The eye-forming field in the diencephalon has been a classical

Generation of Rx Pax6 neural retinal precursors from embryonic stem cells Hanako Ikeda*, Fumitaka Osakada*, Kiichi Watanabe*, Kenji Mizuseki*, Tomoko Haraguchi*, Hiroyuki Miyoshi, Daisuke Kamiya*, Yoshihito Honda, Noriaki Sasai*, Nagahisa Yoshimura, Masayo Takahashi, and Yoshiki Sasai* *Organogenesis and Neurogenesis Group, Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan; Department of Ophthalmology and Visual Sciences, Graduate School of Medicine, Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, and Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8507, Japan; and BioResource Center, RIKEN, Tsukuba 305-0074, Japan Edited by Fred H. Gage, The Salk Institute for Biological Studies, San Diego, CA, and approved June 22, 2005 (received for review January 3, 2005) We report directed differentiaion of retinal precursors in vitro from mouse ES cells. Six3 rostral brain progenitors are generated by culturing ES cells under serum-free suspension conditions (SFEB culture) in the presence of Wnt and Nodal antagonists (Dkk1 and LeftyA), and subsequently steered to differentiate into Rx cells (16%) by treatment with activin and serum. Consistent with the characteristics of early neural retinal precursors, the induced Rx cells coexpress Pax6 and the mitotic marker Ki67, but not Nestin. The ES cell-derived precursors efficiently generate cells with the photoreceptor phenotype (rhodopsin, recoverin ) when cocultured with embryonic retinal cells. Furthermore, organotypic culture studies demonstrate the selective integration and survival of ES cell-derived cells with the photoreceptor phenotype (marker expression and morphology) in the outer nuclear layer of the retina. Taken together, ES cells treated with SFEB Dkk1 LeftyA serum activin generate neural retinal precursors, which have the competence of photoreceptor differentiation. regenerative medicine differentiation photoreceptor induction Six3 The eye-forming field in the diencephalon has been a classical paradigm in experimental biology, and the mechanism of how the eye primordium is specified has attracted the interest of embryologists over many decades. In addition, most retinal degeneration diseases in humans are caused by impairment in the neural retina, particularly in photoreceptor cells (1, 2). Therefore, in vitro generation of neural retinal precursors, if successful, would greatly contribute to medical and pharmaceutical researches for retinal diseases. To date, however, only infrequent expression of photoreceptor markers in ES cell-derived neural tissues has been reported (3, 4). The development of the neural retina involves multiple regulatory steps during embryogenesis (see Fig. 1A). A number of transcription factors have been isolated and implicated in the control of the step-wise differentiation of vertebrate retinal tissues (5). In contrast, relatively little has been known about the molecular nature of extracellular factors that mediate tissue interactions to form the retinal tissues in the exact location. To facilitate the study on retinal development, we have attempted to establish an in vitro differentiation system for neural retinal precursors from mouse ES cells. In our previous reports, it has been shown that retinal pigment epithelial cells can be induced at a moderate efficiency from primate ES cells by coculturing with PA6 cells [stromal cell-derived inducing activity (SDIA) method] (6, 7). However, as described below in this study, the frequency of the generation of neural retinal cells in the SDIA system has proven to be low. In addition to the SDIA method, we have recently established another in vitro culture system that induces efficient neural differentiation from ES cells. In this method, floating aggregates of ES cells are cultured under serum-free conditions in the optimized medium without retinoic acid (RA) or exogenous growth factors (serum-free floating culture of embryoid body-like aggregates, SFEB) (8). A characteristic feature of the SFEB culture is that it efficiently induces differentiation of rostral-most CNS tissues. Particularly, the addition of the Wnt antagonist Dkk1 (9) and the Nodal antagonist LeftyA (10) to the SFEB culture during the first 5 days facilitates both neural differentiation (up to 90%) and rostral specification of neural tissues ( 35% are telencephalic tissues) (8). Here, we report efficient in vitro generation of neural retinal precursors from mouse ES cells by combining the SFEB culture and extracellular inductive signals. The ability of the ES cellderived progenitors to produce photoreceptors is also demonstrated. Materials and Methods Cell Culture. The methods of ES cell maintenance and the differentiation by the SDIA method have been described (11). For the SFEB method, 5 10 4 dissociated ES cells (EB5 line) per milliliter were incubated in a bacterial-grade dish with ES differentiation medium (G-MEM, 5% KSR 0.1 mm nonessential amino acids 1 mm pyruvate 0.1 mm 2-mercaptoethanol) (8). Floating ES cell