Abstract
We previously reported the differentiation of mouse embryonic stem (ES) cells into retinal progenitors. However, these progenitors rarely differentiate into photoreceptors unless they are cultured with embryonic retinal tissues. Here we show the in vitro generation of putative rod and cone photoreceptors from mouse, monkey and human ES cells by stepwise treatments under defined culture conditions, in the absence of retinal tissues. With mouse ES cells, Crx+ photoreceptor precursors were induced from Rx+ retinal progenitors by treatment with a Notch signal inhibitor. Further application of fibroblast growth factors, Shh, taurine and retinoic acid yielded a greater number of rhodopsin+ rod photoreceptors, in addition to default cone production. With monkey and human ES cells, feeder- and serum-free suspension culture combined with Wnt and Nodal inhibitors induced differentiation of Rx+ or Mitf+ retinal progenitors, which produced retinal pigment epithelial cells. Subsequent treatment with retinoic acid and taurine induced photoreceptor differentiation. These findings may facilitate the development of human ES cell–based transplantation therapies for retinal diseases.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Change history
26 February 2008
In the version of this article initially published, one author’s name, the density of ES cells and the composition of RA/T medium are incorrect. The errors have been corrected in the HTML and PDF versions of the article.
References
Rattner, A. & Nathans, J. Macular degeneration: recent advances and therapeutic opportunities. Nat. Rev. Neurosci. 7, 860–872 (2006).
Kaplan, H.J., Tezel, T.H., Berger, A.S., Wolf, M.L. & Del Priore, L.V. Human photoreceptor transplantation in retinitis pigmentosa. A safety study. Arch. Ophthalmol. 115, 1168–1172 (1997).
Takahashi, M., Palmer, T.D., Takahashi, J. & Gage, F.H. Widespread integration and survival of adult-derived neural progenitor cells in the developing optic retina. Mol. Cell. Neurosci. 12, 340–348 (1998).
Aramant, R.B., Seiler, M.J. & Ball, S.L. Successful cotransplantation of intact sheets of fetal retina with retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 40, 1557–1564 (1999).
Radtke, N.D., Aramant, R.B., Seiler, M.J., Petry, H.M. & Pidwell, D. Vision change after sheet transplant of fetal retina with retinal pigment epithelium to a patient with retinitis pigmentosa. Arch. Ophthalmol. 122, 1159–1165 (2004).
MacLaren, R.E. et al. Retinal repair by transplantation of photoreceptor precursors. Nature 444, 203–207 (2006).
Haruta, M. et al. In vitro and in vivo characterization of pigment epithelial cells differentiated from primate embryonic stem cells. Invest. Ophthalmol. Vis. Sci. 45, 1020–1025 (2004).
Haruta, M. et al. Induction of photoreceptor-specific phenotypes in adult mammalian iris tissue. Nat. Neurosci. 4, 1163–1164 (2001).
Sun, G. et al. Retinal stem/progenitor properties of iris pigment epithelial cells. Dev. Biol. 289, 243–252 (2006).
Tropepe, V. et al. Retinal stem cells in the adult mammalian eye. Science 287, 2032–2036 (2000).
Zhao, X., Liu, J. & Ahmad, I. Differentiation of embryonic stem cells into retinal neurons. Biochem. Biophys. Res. Commun. 297, 177–184 (2002).
Hirano, M. et al. Generation of structures formed by lens and retinal cells differentiating from embryonic stem cells. Dev. Dyn. 228, 664–671 (2003).
Ikeda, H. et al. Generation of Rx+/Pax6+ neural retinal precursors from embryonic stem cells. Proc. Natl. Acad. Sci. USA 102, 11331–11336 (2005).
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).
Thomson, J.A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).
Hoffman, L.M. & Carpenter, M.K. Characterization and culture of human embryonic stem cells. Nat. Biotechnol. 23, 699–708 (2005).
Watanabe, K. et al. Directed differentiation of telencephalic precursors from embryonic stem cells. Nat. Neurosci. 8, 288–296 (2005).
Mathers, P.H., Grinberg, A., Mahon, K.A. & Jamrich, M. The Rx homeobox gene is essential for vertebrate eye development. Nature 387, 603–607 (1997).
Furukawa, T., Kozak, C.A. & Cepko, C.L. rax, a novel paired-type homeobox gene, shows expression in the anterior neural fold and developing retina. Proc. Natl. Acad. Sci. USA 94, 3088–3093 (1997).
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).
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).
Kubo, F., Takeichi, M. & Nakagawa, S. Wnt2b inhibits differentiation of retinal progenitor cells in the absence of Notch activity by downregulating the expression of proneural genes. Development 132, 2759–2770 (2005).
Jadhav, A.P., Mason, H.A. & Cepko, C.L. Notch 1 inhibits photoreceptor production in the developing mammalian retina. Development 133, 913–923 (2006).
Yaron, O. et al. Notch1 functions to suppress cone-photoreceptor fate specification in the developing mouse retina. Development 133, 1367–1378 (2006).
Dovey, H.F. et al. Functional gamma-secretase inhibitors reduce beta-amyloid peptide levels in brain. J. Neurochem. 76, 173–181 (2001).
Geling, A. et al. A γ-secretase inhibitor blocks Notch signaling in vivo and causes a severe neurogenic phenotype in zebrafish. EMBO Rep. 3, 688–694 (2002).
Levine, E.M., Fuhrmann, S. & Reh, T.A. Soluble factors and the development of rod photoreceptors. Cell. Mol. Life Sci. 57, 224–234 (2000).
Altshuler, D. & Cepko, C. A temporally regulated, diffusible activity is required for rod photoreceptor development in vitro. Development 114, 947–957 (1992).
