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Holly Chen, Lijing Dong, Anand Swaroop; Development of treatments for CEP290-LCA using three-dimensional retinal organoids. Invest. Ophthalmol. Vis. Sci. 2017;58(8):293.
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© ARVO (1962-2015); The Authors (2016-present)
Leber congenital amaurosis (LCA) is a group of congenital retinal degenerative diseases. Mutations of CEP290, which encodes for a ciliary protein at the connecting cilium of photoreceptors (PRs), are a major cause of LCA. No treatments are available largely due to lack of appropriate in vitro model to investigate disease pathogenesis and to develop therapeutics. Previously, we developed HIPRO protocol to efficiently differentiate mouse pluripotent stem cells into three-dimensional retinal organoids with elongated axoneme of PRs. We hypothesize that PRs differentiated from induced pluripotent stem cells (iPSCs) of rd16, which is the mouse model of CEP290-LCA, have defects in ciliogenesis, based on which corresponding treatments can be developed.
Nrl-GFP wild-type (WT) and rd16 iPSCs were differentiated into retinal organoids using HIPRO protocol. At least 2 clones were used for WT or rd16 iPSCs. For each clone, 30-60 organoids were harvested at specific differentiation stages of rod PRs and subjected to flow analysis to evaluate viability and percentage of rod cells (N=4). Immunohistochemistry (IHC) was performed to investigate PR ciliary morphology (N = 4, 10-20 organoids of each clone were harvested).
At the early stage of rod birth, which is differentiation day (D) 18 and 22 in culture, both WT and rd16 iPSCs showed similar viability. Shortly after the start of ciliogenesis, viability of rd16 organoids started to decrease and was approximately 50% lower than that of WT at the end of differentiation (p<0.05). While percentage of GFP+ rod cells in WT culture steadily rose from D18 to D35, the increase stopped in one rd16 clone and was more slowly in another clone after D28, suggesting pause of differentiation or death of rods. IHC analysis showed consistent results. Rd16 organoids had less ciliated PRs, with one clone having more severe phenotypes than the other. These phenotypes may be caused by defects of ciliogenesis, as rootlets of rd16 PRs were disrupted and outer limiting membrane were missing in both clones.
Rd16 retinal organoids have lower viability compared to WT, which may be caused by defects in development of PR cilia. The underlying mechanisms are being investigated by transcriptome profiling of developing rods and small molecule screening is being performed to maintain the survival of rod cells in rd16 organoids.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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