aggregates spontaneously formed within 1 day under this condition. After 5 days of suspension culture, the cell aggregates were replated en bloc at a density of 1 2 10 2 aggregates per cm 2 on the poly(d-lysine) laminin fibronectin-coated culture slides. Each growth factor or FCS (JRH Biosciences) was added as indicated in the text and Fig. 1G. Recombinant proteins (Dkk1, LeftyA, activin-a, FGF, and Shh) were purchased fromr&dsystems.the ED 50 of the activin protein is 0.5 2 ng ml (the ability to induce hemoglobin expression in K562 cells), and activin was used at sufficient doses (1 100 ng ml) in this study. BrdUrd uptake was examined by culturing cells with 5 g ml BrdUrd in medium for 12 h before fixation. For coculture experiments, SFEB Dkk1 LeftyA FCS activin (DLFA)- or SFEB RA-treated ES cells were dissociated with trypsin into single cells on day 5, labeled with 100 g ml of the rhodamine-labeling reagent [5-(6)-carboxytetramethylrhodamine, succinimidyl ester, which covalently binds to cell surface proteins, Molecular Probes] for 10 min, followed by washing three times with PBS. Anti-rhodamine antibody (see Supporting Text, which is published as supporting information on the PNAS web site) was used for enhancement of the detection when needed. For the genetic labeling using the Venus GFP lentivirus (see below), ES cells were infected with the virus 1 day before the start of the SFEB DLFA culture. These cells were dissociated on day 5 of differentiation and used for coculture experiments. This paper was submitted directly (Track II) to the PNAS office. Abbreviations: SDIA, stromal cell-derived inducing activity; SFEB, serum-free floating culture of embryoid body-like aggregates; RA, retinoic acid; En, embryonic day n; RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; DLFA, Dkk1 LeftyA FCS activin. To whom correspondence should be addressed. E-mail: sasaicdb@mub.biglobe.ne.jp. 2005 by The National Academy of Sciences of the USA DEVELOPMENTAL BIOLOGY www.pnas.org cgi doi 10.1073 pnas.0500010102 PNAS August 9, 2005 vol. 102 no. 32 11331 11336

Immunohistochemistry, in Situ Hybridization, and Statistical Analysis. Immunohistochemistry was performed as described (11, 12). Detailed information on the use of antibodies and on the antibody production is available in Supporting Text. All images of immunostaining (including three-dimensional reconstruction) on ES cellderived cells were obtained with the LSM5 PASCAL (Zeiss) confocal microscope. Numbers of total cells were counted by staining nuclei with TOTO-3 or DAPI (Molecular Probes). The numbers of positive cells were counted in optical slice images prepared with confocal microscopy. Whole mount in situ hybridization was performed as described (13). A fragment of mouse Six3 cdna (116 1175 bp; database accession no. X90871) was amplified by RT-PCR and used as templates for making the RNA probe. For statistical analysis, 100 200 colonies were examined in each experiment, and each experiment was performed at least three times. All statistical analyses were performed with INSTAT software (version 3.0, GraphPad, San Diego). The statistical significance of difference was determined by one-way ANOVA followed by Tukey s test for experiments in Fig. 1H. Data from Fig. 3B were evaluated by an unpaired t test. Probability values 5% were considered significant. Lentivirus Construction and Production. Replication-defective selfinactivating lentivirus vectors were used (14, 15). The DNA region encoding EGFP in the pcs-cag-egfp (a kind gift of K. Tanabe) was replaced with a fragment encoding Ires2-Venus (a kind gift of A. Miyawaki; psin-cag-ires-venus). A 0.9-kb mouse Crx cdna fragment containing the entire Crx coding region was amplified by PCR and subcloned into psin-cag-ires-venus (the psin-cag- Crx-Ires-Venus). These plasmids were cotransfected with the packaging plasmid (pcag-hivgp) and the VSV-G- and Revexpressing plasmid (pcmv-vsv-g-rsv-rev) into 293T cells. High-titer viral solution (5 10 9 units ml) was prepared by ultracentrifugation at 80,000 g. Virus solution was applied to the SFEB DLFA, SFEB ( ), or SFEB RA-ES cells on day 7. The cells were analyzed after additional 10-day culture. Fig. 1. Efficient generation of Rx Pax6 retinal precursors from mouse ES cells by a modified SFEB method. (A) Multiple-step commitment in the development of retinal cells. Markers for respective differentiation steps are boxed. (B D) E10.5 mouse embryos immunostained with anti-rx (red) and anti-pax6 (green) antibodies. (B) Cross-section at the diencephalic region. oc, optic cup. A high-power image of the boxed ventral portion is shown in C. Cells in the ventral floor of the diencephalon express only Rx. (D) Cells in the neural retina express both Rx and Pax6. The pigment epithelium and lens cells are Pax6 and Rx. RPE, retinal pigment epithelium; NR, neural retina; L, lens vesicle. In addition to the neural retina, at this early stage, Rx Pax6 cells are found only transiently in the medial end of the optic stalk (ref. 22; Rx Pax6 expression there is shut