Young, T.L. & Cepko, C.L. A role for ligand-gated ion channels in rod photoreceptor development. Neuron 41, 867–879 (2004).
Hyatt, G.A. & Dowling, J.E. Retinoic acid. A key molecule for eye and photoreceptor development. Invest. Ophthalmol. Vis. Sci. 38, 1471–1475 (1997).
McGinnis, J.F. et al. Unique retina cell phenotypes revealed by immunological analysis of recoverin expression in rat retina cells. J. Neurosci. Res. 55, 252–260 (1999).
Lukats, A. et al. Photopigment coexpression in mammals: comparative and developmental aspects. Histol. Histopathol. 20, 551–574 (2005).
Bora, N., Conway, S.J., Liang, H. & Smith, S.B. Transient overexpression of the Microphthalmia gene in the eyes of Microphthalmia vitiligo mutant mice. Dev. Dyn. 213, 283–292 (1998).
Nguyen, M. & Arnheiter, H. Signaling and transcriptional regulation in early mammalian eye development: a link between FGF and MITF. Development 127, 3581–3591 (2000).
Baumer, N. et al. Retinal pigmented epithelium determination requires the redundant activities of Pax2 and Pax6. Development 130, 2903–2915 (2003).
Burke, J.M. Determinants of retinal pigment epithelial cell phenotype and polarity. in The Retinal Pigment Epithelium (eds. Marmor, M.F. & Wolfensberger, T.J.) 86–102 (Oxford Univ. Press, New York, 1998).
Boulton, M. Melanin and the retinal pigment epithelium. in The Retinal Pigment Epithelium (eds. Marmor, M.F. & Wolfensberger, T.J.) 68–85 (Oxford Univ. Press, New York, 1998).
Martin, M.J., Muotri, A., Gage, F. & Varki, A. Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat. Med. 11, 228–232 (2005).
Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).
Pouton, C.W. & Haynes, J.M. Embryonic stem cells as a source of models for drug discovery. Nat. Rev. Drug Discov. 8, 605–616 (2007).
Suemori, H. et al. Establishment of embryonic stem cell lines from cynomolgus monkey blastocysts produced by IVF or ICSI. Dev. Dyn. 222, 273–279 (2001).
Kawasaki, H. et al. Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity. Proc. Natl. Acad. Sci. USA 99, 1580–1585 (2002).
Suemori, H. et al. Efficient establishment of human embryonic stem cell lines and long-term maintenance with stable karyotype by enzymatic bulk passage. Biochem. Biophys. Res. Commun. 345, 926–932 (2006).
Ueno, M. et al. Neural conversion of ES cells by an inductive activity on human amniotic membrane matrix. Proc. Natl. Acad. Sci. USA 103, 9554–9559 (2006).
Osakada, F. et al. Wnt signaling promotes regeneration in the retina of adult mammals. J. Neurosci. 27, 4210–4219 (2007).
Mizuseki, K. et al. Generation of neural crest-derived peripheral neurons and floor plate cells from mouse and primate embryonic stem cells. Proc. Natl. Acad. Sci. USA 100, 5828–5833 (2003).
Su, H.L. et al. Generation of cerebellar neuron precursors from embryonic stem cells. Dev. Biol. 290, 287–296 (2006).
Suzuki, T. et al. Chondroitinase ABC treatment enhances synaptogenesis between transplant and host neurons in model of retinal degeneration. Cell Transplant. 16, 493–503 (2007).
Acknowledgements
We thank J. Takahashi (Kyoto University, Kyoto, Japan) for providing the monkey ES cell line, H. Suemori and N. Nakatsuji (Kyoto University) for providing the human ES cell line, S. Nakagawa (RIKEN, Japan), T. Kume, H. Katsuki (Kyoto University), M. Haruta and M. Akimoto (Kyoto University Hospital) for valuable comments on this work, S. Nishikawa, M. Osawa and T. Era (RIKEN) for advice on FACS, K. Iseki and S. Yonemura (Riken) for the electron microscopic analysis, T. Yokota, A. Nomori, N. Ishibashi and A. Nishiyama for excellent technical assistance, and members of the Takahashi laboratory, the Sasai laboratory and the Akaike laboratory for discussions. This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, and the Leading Project (M.T. and Y.S.). This study was also supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, and the Mochida Memorial Foundation for Medical and Pharmaceutical Research (F.O.).
Author information
Authors and Affiliations
Contributions
F.O. designed research, performed experiments, wrote the paper and provided financial support. H.I. designed research, performed experiments and wrote the paper. M.M., T.W. and K.W. performed experiments. N.Y., A.A. and Y.S. coordinated the project. M.T. provided financial support and supervised the whole project. All authors discussed the results and commented on the manuscript.
Corresponding author
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–6 and Methods (PDF 3318 kb)
Rights and permissions
About this article
Cite this article
Osakada, F., Ikeda, H., Mandai, M. et al. Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells. Nat Biotechnol 26, 215–224 (2008). https://doi.org/10.1038/nbt1384
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nbt1384
This article is cited by
-
Retinal Organoids: A Next-Generation Platform for High-Throughput Drug Discovery
Stem Cell Reviews and Reports (2024)
-
Comparing the transcriptome of developing native and iPSC-derived mouse retinae by single cell RNA sequencing
Scientific Reports (2023)
-
Application of Human Stem Cell Derived Retinal Organoids in the Exploration of the Mechanisms of Early Retinal Development
Stem Cell Reviews and Reports (2023)
-
Self-organization, quality control, and preclinical studies of human iPSC-derived retinal sheets for tissue-transplantation therapy
Communications Biology (2023)
-
Retinoic acid delays initial photoreceptor differentiation and results in a highly structured mature retinal organoid
Stem Cell Research & Therapy (2022)