Quantification of Drusen-Like Deposits in Human Pluripotent Stem Cell-Derived Retinal Pigment Epithelium Cells Using Confocal Microscopy.

Accessing pathologically relevant models of human retinal degeneration is crucial for understanding, screening, and developing accurate treatment strategies that prevent or slow progression of blinding diseases. Human pluripotent stem cells (hPSCs) are a powerful tool to investigate retinal disease, as cells from selected individuals or specific genetic background can be differentiated into cell types of interest. hPSC-derived retinal pigment epithelium (RPE) cells can produce drusen-like deposits that accumulate between the RPE and the underlying Bruch's membrane. These key pathological markers of specific retinal disorders are detectable using simple microscopy techniques. However, quantification of these deposits often requires slow, low-throughput manual counting of images. This further lends itself to issues including sampling biases while ignoring critical data parameters such as deposit volume and precise localization. To overcome these issues, we developed two methods for quantifying the presence of drusen-like deposits in vitro, using hPSC-derived RPE cultures. Both methods accurately quantify drusen-like deposit numbers, and the mid-throughput pipeline allows for quantification of drusen-like deposit volume, as well as discerns their apical or basal localization on RPE cells. The platform's ability to track changes in drusen-like deposits supports screening experimentation and personalized medicine initiatives, including for gene therapy optimization.