off soon after). (E) In situ hybridization signals for Six3 in SFEB DL aggregates ( 60 70% cells in the positive aggregates were Six3 ). (F)NoSix3 expression in SFEB RA aggregates. (G) Schema of the modified SFEB culture. D, culture day. (H) Percentages of Rx Pax6 colonies (containing more than five double-positive cells) in SFEB-treated ES cells on day 8. The SFEB culture combined with Dkk1, LeftyA, FCS, and activin gave the highest efficiency for the differentiation of Rx Pax6 cells (**, P 0.01, Tukey s test). DL, SFEB combined with Dkk and Lefty; DLF, with Dkk, Lefty, and FCS; DLFA, with Dkk, Lefty, FCS, and activin. (I K) Immunocytochemical analyses of SFEB DLFAtreated ES cells on day 8 (I and J) and day 7 (K) with confocal microscopy. (I)A majority of colonies contained significant numbers of Rx cells (red). Nuclei were counterstained with TOTO-3 (blue). (J) Induced Rx cells frequently coexpressed Pax6 (green). (K) Nearly all Rx (red) cells were Six3 (green). (Scale bar, 100 m indand I and 20 m inj and K.) Retina Reaggregation Pellet and Explant Coculture. Retinas from 10 mouse embryos [embryonic day (E) 17.5] were excised and separated from other ocular tissues in Hanks balanced salt solution (HBSS), and then dissociated by incubation for 5 10 min at 37 C in 0.05% EDTA PBS with 2.5% trypsin. After being washed with retinal culture medium [66% E-MEM-Hepes (Sigma catalog no. M7278) 33% HBSS (Gibco catalog no. 24020-117) 1% FCS supplemented with N2 5.75 mg/ml glucose 200 M L-glutamine 100 units/ml penicillin 100 g/ml streptomycin (Gibco)], 5 10 5 embryonic retinal cells and 1 10 4 rhodamine- or Venus-labeled ES cells (SFEB DLFA, SFEB RA) were mixed and centrifuged to produce reaggregated pellets as described (16). The pellets were cultured on 30-mm membranes (0.4- m diameter pore; Millicell Millipore catalog no. PICM03050) for 12 days at 37 C in a humidified atmosphere of 5% CO 2 with a medium change every other day. Retina explant cultures were prepared as described (17) with minor changes. Briefly, neural retinal tissues from E17.5 mouse embryos were excised and separated from other ocular tissues. About 2 10 5 labeled ES cells were placed on the filter membrane before the retina was positioned on the membrane (with the vitreous side up). The retina was incubated in the retinal culture medium at 34 C in a humidified atmosphere of 5% CO 2 for 12 days. For immunohistochemistry, the retinal pellets or explants were fixed and sectioned (20 m thick). Results and Discussion Generation of Rx Pax6 Cells from ES Cells in Vitro. In early embryogenesis, the retinal primordia form within the rostral-most diencephalic region expressing Six3 (18) (Fig. 1A). The transcription factor Rx (an early bona fide marker for the eye field) plays an 11332 www.pnas.org cgi doi 10.1073 pnas.0500010102 Ikeda et al.

essential role for the specification of the retinal primordium in the Six3 rostral CNS (19 21). During early embryogenesis (E10.5), Rx expression coincides with Pax6 expression (22) in the neural retinal progenitors (Fig. 1 B and D), whereas Rx cells in the floor of the ventral diencephalon are Pax6 (Fig. 1C). The retinal pigment epithelium (RPE) is Rx and Pax6 (Fig. 1D). Thus, the neural retinal lineage during early development is characterized by the Rx Pax6 coexpression (Pax6 expression gradually decreases as the neural retina matures). To understand the suitability of the SFEB-based approach (8) for the induction of retinal tissues from ES cells, we first examined the expression of the rostral-most CNS marker Six3. On culture day 5, strong expression of Six3 was found in SFEB-treated ES cell aggregates cultured in the absence and presence of Dkk1 (100 ng ml) plus LeftyA (500 ng ml) (in 82% and 87% of aggregates, respectively; Fig. 1E and data not shown), but not in those cultured with the caudalizing factor RA (0.2 M, during days 3 5; Fig. 1F; ref. 23). Because retinal progenitors arise from the Six3 rostral CNS tissue, we next attempted to induce Rx Pax6 cells from SFEB-induced neural precursors by modifying the culture conditions. Because extracellular patterning signals that determine the induction of retinal primordia in the embryo have not yet been elucidated, we experimentally searched for soluble factors that induced Rx Pax6 expression by testing a number of candidate factors for such activities (Fig. 1 G and H and data not shown). Among them, the most obvious enhancement was seen with treatment of 5% FCS during days 3 5 (Fig. 1H, lane 4). Furthermore, activin-a treatment (100 ng ml) during days 4 6 increased the induction when combined with Dkk1, LeftyA, and FCS treatments (Fig. 1H, lane 5; little effects were seen with activin treatment alone at 1 100 ng ml, data not shown). We also tested the effects of Shh, Wnt, BMP4, Nodal (without LeftyA), IGF, FGF-1, FGF-2, or FGF antagonists during days 3 6, but observed only marginal effects, if any, on Rx induction in this culture system (data not shown). Hereafter, ES cells treated with SFEB and Dkk1 LeftyA FCS activin are referred to as