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

Nature Biotechnology volume 31, pages 741–747 (2013)Cite this article

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Abstract

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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References

  1. MacLaren, R.E. et al. Retinal repair by transplantation of photoreceptor precursors. Nature 444, 203–207 (2006).

    Article  CAS  Google Scholar 

  2. Pearson, R.A. et al. Restoration of vision after transplantation of photoreceptors. Nature 485, 99–103 (2012).

    Article  CAS  Google Scholar 

  3. Barber, A.C. et al. Repair of the degenerate retina by photoreceptor transplantation. Proc. Natl. Acad. Sci. USA 110, 354–359 (2013).

    Article  CAS  Google Scholar 

  4. Lamba, D.A., Karl, M.O., Ware, C.B. & Reh, T.A. Efficient generation of retinal progenitor cells from human embryonic stem cells. Proc. Natl. Acad. Sci. USA 103, 12769–12774 (2006).

    Article  CAS  Google Scholar 

  5. Osakada, F. et al. Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells. Nat. Biotechnol. 26, 215–224 (2008).

    Article  CAS  Google Scholar 

  6. Eiraku, M. et al. Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature 472, 51–56 (2011).

    Article  CAS  Google Scholar 

  7. Bartsch, U. et al. Retinal cells integrate into the outer nuclear layer and differentiate into mature photoreceptors after subretinal transplantation into adult mice. Exp. Eye Res. 86, 691–700 (2008).

    Article  CAS  Google Scholar 

  8. Lakowski, J. et al. Cone and rod photoreceptor transplantation in models of the childhood retinopathy Leber congenital amaurosis using flow-sorted Crx-positive donor cells. Hum. Mol. Genet. 19, 4545–4559 (2010).

    Article  CAS  Google Scholar 

  9. Pearson, R.A. et al. Targeted disruption of outer limiting membrane junctional proteins (Crb1 and ZO-1) increases integration of transplanted photoreceptor precursors into the adult wild-type and degenerating retina. Cell Transplant. 19, 487–503 (2010).

    Article  CAS  Google Scholar 

  10. Lakowski, J. et al. Effective transplantation of photoreceptor precursor cells selected via cell surface antigen expression. Stem Cells 29, 1391–1404 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Eberle, D., Schubert, S., Postel, K., Corbeil, D. & Ader, M. Increased integration of transplanted CD73-positive photoreceptor precursors into adult mouse retina. Invest. Ophthalmol. Vis. Sci. 52, 6462–6471 (2011).

    Article  CAS  Google Scholar 

  12. Singh, M.S. et al. Reversal of end-stage retinal degeneration and restoration of visual function by photoreceptor transplantation. Proc. Natl. Acad. Sci. USA 110, 1101–1106 (2013).

    Article  CAS  Google Scholar 

  13. Meyer, J.S. et al. Modeling early retinal development with human embryonic and induced pluripotent stem cells. Proc. Natl. Acad. Sci. USA 106, 16698–16703 (2009).

    Article  CAS  Google Scholar 

  14. Hirami, Y. et al. Generation of retinal cells from mouse and human induced pluripotent stem cells. Neurosci. Lett. 458, 126–131 (2009).

    Article  CAS  Google Scholar 

  15. Meyer, J.S. et al. Optic vesicle-like structures derived from human pluripotent stem cells facilitate a customized approach to retinal disease treatment. Stem Cells 29, 1206–1218 (2011).

    Article  CAS  Google Scholar 

  16. Phillips, M.J. et al. Blood-derived human iPS cells generate optic vesicle-like structures with the capacity to form retinal laminae and develop synapses. Invest. Ophthalmol. Vis. Sci. 53, 2007–2019 (2012).

    Article  Google Scholar 

  17. Osakada, F., Ikeda, H., Sasai, Y. & Takahashi, M. Stepwise differentiation of pluripotent stem cells into retinal cells. Nat. Protoc. 4, 811–824 (2009).

    Article  CAS  Google Scholar 

  18. West, E.L. et al. Defining the integration capacity of embryonic stem cell-derived photoreceptor precursors. Stem Cells 30, 1424–1435 (2012).

    Article  CAS  Google Scholar 

  19. Ali, R.R. & Sowden, J.C. Regenerative medicine: DIY eye. Nature 472, 42–43 (2011).

    Article  CAS  Google Scholar 

  20. Martinez-Morales, J.R. & Wittbrodt, J. Shaping the vertebrate eye. Curr. Opin. Genet. Dev. 19, 511–517 (2009).

    Article  CAS  Google Scholar 

  21. Hyatt, G.A., Schmitt, E.A., Fadool, J.M. & Dowling, J.E. Retinoic acid alters photoreceptor development in vivo. Proc. Natl. Acad. Sci. USA 93, 13298–13303 (1996).

