Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs

ARTICLE Received 31 Oct 2013 | Accepted 5 May 2014 | Published 10 Jun 2014 DOI: 10.1038/ncomms5047 Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs Xiufeng Zhong1, Christian Gutierrez1, Tian Xue2,3, Christopher Hampton1, M. Natalia Vergara1, Li-Hui Cao3,w, Ann Peters4, Tea Soon Park4, Elias T. Zambidis4, Jason S. Meyer5, David M. Gamm6, King-Wai Yau1,3 & M. Valeria Canto-Soler1 Many forms of blindness result from the dysfunction or loss of retinal photoreceptors. Induced pluripotent stem cells (iPSCs) hold great potential for the modelling of these diseases or as potential therapeutic agents. However, to fulfill this promise, a remaining challenge is to induce human iPSC to recreate in vitro key structural and functional features of the native retina, in particular the presence of photoreceptors with outer-segment discs and light sensitivity. Here we report that hiPSC can, in a highly autonomous manner, recapitulate spatiotemporally each of the main steps of retinal development observed in vivo and form three-dimensional retinal cups that contain all major retinal cell types arranged in their proper layers. Moreover, the photoreceptors in our hiPSC-derived retinal tissue achieve advanced maturation, showing the beginning of outer-segment disc formation and photosensitivity. This success brings us one step closer to the anticipated use of hiPSC for disease modelling and open possibilities for future therapies. 1 Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA. 2 School of Life Sciences and Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei 230026, China. 3 Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. 4 Institute for Cell Engineering and Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA. 5 Department of Biology, Department of Medical and Molecular Genetics, and Stark Neuroscience Research Institute, Indiana University–Purdue University, Indianapolis, Indiana 46202, USA. 6 Department of Ophthalmology and Visual Sciences, McPherson Eye Research Institute and Waisman Center Stem Cell Research Program, University of Wisconsin, Madison, Wisconsin 53705, USA. w Present address: State Key Laboratory of Biomembrane and Membrane Biotechnology, Center for Quantitative Biology, McGovern Institute for Brain Research, Peking-Tsinghua Center for Life Sciences, College of Life Sciences, Peking University, Beijing 100871, China. Correspondence and requests for materials should be addressed to M.V.C.-S. (email: ). NATURE COMMUNICATIONS | 5:4047 | DOI: 10.1038/ncomms5047 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5047 M any retinal degenerative diseases are characterized by the dysfunction and death of photoreceptor cells, leading to vision loss and eventually total blindness1–3. Despite decades of research, there is currently no cure for these diseases. The establishment of human induced pluripotent stem cell (hiPSC) technology generated considerable excitement due to its potential for developing in vitro biological models and, eventually, therapeutic treatments for such diseases4–9. However, it is still unclear to what extent hiPSC may be capable of recapitulating in vitro the cellular and molecular features of the native retina, especially regarding photoreceptor differentiation and functional maturation. Several studies have shown that under specifically defined culture conditions, embryonic stem (ES) and induced pluripotent stem (iPS) cells can be induced to differentiate along a retinal lineage, including differentiation into photoreceptors10–19. Moreover, it has recently been shown that mouse and human ES cells can develop into a three-dimentional (3D) optic cup in culture that remarkably resembles the embryonic vertebrate eye20,21. Notwithstanding, the structural and molecular characteristics of advanced photoreceptor differentiation, including the formation of outer-segment discs—an essential structural feature for photoreceptor function—have yet to occur in vitro6–9. Perhaps as a consequence, no photoreceptor–light response has been observed in such cultures either. Finally, it remains to be determined whether iPSC can recreate the 3D histo-architecture of the neural retina (NR) in vitro beyond a rudimentary stratification22. Retinal cell differentiation in vivo takes place through sequential cell-fate specification steps, within a very dynamic and complex microenvironment involving highly coordinated cell–cell interactions through direct contact or diffusible signals23,24. Accordingly, in most published studies, differentiation of ES or iPS cells into retinal cells in vitro required an elaborate regime of exogenous factors10–13,15,16,18,20,21,25–27. Some studies, however, suggest that human ES and iPS cells have a certain propensity to differentiate into a retinal lineage14,19,22,28,29. Here we have succeeded in inducing human iPSC to recapitulate the main steps of retinal development and to form fully laminated 3D retinal tissue by exploiting the intrinsic cues of the system to guide differentiation (Supplementary Fig. 1). Moreover, the photoreceptors in our preparations begin to develop outer-segment discs and reach the stage of photosensitivity. This highly autonomous system provides a powerful platform for developmental, functional and translational studies. Results Self-organized eye field domains. Eye development in the embryo’s neural plate begins with the formation of the eye field (EF), a centrally organized domain consisting of a subpopulation of anterior neuroepithelial cells that have become further specified into retinal progenitors23,30 (Supplementary Fig. 1a). The EF is characterized by the expression of a group of transcription factors that includes PAX6, RX, LHX2, SIX3 and SIX6, while the surrounding anterior neuroepithelial cells express PAX6 and SOX1 (refs 30–33). In parallel to the native events, our hiPSCderived aggregates, after 8 days of differentiation (D8) in a chemically defined neural differentiation medium14,22,29 and attached on Matrigel-coated culture dishes (see Methods for details), acquired an anterior neuroepithelial fate expressing PAX6 and SOX1 (Fig. 1a–c). Soon after, retinal progenitor cells expressing LHX2 appeared in the central region of the differentiating aggregates, concomitantly with a downregulation of SOX1 expression (Fig. 1d). By D12, EF-like domains with their 2 characteristic flat, tightly packed appearance could be observed, surrounded by anterior neuroepithelial cells (Fig. 1e,f). Retinal progenitor cells within the EF domains lacked expression of SOX1 (Fig. 1f) and co-expressed the EF transcription factors PAX6, LHX2 and RX (Fig. 1g, (...truncated)