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Deng Pan, Xi-Xi Xia, Hui Liu, Shao-Hui Pan, Mei-Ling Gao, Zi-Bing Jin; The CRX allelic deletion in ES cells rendering delayed maturation of photoreceptors in three-dimensional Retinal organoids differentiation. Invest. Ophthalmol. Vis. Sci. 2018;59(9):569.
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© ARVO (1962-2015); The Authors (2016-present)
Photoreceptors make up 70-80% of the retinal cells. CRX, an established photoreceptor gene in postmitotic photoreceptor precursors, determines precursor cell fate and regulates the development and function of photoreceptors. Herein, we use three-dimensional differentiation system on established CRX allelic deletion ES cells for systemic gene function analysis.
One CRX allele was replaced by tdTomato using CRISPR-Cas9 system in H9 cells. Chromosome aberration in the resulting H9-tdTomato cell line was detected by karyotype analysis. Flow cytometry and immunostaining were used to detect the expression consistency between CRX and tdTomato. Whole transcriptome profiling in retinal organoids were performed at different 3-D differentiation stages. Mature photoreceptor markers, M-/S-opsin and Rhodopsin, were detected by immunostaining.
H9-tdTomato cell line preserved genomic stability through genotyping. Fluorescent cells appeared inside of retinal organoids in early differentiation stage and gradually migrated outward, with exactly the same with expression of CRX in the immunostaining characterization. mRNA level of CRX was significantly decreased on day 90 (P<0.01) but no obvious changes observed on NRL. Immunostaining detection of M-/S-opsin appeared on day 110 in organoids derived by H9 cell line, while it must be delayed up to day 250 in H9-tdTomato cell line differentiation.
Deletion an allele of CRX greatly slowed the photoreceptor maturing process down during this in vitro neural retina organization. This study provided an efficient simulation way to dissect the importance of CRX in the retinal development. Allelic deletion in ES cells and the following differentiation may provide an alternative tool for understanding the role of gene in human organogenesis and occurrence of diseases.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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