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. 2016 Apr 1;57(5):ORSFl1-ORSFl11.
doi: 10.1167/iovs.15-17639.

Treatment Paradigms for Retinal and Macular Diseases Using 3-D Retina Cultures Derived From Human Reporter Pluripotent Stem Cell Lines

Rossukon Kaewkhaw  1 , Manju Swaroop  2 , Kohei Homma  3 , Jutaro Nakamura  3 , Matthew Brooks  3 , Koray Dogan Kaya  3 , Vijender Chaitankar  3 , Sam Michael  2 , Gregory Tawa  2 , Jizhong Zou  4 , Mahendra Rao  5 , Wei Zheng  2 , Tiziana Cogliati  3 , Anand Swaroop  3
Affiliations

Treatment Paradigms for Retinal and Macular Diseases Using 3-D Retina Cultures Derived From Human Reporter Pluripotent Stem Cell Lines

Rossukon Kaewkhaw et al. Invest Ophthalmol Vis Sci. .

Abstract

We discuss the use of pluripotent stem cell lines carrying fluorescent reporters driven by retinal promoters to derive three-dimensional (3-D) retina in culture and how this system can be exploited for elucidating human retinal biology, creating disease models in a dish, and designing targeted drug screens for retinal and macular degeneration. Furthermore, we realize that stem cell investigations are labor-intensive and require extensive resources. To expedite scientific discovery by sharing of resources and to avoid duplication of efforts, we propose the formation of a Retinal Stem Cell Consortium. In the field of vision, such collaborative approaches have been enormously successful in elucidating genetic susceptibility associated with age-related macular degeneration.

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Figures

Figure 1
Figure 1
Development of neural retina and retinal pigment epithelium cells. (A) Neural retina (NR) and retinal pigment epithelium (RPE) cells develop from the optic vesicle (OV) that emerges from the neural tube. Invagination of the optic vesicle, placing the prospective NR alongside the prospective RPE, gives rise to the optic cup. Key transcription factors that regulate each developmental stage and determine the fate of cell types in the retinal layers are listed. (B) Three-dimensional retina in vitro can be generated by floating aggregates of PSCs, which form optic vesicles where NR and pigment mass (PM) develop. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; PR, photoreceptor; GC, ganglion cell.
Figure 2
Figure 2
Donor vectors for insertion of fluorescent reporters at the AAVS1 site using zinc finger nucleases. The use of different color spectra can allow the concomitant detection of more than one reporter.
Figure 3
Figure 3
Strategy for knock-in using gene cleavage–induced homologous recombination. FP, fluorescent protein.
Figure 4
Figure 4
Three-dimensional retina derived from the CRXp-GFP H9 hESC reporter line. (A) Three-dimensional neural retina (NR) is generated from CRXp-GFP hESCs. (B) CRXp-GFP+ photoreceptors in NR can (C) be purified by fluorescence-activated cell sorting (FACS) for RNA isolation and next-generation sequencing. (D) Gene regulatory networks and cell surface molecules can be extracted from global gene expression analysis. PM, pigment mass.
Figure 5
Figure 5
Expression profiles of selected genes in developing human photoreceptors in 3-D cultures. Clustering in the heat maps was performed using affinity propagation. The transcriptome data of d37, d47, d67, and d90 CRX+/GFP+ photoreceptors, shown here for comparison, has been reported recently. (A) Transcription factors. (B) Phototransduction genes. (C) Cell differentiation (CD) markers (≥10 FPKM at d134 or d220). FPKM, fragments per kilobase of transcript per million mapped reads.
Figure 6
Figure 6
In vivo differentiation of CRXp-GFP H9 hESC-derived retinal cells from 3-D cultures. CRX+/GFP+ cultures at d58 were dissociated, and cells were injected in the subretinal space of recipient adult C57BL/6 mice. Eyes were collected 4, 6, and 14 weeks post injection, resulting in combined differentiation times of 12 weeks (w), 14w, and 22w, respectively. (A–E) Most transplanted CRX+/GFP+ cells formed polarized “rosette” structures with the apical side facing inward based on phalloidin staining (arrows) (A). By 22w, a majority of rosettes were labeled with rhodopsin (4D2) antibody in their apical center (arrowheads) (B, C). (D) Occasional, faint S opsin staining could be observed as early as 14w on the apex of CRX+/GFP+ photoreceptor cells (asterisk). (E) A small number of CRX+/GFP+ photoreceptors expressed L/M opsin at 22w (arrows). INL, host inner nuclear layer; ONL, host outer nuclear layer; SRS, host subretinal space; a, apical; b, basal. Scale bars: 20 μm.
Figure 7
Figure 7
Schematic of HTS using 3-D retinal culture of the CRXp-GFP H9 hESC line.
Figure 8
Figure 8
Plate view of small-molecule hit confirmation in 1536-well format. A total of 5600 compounds from four diverse molecular libraries were screened by HTS in a homogenous GFP cell-based assay. Validation of 32 primary actives using an 11-point dose–response titration was done at 3-fold serial dilutions of test compounds dissolved in DMSO at starting concentrations of 10 mM.
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