and 470-570 for lipofuscin. For metabolic analysis, the percentage of bound NAD(P)H was compared between the macula and mid-periphery in each eye. Two sample t-test was used for statistical analysis.

Results : Both eyes showed outer retinal atrophy in the macula and cuboidal RPE cells filled with lipofuscin in the mid periphery. The transplanted cells were filled with melanin granules. In some areas they were monolayered, but in other areas, which corresponded to dense pigmentation on fundus photo, they were multi layered. Metabolic analysis with FLIM measured based on the percentage of bound NAD(P)H showed a predominance of oxidative phosphorylation in both atrophic macula and peripheral retina with more oxidative phosphorylation in the macula compared to the periphery in both transplanted and untransplanted eyes. In the transplanted eye, the percentage of bound NAD(P)H was 58.4+/-2.3 for the macula and 54.1+/-2.3 for the mid periphery (p=0.002); in the untransplanted eye, it was 61.7+/-1.1 for the macula and 57.4+/-3.2 for the mid-periphery (p=0.003).
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Conclusions : FLIM can be used to study the metabolic activity of various regions of the retina even in formalin-fixed eyes. The stem cell transplanted atrophic macula in a Stargardt patient showed more oxidative phosphorylation compared to the peripheral retina, similar to the untransplanted eye.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.