Protective effects of human iPS-derived retinal pigment epithelium cell transplantation in the retinal dystrophic rat

Abstract

Transformation of somatic cells with a set of embryonic transcription factors produces cells with the pluripotent properties of embryonic stem cells (ESCs). These induced pluripotent stem (iPS) cells have the potential to differentiate into any cell type, making them a potential source from which to produce cells as a therapeutic platform for the treatment of a wide range of diseases. In many forms of human retinal disease, including age-related macular degeneration (AMD), the underlying pathogenesis resides within the support cells of the retina, the retinal pigment epithelium (RPE). As a monolayer of cells critical to photoreceptor function and survival, the RPE is an ideally accessible target for cellular therapy. Here we report the differentiation of human iPS cells into RPE. We found that differentiated iPS-RPE cells were morphologically similar to, and expressed numerous markers of developing and mature RPE cells. iPS-RPE are capable of phagocytosing photoreceptor material, in vitro and in vivo following transplantation into the Royal College of Surgeons (RCS) dystrophic rat. Our results demonstrate that iPS cells can be differentiated into functional iPS-RPE and that transplantation of these cells can facilitate the short-term maintenance of photoreceptors through phagocytosis of photoreceptor outer segments. Long-term visual function is maintained in this model of retinal disease even though the xenografted cells are eventually lost, suggesting a secondary protective host cellular response. These findings have identified an alternative source of replacement tissue for use in human retinal cellular therapies, and provide a new in vitro cellular model system in which to study RPE diseases affecting human patients.

Figure 1. Human induced pluripotent stem cells differentiate into retinal pigment epithelial cells.

(A) Photomicrographs showing undifferentiated (left) and differentiating iPS cells (right). (B) iPS-RPE cells form a pigmented monolayer in culture with typical RPE cell cobblestone appearance. (C) Comparison of pigmentation observed in T25 flasks of confluent ARPE-19 cells and iPS-RPE cells. (D) PCR amplification of classic RPE cell markers in iPS-RPE cells. A 100 bp DNA ladder was applied to the gel as an amplicon size reference. (E) Western blot analysis of iPS-RPE protein expression using antibodies against a panel of RPE cell markers. A protein standard was used on each Western blot to determine the correct molecular weight of proteins. (F) Quantitative PCR analysis of RPE and iPS gene expression in iPS, iPS-RPE and foetal RPE cDNAs. (G) iPS reprogramming proteins are reduced after differentiation to iPS-RPE. OCT4 and SOX2 expression is undetectable in iPS-RPE cells. Combined confocal and Nomarski image of iPS-RPE cells are shown on the left with confocal channels to the right. OCT4 and SOX2 (red channel) and DAPI stained nuclei are blue. Scale bars: A, 20 µm; B and G, 50 µm.

Figure 2. Human iPS-RPE cells are polarized and display classic RPE cell morphology.

(A) Electron micrograph of an iPS-RPE cell monolayer. Human iPS-RPE are pigmented cuboidal epithelial cells with cytoplasmic polarization. Indicated are apical microvilli (AMv), melanin-containing melanosomes (red arrows), the basal nucleus (N), desmosomes (white arrows) and basal lamina (black arrows). (B) Densely packed melanosomes: stages II, III and IV of melanosome maturation are labelled. (C) Microvilli (AMv) extend out from the apical surface of iPS-RPE. (D) Adherens junctions (white arrows) between cells are in the apical portion of the cell, whilst the nucleus (N) is basal. (E) Coated pits (asterisk) are found throughout the plasma membrane. (F) iPS-RPE produce their own basal lamina (indicated by black arrows). Scale bars: A, 2 µm; B-F, 1 µm.

Figure 3. Immunocytochemical localization of RPE cell specific proteins in sectioned sheets of iPS-RPE cells.

The left column shows combined Nomarski and confocal images of the pigmented iPS-RPE cell sheet sections; adjacent are images of immunolabelling only. Protein staining is indicated by the colour of the text (red or green) and DAPI stained nuclei are blue. Scale bars: All 50 µm.

Figure 4. iPS-RPE cells phagocytose photoreceptor outer segment material in vitro.

(A) Confocal images showing phagocytosis of isolated FITC-labelled porcine photoreceptor outer segments (POS – green) by iPS-RPE in culture. The nuclei are stained with DAPI (blue). Internalization of POS is observed in a single optical y-axis projection (<1 µm) of pigmented iPS-RPE cells labelled with the apical cell surface marker ATP1B1 (red). (B- i) Electron microscopy image of a porcine photoreceptor outer segment (POS) adjacent to an iPS-RPE cell following 3 hours co-culture with a porcine retina explant. iPS-RPE apical microvilli (red arrows) and coated vesicles (white arrows) are observed proximal to the porcine POS. (ii) Internalized coated pits (black arrow and enlarged inset in blue box) are seen within the cytoplasm of iPS-RPE cells co-cultured with POS. (iii) Lipid deposits (L), a sign of late stage POS phagocytosis, are observed within the iPS-RPE cytoplasm after 12 h co-culture. Scale bars: A, Upper panel 20 µm and lower panel 10 µm; i, iii, 2 µm and ii, 1 µm.

