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Photoreceptor precursors derived from three-dimensional embryonic stem cell cultures integrate and mature within adult degenerate retina


Irreversible blindness caused by loss of photoreceptors may be amenable to cell therapy. We previously demonstrated retinal repair1 and restoration of vision through transplantation of photoreceptor precursors obtained from postnatal retinas into visually impaired adult mice2,3. Considerable progress has been made in differentiating embryonic stem cells (ESCs) in vitro toward photoreceptor lineages4,5,6. However, the capability of ESC-derived photoreceptors to integrate after transplantation has not been demonstrated unequivocally. Here, to isolate photoreceptor precursors fit for transplantation, we adapted a recently reported three-dimensional (3D) differentiation protocol that generates neuroretina from mouse ESCs6. We show that rod precursors derived by this protocol and selected via a GFP reporter under the control of a Rhodopsin promoter integrate within degenerate retinas of adult mice and mature into outer segment–bearing photoreceptors. Notably, ESC-derived precursors at a developmental stage similar to postnatal days 4–8 integrate more efficiently compared with cells at other stages. This study shows conclusively that ESCs can provide a source of photoreceptors for retinal cell transplantation.

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Figure 1: Efficient photoreceptor differentiation in wEB 3D cultures.
Figure 2: Time course of photoreceptor genesis in wEB 3D differentiation system.
Figure 3: Integration and connectivity of ESC-derived photoreceptor precursors.

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This work was supported by the Medical Research Council UK (mr/j004553/1, G0901550), RP Fighting Blindness (GR566), The Miller's Trust and Moorfields Eye Charity through a generous private donation. A.G.-C. is a Wellcome Trust PhD student (087256/Z08/Z). R.A.P. is a Royal Society University Research Fellow. J.C.S. is supported by Great Ormond Street Hospital Children's Charity. R.R.A. is partly funded by the Department of Health's National Institute for Health Research Biomedical Research Centre at Moorfields Eye Hospital and Alcon Research Institute. We thank A. Eddaoudi, A. Rose and T. Adejumo for FACS assistance; S. Azam and S. Haria for virus purification; S. Sharma for performing the Affymetrix microarray; and P. Munro for EM assistance. The mouse EK.CCE ESC line34 (129/SvEv) was a kind gift of E. Robertson. The following mouse lines were kind gifts: Gnat1−/− was provided by J. Lem, Tufts University School of Medicine; Prph2rd2/rd2 by G. Travis, UCLA; Rho−/− by P. Humphries, Trinity College Dublin and Nrlp.GFP+/+ by A. Swaroop, University of Michigan.

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Authors and Affiliations



A.G.-C. and E.L.W. contributed equally to the concept, design, execution and analysis of all experiments and manuscript writing. R.A.P. performed subretinal transplantation and calcium imaging, and contributed to the concept and design of the experiments, funding and manuscript writing. Y.D. performed subretinal transplantations and histological processing. L.S.C., A.G. and J.L. contributed to experimental execution. C.J.C. performed IMARIS reconstruction. A.N. and S.J.I.B. provided technical assistance. M.H. performed microarray data analysis. J.W.B.B., A.J.S., J.C.S. and R.R.A. contributed to the concept and design of the experiments, funding and to manuscript writing.

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Correspondence to Emma L West or Robin R Ali.

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The authors declare no competing financial interests.

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Supplementary Text and Figures

Supplementary Figures 1–11 and Supplementary Tables 1–2 (PDF 2735 kb)

Cellular morphology and alignment of an integrated ESC-derived rod photoreceptor

Following transplantation of day 29 ESC-derived Rhop.GFP+ photoreceptors into Gnat1−/− mice, retinal flatmounts were stained for the ribbon synaptic marker, Ribeye (magenta) and rod a-Transducin (red). 3D reconstruction of a representative confocal image showing the correct position of the integrated cell in the ONL (DAPI, blue), the presence of inner and outer segments expressing rod a-Transducin, as well as an inner process ending in a spherule synaptic button correctly positioned in the OPL, where synapses were located. The 3D confocal image was reconstructed to illustrate these morphologies. The whole synaptic layer is shown first, before highlighting the synapse contacting the spherule. The surface of the entire cell was then rendered to demonstrate correct spatial alignment and morphology between the ribbon synapse in relation to the rod spherule. ONL, outer nuclear layer; OPL, outer plexiform layer. (AVI 6951 kb)

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Gonzalez-Cordero, A., West, E., Pearson, R. et al. Photoreceptor precursors derived from three-dimensional embryonic stem cell cultures integrate and mature within adult degenerate retina. Nat Biotechnol 31, 741–747 (2013).

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