Generation of a ciliary margin-like stem cell niche from self-organizing human retinal tissue

ARTICLE Received 10 May 2014 | Accepted 12 Jan 2015 | Published 19 Feb 2015 DOI: 10.1038/ncomms7286 Generation of a ciliary margin-like stem cell niche from self-organizing human retinal tissue Atsushi Kuwahara1,2,3,*,w, Chikafumi Ozone1,4,*, Tokushige Nakano1,2,3,*, Koichi Saito3, Mototsugu Eiraku5 & Yoshiki Sasai1,2,z In the developing neural retina (NR), multipotent stem cells within the ciliary margin (CM) contribute to de novo retinal tissue growth. We recently reported the ability of human embryonic stem cells (hESCs) to self-organize stratified NR using a three-dimensional culture technique. Here we report the emergence of CM-like stem cell niches within human retinal tissue. First, we developed a culture method for selective NR differentiation by timed BMP4 treatment. We then found that inhibiting GSK3 and FGFR induced the transition from NR tissue to retinal pigment epithelium (RPE), and that removing this inhibition facilitated the reversion of this RPE-like tissue back to the NR fate. This step-wise induction-reversal method generated tissue aggregates with RPE at the margin of central-peripherally polarized NR. We demonstrate that the NR–RPE boundary tissue further self-organizes a niche for CM stem cells that functions to expand the NR peripherally by de novo progenitor generation. 1 Neurogenesis and Organogenesis Group, RIKEN Center for Developmental Biology, 2-2-3 Manatojima-Minamimachi, Chuo, Kobe 650-0047, Japan. 2 Human Stem Cell Technology Unit, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo, Kobe 650-0047, Japan. 3 Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., 3-1-98 Kasugade-naka, Konohana, Osaka 554-8558, Japan. 4 Department of Endocrinology and Diabetes, Graduate School of Medicine, Nagoya University, 65 Tsurumai-cho, Showa, Nagoya 466-8550, Japan. 5 Four-Dimensional Tissue Analysis Unit, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo, Kobe 650-0047, Japan. * These authors contributed equally to this work. w Present address: Regenerative & Cellular Medicine Office, Sumitomo Dainippon Pharma Co., Ltd., 2-2-2, Minatojima-minamimachi, Chuo, Kobe 650-0047, Japan. Correspondence and requests for materials should be addressed to A.K. (email: ) and M.E. (email: ). zDeceased. NATURE COMMUNICATIONS | 6:6286 | DOI: 10.1038/ncomms7286 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. 1 ARTICLE T NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7286 he retina is the main visual sensory tissue in mammals. Its anlage, the optic cup, is derived from the rostral diencephalon and is composed of the inner and outer walls—the neural retina (NR) and retinal pigment epithelium (RPE), respectively1,2. Mammalian NR is known to possess a low regenerative capacity, and spontaneous tissue recovery hardly occurs following substantial damage caused by trauma or degenerative diseases. This stands in sharp contrast to the newt NR, for instance, which can fully regenerate from neighbouring RPE even after total resection (reviewed in refs 3,4). In vertebrate retinogenesis, the NR has two different groups of progenitors present in distinct locations. During early retinal development, epithelial progenitors are widely present in the NR epithelium and function to generate photoreceptors and other types of retinal neurons5. Their differentiation capacity gradually changes as retinogenesis proceeds; they have a tendency to produce ganglion cells and cones at early stages, and then rods and bipolar cells at late stages6,7. Like cortical progenitors, these NR progenitors show the radial glia-like morphology with thin apical and basal processes, characteristic of neuroepithelial progenitors, and they undergo interkinetic nuclear migration during mitotic cycles8,9. (Hereafter, these progenitors are referred to as ‘NR progenitors’). Another kind of progenitor is located at the peripheral margin of the NR, the ciliary margin (CM)5,10,11. The teleost (fish) eye contains an active stem cell system in the CM zone (also called circumferential germinal zone) that contributes to NR expansion by de novo generation of the tissue even in adulthood12. This type of stem cell robustly self-renews and gives rise to all NR cell types in the extending NR periphery, unlike the nonmarginal-NR progenitor, whose developmental competence gradually becomes confined to the generation of limited cell types during retinogenesis as mentioned above. In the chick embryo, the stem cell system at the CM also plays an active role during eye development, and stem cells isolated from this region can form NR tissue in floating aggregate culture13. The mammalian foetal eye also contains a peripheral NR zone resembling the chick embryonic CM in molecular marker expression, and this zone contains a substantial number of stem cells10,14,15 (Hereafter, these cells are referred to as ‘CM stem cells’). The presence of a small number of retinal stem cells at the ciliary region in the adult mammalian eye has also been reported16–20, although the extent of their in vivo contribution to the maintenance and regeneration of adult retinal tissue remains elusive. Recent advancement in stem cell research has enabled in vitro differentiation of retinal progenitors and their derivatives from pluripotent stem cells21–29. Moreover, mouse and human embryonic stem cells (hESC) aggregate have been shown to possess the potential to self-form optic cups in three-dimensional (3D) culture when retinal differentiation efficiency is sufficiently high30–32. Notably, the ESC-derived NR self-organizes the formation of multiple retinal layers reminiscent of the postnatal retina. NR progenitors in this culture exhibit radial glia-like epithelial morphology, frequently proliferate with interkinetic nuclear migration and give birth to photoreceptors and other retinal neurons in a stage-dependent manner30,32, a process which largely recapitulates the activity of NR progenitors in vivo. Moreover, a recent report using mouse ES cells showed that self-forming NR tissue can give rise to functional photoreceptors capable of light response after grafting in the murine eye33. In the self-organized optic cup, the boundary domain between NR and RPE, called the hinge domain, contains cells with a morphology distinct from that of NR and RPE cells. However, whether CM-type stem cells are generated at this region in longterm culture has remained unexplored. In the present study, we 2 have uncovered the formation of CM-like zones in self-organizing retinal culture. First, we made substantial improvements to the 3D culture method of hESCs for promoting retinal differentiation. As reported previously, the formation of optic cup in hESC culture is less frequent (B10% of aggregates)32 than in mouse ESC culture. Its efficacy is also influenced by minute changes in culture conditions. In particular, our previous culture included Matrigel, a crude extract (...truncated)