Figure 5. iPS-RPE maintain RPE cell markers and phagocytose host photoreceptor outer segment material following transplantation into the subretinal space of dystrophic RCS rats.

(A) A schematic of retinal cell organisation. The nuclear layers are indicated. iPS-RPE cells were injected into the subretinal space between the host RPE and photoreceptor cells (B) A layer of iPS-RPE cells in the subretinal space of the dystrophic RCS rat 20 hours following transplantation. (C) Retention of RPE markers by iPS-RPE cells in vitro after dissociation, re-plating and culturing for 8 days. Protein staining is indicated by the text colour. (D) Expression of the same RPE cell markers is maintained in vivo by iPS-RPE (white arrows) 8 days following transplantation into the subretinal space of the RCS rat. (E) Rhodopsin-positive material (red) is present within the cell membrane of human specific marker (HSM)-labelled iPS-RPE (green) 8 days post-transplantation and in the tips of the host outer segment (OS) layer. DAPI (blue) stains nuclei. Indicated are the outer and inner nuclear layer (ONL and INL respectively) of the retina. Scale bars: B-D, 50 µm; E, 50 µm and 20 µm in magnified view.

Figure 6. Preservation of visual function following iPS-RPE transplantation into the RCS rat.

(A) Preservation of optokinetic head-tracking response to a rotating vertical stimulus in 16-week-old RCS dystrophic rats following transplantation of iPS-RPE. Mean visual acuity (±S.E.M.) of the transplanted eye versus control sham-injected eye and non-transplanted dystrophic eye. Spatial frequency is indicated in cycles per degree (c/d).

Figure 7. Preservation of the photoreceptor cell layers after transplantation of iPS-RPE.

(A) Extensive preservation of the nuclear photoreceptor layers in the dorsal retina of the dystrophic RCS rat 13 weeks following transplantation of iPS-RPE cells (DAPI stained nuclei). Inset shows higher resolution confocal images of photoreceptor cell nuclear layers (DAPI blue) and rhodopsin expression (red) in the dystrophic control (left inset) and dystrophic with iPS-RPE transplant (right inset) RCS rat. (B) Confocal images showing HSM-positive staining (green, indicated with white arrow) within the subretinal space 13 weeks post-transplantation. Scale bars: A 500 µm; inset, 50 µm; B 50 µm.

Figure 8. Macrophages are present in the subretinal space of the dystrophic RCS following transplantation of iPS-RPE cells.

(A) Coronal confocal projection showing confocal channels and Nomarski images of iPS-RPE transplanted eye containing non-pigmented CD68-positive cells (green) 8 days following transplantation. The white box contains a CD68-positive cell with rhodopsin-positive cytoplasmic inclusions (red) which is magnified and shown in serial confocal slices in B. (C) Co-labelling of non-pigmented CD68 and rhodopsin-positive cells at 13 weeks post-graft. Note the presence of an intact outer segment and outer nuclear layer at this stage. (D) Large pigmented cells are observed in the subretinal space at 13 week post-graft. The white box indicates a CD68-positve cell containing rhodopsin-positive inclusions, shown magnified and in serial confocal slices in (E). Scale bars: All 20 µm.

Figure 9. iPS-RPE transplantation preserves the light induced c-Fos response in the RCS dystrophic rat retina.

(A) Age-matched non-dystrophic control, dystrophic no-transplant control and dystrophic rats with iPS-RPE grafts were dark-adapted overnight and sacrificed in the dark or after 90 min exposure to white light (250 µW/cm²). Coronal sections through the retina of dystrophic transplanted, dystrophic non-transplanted and non-dystrophic control RCS rats showing the inner and outer nuclear layers (ONL and INL respectively) and the ganglion cell layer (GCL). Preservation of ONL 13 weeks after iPS-RPE transplant correlates with preservation of the light-induced c-Fos expression (red) in coronal sections of the retina and in representative dorsal whole-mount preparations of the inner nuclear and ganglion cell layers. Note absence of activity in darkness and responsivity to light in the normal and transplanted eyes. c-Fos positive cells in the ganglion cell layer of unoperated RCS eyes match the distribution expected for intrinsically light-responsive melanopsin-containing ganglion cells. DAPI-stained nuclei are shown in blue in the coronal section and the autofluorescent debris zone (dz) is indicated. (B) Light-induced c-Fos activation in the transplanted eye of RCS rats is preferentially preserved in the dorsal retina (the region of the transplant), corresponding with preservation of photoreceptors (ONL) by the iPS-RPE graft. No such preservation is observed in the ventral retina of the transplanted animal. Scale bars: Coronal sections, 50 µm; whole-mount images of the inner nuclear and retinal ganglion cell layers, 200 µm.