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Michelle G Zhang, Zenith Acosta Torres, Dawn Owens, Robert Suter, Nagi Ayad, J William Harbour, Daniel Pelaez; Lineage tracing in retinal organoids as a platform for studying retinoblastoma. Invest. Ophthalmol. Vis. Sci. 2020;61(7):3815.
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© ARVO (1962-2015); The Authors (2016-present)
Retinoblastoma is the most common primary intraocular malignancy in children. Approximately 25% of the cases fail first line chemotherapy, and the most significant barrier to the treatment strategy is the gap in knowledge regarding biological mechanisms driving retinoblastoma progression. The purpose of this project is to characterize the susceptibility to RB1 loss in the terminal differentiation of the various retinal phenotypes.
We employ an iPSC-derived 3-Dimensional retinal organoid platform, along with CRISPR gene editing, CellTag barcoding libraries for lineage tracing, and single cell RNA-sequencing (scRNA-seq) to analyze the development of retinal phenotypes from RB1 wild-type (RB1-WT) and RB1 knockout (RB1-KO) retinal organoids. This approach allows us to trace shared retinal cell lineages and identify developmental bifurcations affected by RB1 loss. WT and RB1-KO organoids were established from dental pulp stem cells and photoreceptor markers were probed for in Week 20 using immunofluorescence. At Week 14, both organoids were scRNA-sequenced and analyzed for changes in differentiation markers specific to each major retinal cell type. After differences were confirmed, organoid microinjections were performed with GFP-lentivirus to ensure the feasibility of capturing a diverse representation of retinal cells for downstream analyses. Meanwhile, CellTag libraries were amplified, sequenced for quality control, and packaged into lentivirus. WT and RB1-KO organoids were then marked at three different timepoints using these lentivirus in preparation for scRNA-seq.
Retinal organoids form an ordered structure that can be maintained as far as Week 20, and photoreceptor markers CRX and RCVRN are present along the periphery. By Week 14, there is already an imbalance in the distribution of early-stage and late-stage differentiation markers caused by RB1 loss. GFP-lentivirus microinjections allow for a deeper penetration of lentivirus while keeping the organoids viable. CellTag barcoding libraries sequenced for quality control show a diverse number of unique identifiers.
These preliminary results demonstrate the feasibility of using this platform as a model for modeling retinoblastoma progression and identifying the developmental effects of RB1 loss in the distribution of nascent retinal phenotypes.
This is a 2020 ARVO Annual Meeting abstract.
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