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.2014 Apr 24;2(5):662-74.
doi: 10.1016/j.stemcr.2014.03.011. eCollection 2014 May 6.

Transplantation of embryonic and induced pluripotent stem cell-derived 3D retinal sheets into retinal degenerative mice

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Transplantation of embryonic and induced pluripotent stem cell-derived 3D retinal sheets into retinal degenerative mice

Juthaporn Assawachananont et al. Stem Cell Reports..

Abstract

In this article, we show that mouse embryonic stem cell- or induced pluripotent stem cell-derived 3D retinal tissue developed a structured outer nuclear layer (ONL) with complete inner and outer segments even in an advanced retinal degeneration model (rd1) that lacked ONL. We also observed host-graft synaptic connections by immunohistochemistry. This study provides a "proof of concept" for retinal sheet transplantation therapy for advanced retinal degenerative diseases.

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Graphical abstract
Figure 1
Figure 1
Efficient and Reproducible Generation of Neural Retina from mESCs and iPSCs (A) Schematic diagram of the modified protocol used for 3D differentiation of mESC- and miPSC-derived neural retinal sheets. (B) Enhancing effect of retinoic acid receptor antagonist onRx-GFP induction. (C) Colonies ofRx-GFP mESCs. (D and E) DD8 aggregate withRx-GFP optic-vesicle-like structures. (F–H) Immunostaining of DD7Rx-GFP mESC-derived neural retina shows coexpression of theRx-GFP and PAX6, indicating retinal progenitors. (I) Green histogram: percentage ofRx-GFP+ cells among DD9 mESC-derived spheres. Red open-peak histogram: undifferentiatedRx-GFP mESCs (isotype control). (J) Red histogram: percentage of PAX6+ cells amongRx-GFP+ cells of DD8 mESC-derived spheres. Black open-peak histogram: isotype control. Almost all of theRx+ cells were PAX6+, and PAX6 expression was observed outside the vesicles as well (Figure S1A). (K) PCR shows downregulation of the pluripotent genes on DD10 with increased gene expression ofRx in all three lines tested. (L) Immunostaining of DD10Nrl-GFP miPSC-derived neural retina for LAMININ (basement membrane) and ISLET1 (ganglion cells). (M–Q) Retina marker expression of DD24Nrl-GFP miPSC-derived neural retina, rod photoreceptors (RHODOPSIN), amacrine cells (CALRETININ), horizontal cells (CALBINDIN), photoreceptor precursors (CRX, RECOVERIN), Muller glia (GS), and external limiting membrane (ZO1). All of these markers were similarly expressed in both mESCs and miPSCs, and were also correlated with the developing retina (Figures S1B and S1C). Scale bars, 200 μm (B–E) and 50 μm (F–H and L–Q). See also Tables S1 and S2.
Figure 2
Figure 2
Transplanted Retina-like Sheets Show a Well-Developed Stratified Retinal Layer (A) DD14 excised optic-vesicle-like tissue fromRx-GFP mESCs. (B) DD18 excised optic-vesicle-like tissue fromNrl-GFP miPSCs with developingNrl-GFP+ photoreceptor precursors from the surface area of a sphere. (C) DD22Rx-GFP mESC-derived retina sheet. (D) DD18Nrl-GFP miPSC-derived retina sheet. (E) Scheme showing an image of subretinal transplantation. (F and G) The DD17Nrl-GFP miPSC-derived sheet survived in the subretinal space of an rd1 mouse that had no remaining photoreceptors at 3 weeks posttransplantation. The RHODOPSIN+ cell mass (F) was only present in the graft area with colocalizedNrl-GFP expression (G). (H–K) The DD14Nrl-GFP miPSC-derived retinal sheet developed a stratified neural retina layer that included ONL, INL, and IPL (group A). (L) DD16Nrl-GFP miPSC-derived retinal graft showed well-structured ONL (group B). (M) DD18Rx-GFP mESC-derived retinal graft in a disorganized pattern (group C). (N) Bar graph shows the subsequent graft patterns of short (2–4 weeks) and midterm (1–3 months) posttransplantation periods, comparing young grafts (DD11–17) and older grafts (DD18–24). The subsequent graft integrity was categorized into three groups: group A, retinal sheet with INL/IPL and ONL (H–K); group B, retinal sheet with ONL and the remaining INL cells (L); and group C, disorganized structure (M). The sample numbers of each group are indicated below. H, host; G, graft; IPL, inner plexiform layer. Scale bars, 200 μm (A–C and F-H), 100 μm (D), 50 μm (I–K), and 20 μm (L and M). See also Figure S2 and Table S1.