SFEB DLFA cells. As a whole, Rx cells were found in 15.6 0.1% of SFEB DLFA cells, whereas 1% Rx cells were generated in SFEB DL-treated ES cells without FCS and activin. At the colony level, 53.5 8.4% colonies of SFEB DLFA cells were Rx (Fig. 1I) and 28.3 5.0% colonies contained Rx Pax6 cells (Fig. 1H). In the Rx Pax6 colonies, 27.3 1.3% were Rx Pax6 cells on average (Fig. 1J), whereas Rx Pax6 cells occupied 8.7 1.8%. In all, 6.4 0.3% cells of the culture were strongly positive for both Rx and Pax6. Such frequent induction of Rx Pax6 cells is not commonly seen in other ES cell differentiation systems. For instance, ES cell-derived neural precursors induced by coculture with PA6 cells (SDIA method) (11) generated few Rx Pax6 colonies ( 3%) even upon treatment with serum or activin (data not shown). These findings show that the SFEB DLFA treatment preferentially induces differentiation of Rx Pax6 retinal progenitor-like cells from ES cells. SFEB DLFA-Induced Rx Cells Exhibit a Marker Expression Profile Indistinguishable from That of Embryonic Neural Retinal Progenitors. Consistent with the idea of retinal progenitor differentiation, most of ES cell-derived Rx cells coexpressed Six3 on day 7 (Fig. 1K), which is strongly expressed in the developing neural retina of the E11 mice (18). Next, we further analyzed the nature of Rx cells present in SFEB DLFA cells with multiple eye markers. Otx2 (22) is coexpressed with Rx in the embryonic neural retina (Fig. 2A and data not shown), whereas the RPE is Rx Otx2. Consistent with the in vivo coexpression, nearly all Rx cells in SFEB DLFA cells were Otx2 (95.8 1.0%; Fig. 2B). In the early neural retina (E10.5; Fig. 2C), most Rx cells are proliferating progenitors, which are positive for the mitotic marker Ki67. Similarly, Rx cells in SFEB DLFA cells were Ki67 (95.6 1.3%; Fig. 2D), TuJ1 (99.9 0.1%; postmitotic neuronal marker; Fig. 2E), and positive Fig. 2. Rx cells induced by SFEB DLFA exhibit typical marker characteristics of retinal progenitors. Immunostaining of E10.5 mouse optic cups (A, C, G, I, and K) and SFEB DLFA-treated ES cells (day 8) (B, D F, H, J, and L; confocal images) with anti-rx antibodies (red) [raised in mice (A, B, I, and J) and rabbits (C H, K, and L)] and anti-otx2 (A and B), Ki67 (C and D), TuJ (E), BrdUrd (F), Nestin (G and H), Sox1 (I and J), or Mitf (K and L) antibodies (green). (A) The neural retinal cells (especially in distal portion) coexpress Rx and Otx2. (B) Most ES cell-derived Rx cells coexpressed Otx2. (C and D) Ki67 expression in Rx neural retinal cells (C) and Rx ES cells (D). (E) ES cell-derived Rx cells were negative for TuJ1. (F) Most ES cellderived Rx cells were BrdUrd after 12 h of BrdUrd exposure. (G and H) Lack of Nestin staining in Rx cells of the retina (G) and the ES cell-derived Rx cells (H). (I)Rx retinal cells are Sox1, whereas the cells in the lens vesicle and the neural epithelium of the adjacent diencephalon (arrow) are Sox1.(J)Rx cells induced by SFEB DLFA were negative for Sox1. (K) Mitf cells are Rx except for some cells in the peripheral marginal zone of the embryonic retina on E10.5 (doublepositive cells are more abundant in earlier days). (L) A large portion of the Mitf cells induce by SFEB DLFA were negative for Rx. (Scale bar in K, 100 m for A, C, G, I, and K;inL,20 m, for B, D-F, H, J, and L.) for BrdUrd uptake (85.2 1.3% after 12 h exposure; Fig. 2F), indicating that Rx cells produced by SFEB DLFA are mitotically active. In the embryo, unlike most of the neural progenitors in the CNS, Rx neural retinal precursors do not express Nestin (24) or DEVELOPMENTAL BIOLOGY Ikeda et al. PNAS August 9, 2005 vol. 102 no. 32 11333

Sox1 (25) (Fig. 2 G and I). Similarly, most Rx cells in SFEB DLFA cells were Nestin (Fig. 2H) and Sox1 (Fig. 2 J), although they were mitotically active. Collectively, a high proportion of Rx cells produced by SFEB DLFA exhibit characteristics consistent with those of progenitors in the developing neural retina, at least with regard to the marker expression profile tested here. We next asked whether differentiation of another component of the retina, the RPE, was observed in the SFEB DLFA culture, and examined the expression of the early RPE marker Mitf (22) (Fig. 2K). In the SFEB DLFA culture, 17.1 3.7% of ES cell aggregates were positive for Mitf, whereas 1.6 0.7% of ES cell aggregates treated with SFEB alone were Mitf. Among Mitf SFEB DLFAtreated cell aggregates, 5.2 0.2% cells expressed Mitf (i.e., 1% of total cells in culture). Consistent with the in vivo expression profile of RPE markers (22), most Mitf cells in the SFEB DLFA culture were Pax6 (Fig. 6, which is published as supporting information on the PNAS web site). In the SFEB DLFA culture, the majority of Mitf cells were Rx on day 8, although Rx cells were frequently found in the close vicinity of Mitf cell clusters (Fig. 2L). None of Mitf cells on day 12 coexpressed Rx (data not shown). Induction of Rhodopsin Expression by Crx Overexpression in SFEB DLFA-Treated ES Cells. Next, we asked whether SFEB DLFAtreated