3D organelle map of polarized iPSC-RPE using high-content imaging and volumetric segmentation

Abstract

Purpose : The retinal pigment epithelium (RPE) faces two different environments – the retinal photoreceptors apically and the choroid basally. To perform different functions on each side, RPE cells must undertake a rearrangement of intracellular structures during development and polarize along the apical-basal axis. Failure to acquire a mature phenotype leads to retinal degeneration, as seen in some ciliopathies. In this study, we generated a three-dimensional reference map of induced pluripotent stem cell-derived RPE (iPSC-RPE) organelles and cytoskeletal structures to better understand the process of RPE polarization, using high-content imaging and object segmentation.

Methods : RPE cells were derived from 16 induced pluripotent stem cell reporter lines, each expressing a fluorescently tagged protein to highlight specific intracellular structures. About 10,000 images of iPSC-RPE were taken at different timepoints during maturation. iPSC-RPE cells treated with HPI4, a compound known to disrupt apical-basal polarity, were included as control. A machine-learning algorithm was trained to identify and segment cells and nuclei borders. Each organelle or subcellular structure was segmented using classic segmentation algorithms. Three rounds of segmentation with the human in the loop were carried out to ensure the accuracy of the results.

Results : Cells and nuclei became more compact horizontally and elongated on the vertical axis during polarization. Moreover, nuclei developed invaginations of the nuclear envelope. The volume of endoplasmic reticulum (ER) and mitochondria increased with maturation, while the volume of Golgi apparatus remained constant. Among endosomes, autophagosomes, peroxisomes and lysosomes, the latter was the only organelle that displayed a decrease in volume when RPE polarization was blocked (0.45 vs 0.2 mm²). The ER, mitochondria, and lysosomes moved toward the center of the cell during maturation. The average number of lysosomes decreased with cell polarization (30 vs 20/cell), while the number of peroxisomes increased (70 vs 90/cell).

Conclusions : This work provides a three-dimensional reference map of organelle number, morphology, and distribution during normal development of RPE apical-basal polarity. Abnormal RPE phenotypes occurring in different diseases can now be compared to this reference map to understand the underlying subcellular mechanisms of action.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.