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Claire Bell, Weixiang Fang, Cynthia Berlinicke, Aniruddhah Kaushik, Pengfei Zhang, Tza-Huei Wang, Reza Kalhor, Hongkai Ji, Donald J Zack; Single-cell lineage tracing of developing retinal systems. Invest. Ophthalmol. Vis. Sci. 2020;61(7):4014.
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© ARVO (1962-2015); The Authors (2016-present)
Studies of mammalian retinal development have mostly been done using model organisms, and thus the mechanisms and lineage relationships underlying human retinal development are much less well understood. Human stem cell-derived retinal organoids closely follow the human retinal development timeline while generating all of the known classes of cells in self-organizing layers, allowing human retinal cells to be studied in unprecedented detail. Using single cell methods for analyzing combined transcriptome and recorded cell lineage data, we are working to create high-resolution maps of both human retinal organoid and in vivo mouse retinal development.
To trace retinal lineages, we are using homing guide RNAs (hgRNAs) that are edited by Cas9 such that they become randomly mutated. The mutations are inherited by daughter cells, allowing lineages to be reconstructed by comparing the collection of mutations between different cells. We have developed a novel, droplet-based method for the parallel capture of genomic sequences and the transcriptome of thousands of single cells in a given experiment enabling us to couple the recorded lineage information with gene expression at the single cell level. Using an hgRNA mouse line (Kalhor et al. Science 2018) and retinal organoids differentiated from an hPSC line with randomly integrated hgRNAs, we will analyze multiple time points through the course of retinal development to establish high-resolution lineage maps.
We have developed the methodology, and engineered and validated the cell line containing the hgRNAs from which to differentiate retinal organoids. We have begun to develop the bioinformatics tools needed for the analysis of this unique dataset: using in silico modeling we are able to reconstruct lineage relationships from single-cell sequences. We are now collecting data on human retinal organoid and mouse retinas at various developmental time-points.
Combining lineage tracing with the transcriptomic signature of a cell will make it possible to assess retinal developmental processes at an unprecedented level of resolution, enabling the identification of unique, transient cell-types that lead to the fully differentiated retina. The ability to pinpoint and modify the factors that dictate retinal development at a cellular level will build on what we already know about mouse retinal development and provide newfound insight into normal human retinal development.
This is a 2020 ARVO Annual Meeting abstract.
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