ES cells could generate the photoreceptor cell, a key component of the neural retina, after a long period culture [adherent culture on poly(d-lysine) laminin fibronectin-coated culture slides in the differentiation medium]. On day 12, 6.6 1.2% of the colonies contained small clusters of cells positive for the early photoreceptor precursor marker Crx (26, 27) ( 0.5% of total cells in culture; Fig. 7 A and B, which is published as supporting information on the PNAS web site). These Crx cells coexpressed Otx2, consistent with the colocalization in the developing retina (28) (Fig. 7C). On day 17, 10.8 1.6% of the colonies contained small cell clusters positive for the late photoreceptor marker rhodopsin ( 0.5% of total cells; Fig. 7D). We tested several soluble factors that reportedly have positive effects on in vitro differentiation of late embryonic or neonatal retinal progenitors into photoreceptors (refs. 29 and 30; Shh, FGF-1, FGF-2, Taurine, and RA). No significant enhancement of Crx rhodopsin induction in SFEB DLFA cells was observed with these factors (data not shown; each added from day 9 in serum-free differentiation medium or in 1% FCS containing retinal culture medium). We then asked whether the inefficient Crx rhodopsin induction reflected a small number of progenitors with the competence of photoreceptor differentiation or the lack of proper inductive cues in culture. To date, the exact molecular nature of in vivo signals that induce Crx photoreceptor precursors from Rx retinal progenitors remains elusive. Therefore, we first tested whether SFEB DLFA cells contained sensitized precursors that had the competence to express rhodopsin in response to Crx overexpression. Previous studies have indicated that Crx overexpression induces photoreceptor differentiation in immature retinal tissue (27) and iris-derived precursors (31). Lentivirus-mediated Crx gene transfer (Fig. 3A) remarkably increased the appearance of rhodopsin cells in SFEB DLFA-treated ES cell colonies (21.0 1.0% of Crx virus-infected cells vs. 0.8 0.4% of control Venus-GFP (32) virus-infected cells, P 0.001; Fig. 3 B D). In contrast, forced Crx expression generated only a few rhodopsin cells in SFEB-treated (5.0 1.3% of infected cells; Fig. 3B) and SFEB RA-treated (1.0 1.1%; Fig. 7 E and F) ES cells. No detectable levels of rhodopsin proteins were induced in COS-7 or 293T cells by Crx overexpression. (10 days after infection of the Crx virus; data not shown). These findings demonstrate that the SFEB DLFA-induced progenitors contain sensitized precursors with the ability to express rhodopsin in the presence of Crx, as do immature retinal cells. Fig. 3. Overexpression of Crx efficiently induces rhodopsin cells in SFEB DLFA cells. (A) Lentivirus vectors. CAG, chicken -actin promoter with CMV-IE enhancer; IRES, internal ribosomal unit; RRE, rev responsive element; cppt, central polypurine tract; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element. (B D) ES cells treated with SFEB DLFA (C and D) or SFEB ( ) were infected with the control (Venus only, C) or Crx Venusexpressing (D) lentivirus. (B) Proportions of the rhodopsin cells per Venus cells. The data represent the averages of three independent experiments (***, P 0.001, t test). (C) SFEB DLFA-treated ES cells infected with Venusexpressing virus (green) rarely expressed rhodopsin (red). (D) Rhodopsin induction in SFEB DLFA-treated ES cells infected with the Crx Venus-expressing virus. (Scale bar in D, 20 m, for C and D.) Photoreceptor Differentiation in SFEB DLFA-Treated ES Cells by Coculture with Embryonic Retinal Cells. To obtain further evidence for the generation of neural retinal progenitors from ES cells, we tested whether photoreceptor differentiation could be efficiently induced by mimicking the in vivo environment in culture. We first performed the reaggregation coculture assay (16). In this assay, when cocultured with more mature retinal tissues in vitro, early embryonic retinal progenitors efficiently differentiate into photoreceptors in a fashion similar to in vivo development (16). SFEB DLFAtreated ES cells (1 10 4 cells) were dissociated on day 5, labeled with the rhodamine dye, mixed with dissociated E17.5 mouse retinal cells (1:50), and spun down to make coaggregates of cells (Fig. 4A). The reaggregated cell pellets were subsequently cultured for 12 days on the culture insert (porous filter membrane) in E-MEM HBSS 1% FCS N2 medium. Under these conditions, 3.9 0.5 10 3 surviving cells labeled with rhodamine were found in the cell pellets. In these cell aggregates (where endogenous photoreceptors spontaneously became sorted together and made clusters), 38 6% of the surviving SFEB DLFA cells were found in the areas rich in embryo-derived photoreceptor cells. Interestingly, confocal microscopic analysis showed that 36 3% of ES cell-derived cells in these areas expressed the photoreceptor markers rhodopsin and recoverin (33) (Fig. 4 B F),whereas these markers were rarely observed 11334 www.pnas.org cgi doi 10.1073 pnas.0500010102 Ikeda et al.

