Identification and enrichment of human retinal organoid-derived red/green cone-competent precursors with enhanced...

Cell replacement therapies aimed at restoring foveal vision require a robust source of red/green (long/medium wavelength, or L/M) cone photoreceptors with intrinsic properties conducive to functional integration into host retina. Recent evidence has shown that cones present within mature human retinal organoids (ROs) can generate light responses comparable to macaque foveal cones. However, only cone precursors from early developing ROs possess a capacity for cell-autonomous axonogenesis. Therefore, we sought to identify and enrich for a population of early L/M cone-competent precursors with intrinsically superior axon dynamics that would provide an ideal donor cell source for future foveal reconstruction efforts.

We developed a dual L/M cone/rod reporter (L/M-CRR) line to unequivocally identify early L/M cone-competent precursors from human ROs. To do so, we used CRISPR/Cas9 to link a tdTomato transgene to the endogenous THRB2 promoter in the WA09 NRL^+/eGFP rod reporter line. Differentiated ROs were characterized at early (day 50) and intermediate (day 100) developmental stages using a combination of live fluorescence imaging, immunocytochemistry, and flow cytometry, followed by fluorescence-activated cell sorting and bulk RNAseq analysis to delineate the unique molecular signature of early L/M cone-competent precursors.

THRB2-driven tdTomato fluorescence faithfully demarcated L/M cone-competent precursors throughout RO development, although fluorescence declined in later ROs as L/M cones matured. Transcriptomic profiling revealed that day 50 sorted tdTomato⁺ cells were specifically enriched for genes associated with neural development, axon extension and guidance, and cell migration, which included a gene encoding the cell surface protein CD166/ALCAM. Magnetic-activated cell sorting using an anti-CD166 antibody resulted in specific enrichment of early, highly axonogenic L/M cone-competent precursors assessed by time-lapse imaging.

The L/M-CRR line enables definitive identification and transcriptomic characterization of L/M cone-competent precursors throughout early to mid-stage RO development. Our investigation also revealed that CD166/ALCAM can be used to independently identify and enrich for a subset of early L/M cone-competent precursors that selectively display axon dynamicity conducive for retinal circuit integration. Our studies provide the first insights into early human L/M cone development and establish a method to isolate L/M cone-competent precursors with enhanced axon dynamics, which constitutes a compelling cell population for treating central vision loss caused by photoreceptor degeneration.