Figure 3
Figure 3
IS/OS Formation in Transplanted Retina-like Sheets (A–C) DD14Nrl-GFP miPSC-derived retinal graft showed IS/OS. IS was labeled with RECOVERIN; OS was labeled with RHODOPSIN (C). (D–D″)Rx-GFP mESC-derived retinal culture at DD26 showed short IS/OS colabeled with RECOVERIN and RHODOPSIN. (E–J) DD17Nrl-GFP miPSC-derived retinal graft with IS/OS contact host RPE (red arrowhead) more than 4 months posttransplantation. (F) Electron microscopy showed that rod nuclei in ONL contained a compact mass of heterochromatin, ELM, IS/OS, and RPE. (G and H) IS with mitochondria (white arrowheads) (G) and a well-aligned stack of discs in OS on top of RPE adjacent to microvilli of RPE (H). (I) RPE microvilli in close connection with OS. H, host; G, graft; ELM, external limiting membrane; IS, inner segment; OS, outer segment; RPE, retinal pigment epithelium; MV, microvilli. Scale bars, 100 μm (A), 50 μm (D–D″ and E), 20 μm (B and F), 10 μm (C), 2 μm (I), and 1 μm (G and H). See also Table S1.
Figure 4
Figure 4
Transplanted Retina-like Sheets Show Integration and Synaptic Connection with rd1 Host Retina Schematic diagrams show three typical patterns of integration with rd1 host retina of the transplanted grafts. (A) Pattern 1: laminar interception. Graft INL was present between host INL and graft ONL. (B) Pattern 2: direct contact. The graft ONL was adjacent to the host INL. (C) Pattern 3: cell integration. The graft ONL structure was disorganized, similar to what was observed for cell transplantation. (D) A typical image of pattern 1. RHODOPSIN+ photoreceptors from DD16Nrl-GFP miPSC-derived retinal sheets migrate toward the host retina (white arrowhead). H, host; G, graft. (E) A typical image of pattern 2. DD16Nrl-GFP miPSC-derived retinal sheets show structured ONL directly contacting host INL (same sample as Figure 2L). H, host; G, graft. (F) A typical image of pattern 3. DD18 Rx-GFP mESC-derived retinal grafts show disorganized patterns similar to those observed for cell transplantation (same sample as Figure 2M). H, host; G, graft. (G) Classification of integration patterns of surviving mESC- and iPSC-derived retinal sheets in the subretinal space of rd1 mice, comparing young grafts (DD11–17) and older grafts (DD18–24). (H) Classification of integration patterns of surviving mESC- and iPSC-derived retinal sheets in the subretinal space of rd1 mice, comparing the structural integrity of groups A (INL+ONL) and B (ONL). (I) A few CtBP2+ synaptic terminals remained in the area of bipolar dendrites in the nongrafted area in rd retina (arrows). (J–L) DD11 Nrl-GFP miPSC-derived retinal sheets integrated into rd1 host retina. The graft shows dense synaptic (CtBP2) connection clusters (red arrow) between the host-graft interfaces. (J) A representative host bipolar cell (asterisk) shows CtBP2 gathering at the tip of dendritic processes. (K and K′) Reconstruction image of a couple of host bipolar (PKC-α) cells with CtBP2 assembled at synaptic tips (K) colocalized withNrl-GFP photoreceptors (K′). (L) Orthogonal view of the reconstructed images of K and K’ (see related images in Figures S3C, S3C′, and S3D). (M and N) Electron micrographs show a ribbon synapse from the DD18 Rosa26-TdTomatoRx-GFP mESC-derived retinal graft in the graft-host interface area (M; Figures S4A–S4D) compared with the intragraft ribbon synapse from the DD17Nrl-GFP miPSC-derived retinal graft (N; Figures S3E–S3J). H, horizontal cell. Scale bars, 50 μm (D–F), 20 μm (I), 10 μm (J), 7 μm (L), 5 μm (K and K′), 500 nm (N), and 200 nm (M). See also Table S1 and Movie S1.
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