Fig. 4. Efficient differentiation of rhodopsin cells by coculture with embryonic retinal tissues. Coculture of the SFEB DLFA-treated ES cells with embryonic retinal cells under the reaggregation pellet coculture. (B H) A number of rhodopsin (green) SFEB DLFA cells (rhodamine labeled; red) were found (B; magnified view, C and D) in the rhodopsin cell-rich cluster of the embryonic retinal cells. Large arrowheads indicate ES cell derivatives that were not incorporated in the rhodopsin cluster of the recipient retinal cells (B). (E and F) Recoverin expression (green; overlay in F) in rhodamine SFEB DLFA cells (red) after reaggregation coculture. (G and H) SFEB DLFA-treated ES cells (red) were cocultured with embryonic retinal cells (expressing GFP). Rhodopsin (blue)-expressing ES cells (red) in G did not express GFP (green) in H.(I L) ES cell-derived cells genetically labeled with Venus were detected with anti-gfp antibody (green). (I and J) Some Venus (green) rhodopsin (red) ES-derived cells had processes (small arrows; overlay in J). White lines mark the direction of the reslicing done along the z axis (I and J ). (K and L) ES-derived cells (green) also expressed recoverin (red; overlay in L). Small arrowheads in C, H, and I indicate the margin of the ES cell. Note that rhodopsin (membrane protein) was localized in the periphery of the cell. All pictures were obtained with confocal microscopy. (Scale bar, 20 m inb; and 10 m inl, for C L.) in rhodamine-labeled cells staying away from the photoreceptorrich regions (arrowheads in Fig. 4B). These observations are consistent with a previous coculture study with neonatal retinal tissues (30) showing that E11 mouse retinal progenitors differentiate into photoreceptors only when they are located closely to neonate-derived rod cell clusters. In contrast to SFEB DLFA cells, the reaggregate coculture experiments using SFEB RA-treated ES cells (containing no Rx Pax6 cells) did not result in significant generation of rhodopsin cells (2.0 2.0% in the embryo-derived photoreceptor-rich areas). Fig. 5. Integration of rhodopsin cells into the outer nuclear layer. (A) Confocal images of organotypic coculture using SFEB DLFA cells (red) and embryonic retinal explants. (B) Integration of ES cell (red, arrows)-derived rhodopsin (green) cells into the ONL. Retinal explants were counterstained with TOTO-3 (blue). Large arrowheads indicate ES cells that were not integrated into the retinal layers. (C and D) High-magnification pictures of the ES-derived cell indicated with the white arrow in B (C, and overlay D). Small arrowheads indicate rhodopsin signals in the periphery of the cell body, and the bracket indicates strong localization of rhodopsin in the outer segmentlike structure. (E) The outer segment-like morphology (indicated with the bracket) and axon-like structure (indicated with a small arrow) in the ES cell derivative in the ONL. (F) Expression of glutamine synthetase (a marker for Müller glia, green) in the ES cell-derived cell of the INL. (G) Proportions of SFEB DLFA cells integrated into each layer of the retinal explant. Few cells were found in the OPL and IPL. G, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. (Scale bar, 20 m inband F; and 10 m inc E.) These observations show that cells with the photoreceptor-like phenotype are efficiently generated from SFEB DLFA cells in the presence of retina-derived environmental cues (36% of SFEB DLFA cells in the vicinity of photoreceptor clusters, 14% in the whole reaggregates), but not in the absence of them ( 0.5%). Rhodopsin cells did not appear to be generated by cell fusion, because none of these cells showed double (or large) nuclei (n 50). Consistently, when the embryonic retinal tissues were obtained from mice that constitutively expressed GFP (a kind gift of S. Yamada; ref. 34), embryo-derived GFP signals were not colocalized with the rhodamine tracer (green and red in Fig. 4 G and H). In addition, to rule out the possibility of artifacts caused by the rhodamine dye, we confirmed the cell lineage and expression of photoreceptor markers by using ES cells prelabeled with Venus- GFP. Confocal microscopic analysis showed that the ES cellderived Venus signals were detected in rhodopsin cells (Fig. 4 I and J; I and J show reconstructed slice images along the z axis) and in recoverin cells (Fig. 4 K and L). Integration of ES Cell-Derived Photoreceptors into the Proper Retinal Layer by Organotypic Culture. Finally, we performed organotypic culture experiments to test whether ES cell-derived rhodopsin DEVELOPMENTAL BIOLOGY Ikeda et al. PNAS August 9, 2005 vol. 102 no. 32 11335

