Generation of Rx+/Pax6+ neural retinal precursors from embryonic stem cells

Abstract

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.

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. ; 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) No Six3 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/DLFA-treated 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 μmin D and I and 20 μmin J and K.)

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 cell-derived 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 (double-positive 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 μm for A, C, G, I, and K; in L, 20 μm, for B, D-F, H, J, and L.)

Fig. 3.

Overexpression of Crx efficiently induces rhodopsin⁺ cells in SFEB/DLFA cells. (A) Lentivirus vectors. CAG, chicken β-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/Venus-expressing (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 Venus-expressing 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 μm, for C and D.)

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 μmin B; and 10 μmin L, for C–L.)

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 segment-like 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 μmin B and F; and 10 μmin C–E.)