1. Human ESC–derived retinal epithelial cell sheets potentiate rescue of photoreceptor cell loss in rats with retinal degeneration
by Karim Ben M’Barek, Walter Habeler, Alexandra Plancheron, Mohamed Jarraya, Florian Regent, Angélique Terray, Ying Yang, Laure Chatrousse, Sophie Domingues, Yolande Masson, José-Alain Sahel, Marc Peschanski, Olivier Goureau, and Christelle Monville
Sci Transl Med
Volume 9(421):eaai7471
December 20, 2017
Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

2. Fig. 1 Characterization of hESC-RPE cells.
Characterization of hESC-RPE cells. (A) Timeline of the differentiation protocol for generating retinal pigment epithelial (RPE) cells from human embryonic stem cells (hESCs). (B) Quantitative reverse transcription polymerase chain reaction analysis was performed to measure mRNA expression of RPE markers [paired box 6 (PAX6), retinal pigment epithelium–specific 65-kDa protein (RPE65), bestrophin 1 (BEST1), and microphthalmia-associated transcription factor (MITF)] and pluripotency markers [POU class 5 homeobox 1 (POU5F1) and nanog (NANOG)] in three hESC-RPE cell differentiation batches at passage 1. Expression is presented relative to expression in undifferentiated hESCs. (C to E) Confocal images (maximal projections of zx planes) of hESC-RPE cells after immunostaining for the markers ZO-1 (zonula occludens-1) and BEST1 (C), PAX-6 and EZRIN (D), and MERTK and BEST1 (E). Nuclei were counterstained with DRAQ5 (white). Scale bars, 10 μm. (F) Evaluation by flow cytometry of the number of tyrosinase-related protein 1 (TYRP1)–positive and LIN28-positive cells before hESCs and after differentiation into RPE cells (hESC-RPE cells). Four hESC-RPE cell batches were tested in total. (G) Representative images of hESC-RPE cells after 3 hours of exposure or no exposure to fluorescein isothiocyanate (FITC)–labeled pig photoreceptor cell outer segment (FITC-POS; green). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (white). Scale bars, 10 μm. (H) Quantification of vascular endothelial growth factor (VEGF) secreted by hESC-RPE cells at different time points during culture using an enzyme-linked immunosorbent assay. Values plotted are means ± SD.
Karim Ben M’Barek et al., Sci Transl Med 2017;9:eaai7471
Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

3. Fig. 2 In vitro assessment of hESC-RPE cell sheets.
In vitro assessment of hESC-RPE cell sheets. (A) Macroscopic photographic images of a hESC-RPE cell sheet after 6 weeks in culture. The right-hand image shows the hESC-RPE cells on the human amniotic membrane (hAM) scaffold. Scale bar, 50 μm. (B) Section of the hESC-RPE cell sheet illustrating the monolayer organization of hESC-RPE cells. Cells were stained for TYRP1 expression (red) and counterstained with DAPI (blue). The hAM scaffold is indicated. Scale bar, 50 μm. (C) hESC-RPE cells cultured on the hAM scaffold and stained for specific RPE markers. Top: ZO-1, green; MITF, red. Bottom: DAPI, blue; TYRP1, red. Scale bars, 50 μm. (D) Transmission electron microscopy image of the hESC-RPE cell sheet. Scale bar, 5 μm. (E) Scanning electron microscopy images of the hESC-RPE cell sheet showing hESC-RPE cells on the hAM scaffold at different magnifications. The bottom image (a magnification of the area indicated by the rectangle in the top image) shows the basement membrane and extracellular matrix fibers of the hAM scaffold. Scale bars, 5 μm (top) and 1 μm (bottom). Three different hESC-RPE cell batches were tested.
Karim Ben M’Barek et al., Sci Transl Med 2017;9:eaai7471
Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