cells had the ability to integrate into the proper location of the retina. Explants of the embryonic neural retina (E17.5) were cultured on a porous filter insert with the vitreous side up as previously (17). A drop of single cell suspension of SFEB DLFAtreated ES cells (day 5; rhodamine-labeled) was placed between the explant and the filter (Fig. 5A). After 12 days of organotypic coculture, a number of ES cell-derived rhodamine cells (172 124 cells per explant) had integrated in various layers of the retinal explant. A total of 10% of the ES cell-derived cells that had integrated into the retinal tissue were located in the outer nuclear layer (ONL), where endogenous photoreceptors lay. Interestingly, a significant proportion of the rhodamine cells present in the outer nuclear layer expressed rhodopsin (37%; Fig. 5B), whereas few rhodopsin cells were found in the other layers. Similar observations were obtained when ES cells were labeled genetically with Venus-GFP, indicating that these findings were not artifacts caused by the dye labeling (Fig. 8, which is published as supporting information on the PNAS web site). Interestingly, the rhodamine rhodopsin cells possessed a characteristic protrusion on the outer side, which is reminiscent of the photoreceptor outer segment (brackets in Fig. 5 B, D, and E). These findings suggest that ES cell-derived rhodopsin photoreceptor-like cells are preferentially incorporated into the exact layer expected for photoreceptor cells and survive there. Another interpretation could be that immature retinal progenitors are preferentially induced to differentiate into photoreceptors after incorporation into the ONL. Taken together with the studies of differentiation markers (Figs. 1 and 2), these observations indicate that SFEB DLFA-treatment promote ES cell differentiation into neural retinal precursors, which have the competence of generating cells with the photoreceptor phenotype. In addition to the ONL, a large proportion ( 75%) of surviving SFEB DLFA cells were found in the inner nuclear layer (INL; Fig. 5G). ES cell derivatives incorporated in the INL expressed the Müller glia marker glutamine synthetase (GS) (26% of cells in the INL; Fig. 5F; ref. 35) and the rod bipolar cell marker PKC (10% of cells in the INL; refs. 35 and 36). In contrast, a relatively small number of surviving SFEB DLFA cells in the ganglion cell layer expressed the ganglion cell marker Islet-1 (37) (9% in this layer and 1% in the explant, data not shown). The relatively low frequency may reflect the fact that ganglion cell differentiation starts much earlier in the embryo than on E17.5 (38). Prospects. In this study, we have demonstrated that Rx Pax6 neural retinal precursors are generated at a significant frequency from ES cells in the SFEB culture combined with Dkk1, LeftyA, FCS, and activin treatment. By applying extracellular inductive signals (or environment), ES cells were steered to differentiate stepwise into Six3 rostral CNS progenitors (Fig. 1E), Rx Pax6 neural retinal progenitors (Fig. 1J), and photoreceptor precursors (Figs. 4 and 5). This report demonstrates systematic induction of neural retinal precursors from ES cells. The signaling mechanism of FCS and activin and their in vivo relevance to the induction of the retinal primordia should be interesting in future investigation. It is important to understand whether these factors act mainly in the induction of Rx Pax6 progenitors or in their growth and maintenance (or both). Further analysis on the effects of additional signals should be crucial for obtaining more selective induction of Rx Pax6 progenitors. Successful generation of ES cell-derived neural retinal progenitors raises the future possibility for its applications in both basic and medical researches on the retina. To this end, it is challenging but important to further improve the efficiency of photoreceptor induction in chemically defined culture by identifying the molecular nature of retina-derived promoting factors (39) for photoreceptor differentiation. In addition, for future functional analysis and transplantation studies, the establishment of a selective purification system of photoreceptor precursors should be crucial. We are grateful to members of the Sasai laboratory for discussion; to S. Nakagawa and H. Kawasaki for comments on this work; to A. Miyawaki (RIKEN), H. Enomoto (RIKEN), and K. Tanabe (RIKEN) for expression plasmids and advice; to F. Vaccarino (Yale University, New Haven, CT) for Otx2 antibody; to S. Yamada (Kyoto University) and T. Suzuki (Kyoto University) for GFP mice; to R. Ladher for advice on rhodamine labeling; to T. Akagi for advice on organotypic culture; and to A. Nishiyama, T. Katayama, M. Kawada, Y. Nakazawa, M. Matsumura, and K. Fukushima for excellent technical assistance. This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology (Japan) (to Y.S. and M.T.), the Kobe Cluster Project (to Y.S.), and the Leading Project (to Y.S. and M.T.). 