    Article  CAS  Google Scholar 

  22. Kelley, M.W., Williams, R.C., Turner, J.K., Creech-Kraft, J.M. & Reh, T.A. Retinoic acid promotes rod photoreceptor differentiation in rat retina in vivo. Neuroreport 10, 2389–2394 (1999).

    Article  CAS  Google Scholar 

  23. Lombardini, J.B. Taurine: retinal function. Brain Res. Brain Res. Rev. 16, 151–169 (1991).

    Article  CAS  Google Scholar 

  24. Chen, S. et al. Crx, a novel Otx-like paired-homeodomain protein, binds to and transactivates photoreceptor cell-specific genes. Neuron 19, 1017–1030 (1997).

    Article  CAS  Google Scholar 

  25. Furukawa, T., Morrow, E.M. & Cepko, C.L. Crx, a novel otx-like homeobox gene, shows photoreceptor-specific expression and regulates photoreceptor differentiation. Cell 91, 531–541 (1997).

    Article  CAS  Google Scholar 

  26. Blackshaw, S. et al. Genomic analysis of mouse retinal development. PLoS Biol. 2, e247 (2004).

    Article  Google Scholar 

  27. Calvert, P.D. et al. Phototransduction in transgenic mice after targeted deletion of the rod transducin alpha -subunit. Proc. Natl. Acad. Sci. USA 97, 13913–13918 (2000).

    Article  CAS  Google Scholar 

  28. Lamba, D.A., Gust, J. & Reh, T.A. Transplantation of human embryonic stem cell-derived photoreceptors restores some visual function in Crx-deficient mice. Cell Stem Cell 4, 73–79 (2009).

    Article  CAS  Google Scholar 

  29. Koulen, P., Kuhn, R., Wässle, H. & Brandstätter, J.H. Modulation of the intracellular calcium concentration in photoreceptor terminals by a presynaptic metabotropic glutamate receptor. Proc. Natl. Acad. Sci. USA 96, 9909–9914 (1999).

    Article  CAS  Google Scholar 

  30. Koulen, P. & Brandstätter, J.H. Pre- and postsynaptic sites of action of mGluR8a in the mammalian retina. Invest. Ophthalmol. Vis. Sci. 43, 1933–1940 (2002).

    PubMed  Google Scholar 

  31. West, E.L. et al. Pharmacological disruption of the outer limiting membrane leads to increased retinal integration of transplanted photoreceptor precursors. Exp. Eye Res. 86, 601–611 (2008).

    Article  CAS  Google Scholar 

  32. Tucker, B.A. et al. Transplantation of adult mouse iPS cell-derived photoreceptor precursors restores retinal structure and function in degenerative mice. PLoS ONE 6, e18992 (2011).

    Article  CAS  Google Scholar 

  33. Nakano, T. et al. Self-formation of optic cups and storable stratified neural retina from human ESCs. Cell Stem Cell 10, 771–785 (2012).

    Article  CAS  Google Scholar 

  34. Evans, M.J. & Kaufman, M.H. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156 (1981).

    Article  CAS  Google Scholar 

  35. Gao, G.-P. et al. Rep/Cap gene amplification and high-yield production of AAV in an A549 cell line expressing Rep/Cap. Mol. Ther. 5, 644–649 (2002).

    Article  CAS  Google Scholar 

  36. Davidoff, A.M. et al. Purification of recombinant adeno-associated virus type 8 vectors by ion exchange chromatography generates clinical grade vector stock. J. Virol. Methods 121, 209–215 (2004).

    Article  CAS  Google Scholar 

  37. Luhmann, U.F.O. et al. Differential modulation of retinal degeneration by Ccl2 and Cx3cr1 chemokine signalling. PLoS ONE 7, e35551 (2012).

    Article  CAS  Google Scholar 

  38. Tschernutter, M. et al. Long-term preservation of retinal function in the RCS rat model of retinitis pigmentosa following lentivirus-mediated gene therapy. Gene Ther. 12, 694–701 (2005).

    Article  CAS  Google Scholar 

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Acknowledgements

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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  1. Anai Gonzalez-Cordero and Emma L West: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Genetics, UCL Institute of Ophthalmology, London, UK

    Anai Gonzalez-Cordero, Emma L West, Rachael A Pearson, Yanai Duran, Livia S Carvalho, Colin J Chu, Arifa Naeem, Samuel J I Blackford, Anastasios Georgiadis, Alexander J Smith, James W B Bainbridge & Robin R Ali

  2. Developmental Biology Unit, Institute of Child Health, University College London, London, UK

    Jorn Lakowski & Jane C Sowden

  3. UCL Genomics Institute of Child Health, University College London, London, UK

    Mike Hubank

  4. Molecular Immunology Unit, Institute of Child Health, University College London, London, UK

    Robin R Ali

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  1. Anai Gonzalez-Cordero
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Contributions

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.

Corresponding authors

Correspondence to Emma L West or Robin R Ali.

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The authors declare no competing financial interests.

Supplementary information

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). https://doi.org/10.1038/nbt.2643

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