4. Fig. 3 hESC-RPE cell sheet transplantation and validation of the surgical method.
hESC-RPE cell sheet transplantation and validation of the surgical method. (A) Steps for the preparation and loading of the hESC-RPE cell sheet on the hAM scaffold into the injection device. (B) Image of the hESC-RPE cell sheet embedded in gelatin. A small piece (2 to 3 mm2) was cut for injection into the subretinal space of the rat eye (black rectangle). (C) Image showing loading of the hESC-RPE cell sheet (black rectangle) into the head of the injection device. (D) OCT analysis of the retina of a wild-type control rat and a dystrophic RCS rat after transplantation of the hESC-RPE cell sheet (red line, dystrophic + graft). (E to G) Representative immunofluorescence confocal microscopic images of sections of retina from athymic nude rats 10 days after transplantation with either a hESC-RPE cell sheet (left) or a hESC-RPE cell suspension (right). Sections were stained for human MTCO2 and human ZO-1 (E), human MERTK and human MTCO2 (F), or human MERTK and human collagen IV (G). Nuclei were visualized with a DAPI counterstain. Three rat retinas transplanted with hESC-RPE cell suspensions and four rat retinas transplanted with hESC-RPE cell sheets were analyzed. White box indicates regions that are enlarged. Images correspond to maximal projections of z stacks. Scale bars, 50 μm and 10 μm (higher magnification). IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OLM, outer limiting membrane.
Karim Ben M’Barek et al., Sci Transl Med 2017;9:eaai7471
Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

5. Fig. 4 Improved visual recovery after hESC-RPE cell sheet transplantation into RCS rats.
Improved visual recovery after hESC-RPE cell sheet transplantation into RCS rats. (A) Visual representation of the optokinetic test. Animals were placed on an elevated platform and exposed to a rotating stimulus (on four screens) consisting of vertical black and white lines of varying widths. (B) Bar graphs showing the quantification of visual acuity using the optokinetic test in RCS rats at different time points after transplantation (4, 6, and 13 weeks). (C to H) Electroretinogram responses were recorded at 5, 9, and 12 weeks after surgery in RCS rats transplanted with hESC-RPE cell sheets (yellow), gelatin alone (sham, black), or hESC-RPE cell suspensions (green) compared to untreated RCS rats (blue). Results are presented as average response (b-wave) curves (under dim light exposure) to flashing lights of increasing intensity (C, E, and G) and as area under the curve (AUC) measurements for the corresponding graphs for each group (D, F, and H). Six to nine animals per time point. Analysis of variance (ANOVA) followed by Fisher’s protected least squares difference (PLSD) test; *P < 0.05, **P < 0.01, and ***P < Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test.
Karim Ben M’Barek et al., Sci Transl Med 2017;9:eaai7471
Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

6. Fig. 5 OCT analysis of RCS rat retinas 12 weeks after hESC-RPE cell sheet transplantation.
OCT analysis of RCS rat retinas 12 weeks after hESC-RPE cell sheet transplantation. (A) Reconstructed OCT image of a retina from a wild-type rat (top left). The heat maps illustrate the thickness of the ONL for each condition. Wild-type and dystrophic RCS rat retinas are shown in the top panels, and dystrophic RCS rat retinas transplanted with gelatin only (sham), hESC-RPE cell sheets, or cell suspensions are shown in the bottom panels. White squares represent the optic nerve of each animal. The horizontal and vertical lines (denoted a′ and b′, respectively) correspond to the location of the two widths selected to illustrate ONL thickness. (B) ONL thickness in the temporonasal axis [indicated by double-headed arrows in (A, a′)]. (C) ONL thickness in the dorsoventral axis [indicated by the double-headed arrows in (A, b′)]. Five to six animals per condition. Scale bar, 100 μm. (D) Histogram showing the mean ONL thickness in RCS rat retinas after transplantation with gelatin only (sham), hESC-RPE cell sheets, or cell suspensions (mean of 420 individual measurements per eye; five to six animals per condition). ANOVA followed by Fisher’s PLSD test; *P < 0.05 and **P < Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test.
Karim Ben M’Barek et al., Sci Transl Med 2017;9:eaai7471
Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

7. Fig. 6 Photoreceptor cell survival in the RCS rat retina after transplantation with hESC-RPE cell sheets.
Photoreceptor cell survival in the RCS rat retina after transplantation with hESC-RPE cell sheets. Immunofluorescence staining for recoverin (green) and rhodopsin (red) in sections of RCS rat retinas 3 months after transplantation with gelatin alone (sham), hESC-RPE cell sheets, or hESC-RPE cell suspensions is shown. Nuclei were counterstained with DAPI (white). White boxes in (A) indicate area of enlargement shown in (B) (recoverin), (C) (rhodopsin), and (D) (merge + DAPI). The asterisks in (D) indicate the position of the ONL. Images correspond to maximal projections of z stacks. Scale bars, 50 μm (top row) and 10 μm (bottom row).
Karim Ben M’Barek et al., Sci Transl Med 2017;9:eaai7471
Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works