1. Adler, R., Curcio, C., Hicks, D., Price, D. & Wong, F. (1999) Mol. Vis. 5, 31. 2. Delyfer, M.-N., Léveillard, T., Mohand-Säid, S., Hicks, D., Picaud, S. & Sahel, J.-A. (2004) Biol. Cell 96, 261 269. 3. Zhao, X., Liu, J. & Ahmad, I. (2002) Biochem. Biophys. Res. Commun. 297, 177 184. 4. Hirano, M., Yamamoto, A., Yoshimura, N., Tokunaga, T., Motohashi, T., Ishizaki, K., Yoshida, H., Okazaki, K., Yamazaki, H., Hayashi, S., et al. (2003) Dev. Dyn. 228, 664 671. 5. Chow, R. L. & Lang, R. A. (2001) Annu. Rev. Cell Dev. Biol. 17, 255 296. 6. Kawasaki, H., Suemori, H., Mizuseki, K., Watanabe, K., Urano, F., Ichinose, H., Haruta, M., Takahashi, M., Yoshikawa, K., Nishikawa, S.-I., et al. (2002) Proc. Natl. Acad. Sci. USA 99, 1580 1585. 7. Haruta, M., Sasai, Y., Kawasaki, H., Amemiya, K., Ooto, S., Kitada, M., Suemori, H., Nakatsuji, N., Ide, C., Honda, Y., et al. (2004) Invest. Ophthalmol. Vis. Sci. 45, 1020 1025. 8. Watanabe, K., Kamiya, D., Nishiyama, A., Katayama, T., Nozaki, S., Kawasaki, H., Watanabe, Y., Mizuseki, K. & Sasai, Y. (2005) Nat. Neurosci. 8, 288 296. 9. Glinka, A., Wu, W., Delius, H., Monaghan, A. P., Blumenstock, C. & Niehrs, C. (1998) Nature 391, 357 362. 10. Sakuma, R., Ohnishi, Y., Meno, C., Fujii, H., Juan, H., Takeuchi, J., Ogura, T., Li, E., Miyazono, K. & Hamada, H. (2002) Genes Cells 7, 401 412. 11. Kawasaki, H., Mizuseki, K., Nishikawa, S., Kaneko, S., Kuwana, Y., Nakanishi, S., Nishikawa, S.-I. & Sasai, Y. (2000) Neuron 28, 31 40. 12. Mizuseki, K., Sakamoto, T., Watanabe, K., Muguruma, K., Ikeya, M., Nishiyama, A., Arakawa, A., Suemori, H., Nakatsuji, N., Kawasaki, H., et al. (2003) Proc. Natl. Acad. Sci. USA 100, 5828 5833. 13. Chitnis, A., Henrique, D., Lewis, J., Ish-Horowicz, D. & Kintner, C. (1995) Nature 375, 761 766. 14. Miyoshi, H., Takahashi, M., Gage, F. H. & Verma, I. M. (1997) Proc. Natl. Acad. Sci. USA 94, 10319 10323. 15. Miyoshi, H., Blömer, U., Takahashi, M., Gage, F. H. & Verma, I. M. (1998) J. Virol. 72, 8150 8157. 16. Watanabe, T. & Raff, M. C. (1990) Neuron 4, 461 467. 17. Hatakeyama, J., Tomita, K., Inoue, T. & Kageyama, R. (2001) Development (Cambridge, U.K.) 128, 1313 1322. 18. Oliver, G., Mailhos, A., Wehr, R., Copeland, N. G., Jenkins, N. A. & Gruss, P. (1995) Development (Cambridge, U.K.) 121, 4045 4055. 19. Lagutin, O. V., Zhu, C. C., Kobayashi, D., Topczewski, J., Shimamura, K., Puelles, L., Russell, H. R. C., McKinnon, P. J., Solnica-Krezel, L. & Oliver, G. (2003) Genes Dev. 17, 368 379. 20. Mathers, P. H., Grinberg, A., Mahon, K. A. & Jamrich, M. (1997) Nature 387, 603 607. 21. Furukawa, T., Kozak, C. A. & Cepko, C. L. (1997) Proc. Natl. Acad. Sci. USA 94, 3088 3093. 22. Bäumer, N., Marquardt, T., Stoykova, A., Spieler, D., Treichel, D., Ashery-Padan, R. & Gruss, P. (2003) Development (Cambridge, U.K.) 130, 2903 2915. 23. Durston, A. J., van der Wees, J., Pijnappel, W. W. M. & Godsave, S. F. (1998) Curr. Top. Dev. Biol. 40, 111 175. 24. Yang, J., Bian, W., Gao, X., Chen, L. & Jing, N. (2000) Mech. Dev. 94, 287 291. 25. Collignon, J., Sockanathan, S., Hacker, A., Cohen-Tannoudji, M., Norris, D., Rastan, S., Stevanovic, M., Goodfellow, P. & Lovell-Badge, R. (1996) Development (Cambridge, U.K.) 122, 509 520. 26. Chen, S., Wang, Q.-L., Nie, Z., Sun, H., Lennon, G., Copeland, N. G., Gilbert, D. J., Jenkins, N. A. & Zack, D. J. (1997) Neuron 19, 1017 1030. 27. Furukawa, T., Morrow, E. M. & Cepko, C. L. (1997) Cell 91, 531 541. 28. Nishida, A., Furukawa, A., Koike, C., Tano, Y., Aizawa, S., Matsuo, I. & Furukawa, T. (2003) Nat. Neurosci. 6, 1255 1263. 29. Levine, E. M., Fuhrmann, S. & Reh, T. A. (2000) Cell. Mol. Life Sci. 57, 224 234. 30. Reh, T. A. (1992) J. Neurobiol. 23, 1067 1083. 31. Haruta, M., Kosaka, M., Kanegae, Y., Saito, I., Inoue, T., Kageyama, R., Nishida, A., Honda, Y. & Takahashi, M. (2001) Nat. Neurosci. 4, 1163 1164. 32. Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K. & Miyawaki, A. (2002) Nat. Biotechnol. 20, 87 90. 33. McGinnis, J. F., Stepanik, P. L., Chen, W., Elias, R., Cao, W. & Lerious, V. (1999) J. Neurosci. Res. 55, 252 260. 34. Ikawa, M., Yamada, S., Nakanishi, T. & Okabe, M. (1999) Curr. Top. Dev. Biol. 44, 1 20. 35. Haverkamp, S. & Wässle, H. (2000) J. Comp. Neurol. 424, 1 23. 36. Greferath, U., Grünert, U. & Wässle, H. (1990) J. Comp. Neurol. 301, 433 442. 37. Cayouette, M., Barres, B. A. & Raff, M. (2003) Neuron 40, 897 904. 38. Marquardt, T. & Gruss, P. (2002) Trends Neurosci. 25, 32 38. 39. Watanabe, T. & Raff, M. C. (1992) Development (Cambridge, U.K.) 114, 899 906. 11336 www.pnas.org cgi doi 10.1073 pnas.0500010102 Ikeda et al.