Research ArticleRetinal Disease

Clinical-grade stem cell–derived retinal pigment epithelium patch rescues retinal degeneration in rodents and pigs

See allHide authors and affiliations

Science Translational Medicine  16 Jan 2019:
Vol. 11, Issue 475, eaat5580
DOI: 10.1126/scitranslmed.aat5580
  • Fig. 1 Generation of clinical-grade iRPE cells.

    (A) Workflow illustrating a pipeline to manufacture and test autologous clinical-grade iRPE patches with the goal of filing a phase 1 clinical trial investigational new drug (IND) application to the U.S. Food and Drug Administration. (B) Time line of clinical-grade iRPE differentiation. Clinical-grade iRPE differentiation takes 77 days, is initiated with monolayer iPSCs, and is performed using xeno-free reagents. NEIM, neuroectoderm induction medium; RPEIM, RPE induction medium; RPECM, RPE commitment medium; RPEGM, RPE growth medium; RPEMM, RPE maturation medium. (C) Coding region sequencing of 223 oncogenes at 2000× depth for all nine clinical-grade AMD iPSC clones. (D and E) Flow cytometry analysis of clinical-grade iRPE derived from three AMD patients, performed at the RPE progenitor stage (D17), RPE commitment stage (D27), and immature RPE stage (D42) (n = 6). Analysis of variance (ANOVA) was performed to determine changes in percent positive cells; ***P = 0.0001 for PAX6/MITF and ***P = 8.9 × 10−14 for MITF; Dunn’s test was performed for pairwise comparisons; P values: 2B/2C-3C/3D = 0.909, 2B/2C-4B/4C = 0.400, and 3C/3D-4B/4C = 0.319. ns, not significant. (F) RPE-specific gene expression from D5 to D42 of clinical-grade iRPE differentiation (n = 6). Dunn’s test was performed for pairwise comparisons; P values: 2B/2C-3C/3D = 0.721, 2B/2C-4B/4C = 0.719, and 3C/3D-4B/4C = 0.999.

  • Fig. 2 Generation of functionally mature AMD iRPE patch.

    (A) Young’s modulus of different PLGA scaffolds. **P < 0.01, two-tailed t test. (B) SEM showing surface topology of single-layer fused PLGA scaffold. (C) Top panel: Representative immunostaining for mature RPE marker RPE65 (red) and human-specific antigen STEM121 (green). Middle panel: RPE pigmentation protein GPNMB (red) and Bruch’s membrane protein COLLAGEN IV (green). Bottom panel: COLLAGEN VIII and Bruch’s membrane marker (red) (n = 3). DAPI, 4′,6-diamidino-2-phenylindole. (D) Representative transmission electron microscopy of iRPE patch on transwell membrane or the PLGA scaffold. Basal infoldings can be seen in the case of PLGA scaffold (inset) (n = 3). (E) ∆CT values of RPE-specific genes are displayed for all eight iRPE patches from three AMD donors (n = 8). Dunn’s test was performed to determine pairwise comparisons; P values: 2B/2C-3C/3D = 0.999, 2B/2C-4B/4C = 0.150, and 3C/3D-4B/4C = 0.094. (F) Live TER measurement during the last 3 weeks (D54 to 77) of iRPE patch maturation. Representative data from three clones (3A, 3D, and 4A) are displayed (n = 8). Dunn’s test was performed to determine changes in TER overtime; P values: 3A-3D = 0.630, 3A-4A = 0.845, and 3D-4A = 0.968. (G) Graphs show phagocytosis ratio for AMD iRPE patches (n = 8). Dunn’s test was performed to compare iRPE from different donors; P values: 2B/2C-3A/3C/3D = 0.005, 2B/2C-4A/4B/4C = 0.395, and 3A/3C/3D -4A/4B/4C = 0.63. (H and I) PCA combining data from the following assays (morphometric, gene expression, TER, and phagocytosis) showing variation between clones across PC1. PCA was performed based on k-nearest neighbors, and bootstrap hierarchical clustering was performed to determine differences between iRPE samples; *P < 0.05. (H) PCA plotted without D2B (I) (n = 7 to 8).

  • Fig. 3 Safety and efficacy assessment of clinical-grade AMD iRPE patch in rodent models.

    (A to C) Representative en face infrared image (A), OCT (B), and immunohistochemistry for human antigen (C; STEM121, red) showing subretinal location and integration of the 0.5-mm-diameter clinical-grade AMD iRPE patch (red arrowheads). Black arrowheads mark rat RPE cells (see inset for higher magnification) in the subretinal space of immunocompromised rat at 10 weeks after surgery (n = 20). (D) Representative immunohistochemistry for STEM121 (purple) (red arrowheads, see inset for higher magnifications) confirms the presence of clinical-grade AMD iRPE cells injected in the rat eye. Note that purple color in POS is due to hematoxylin staining. Rat RPE cells are not positive for STEM121 (black arrowheads, see inset for higher magnification). (E) Representative STEM121 (red) immunostaining (red arrowheads) showing integration of a small number of human cells in the rat RPE (black arrowheads; see inset for higher magnification) (n = 10). (F) Representative Ki67 immunostaining showing lack of positivity. Human cells are indicated by red arrowheads (see inset for higher magnification; rat RPE is marked with black arrowheads). (G and I) Representative photomontage of RCS rat retina showing ONL (arrowheads) with transplanted iRPE patch (~10,000 cells on a 1-mm-diameter patch) (G) or iRPE cell suspension (100,000 cells) (I), compared with the degenerated ONL in nontransplanted areas (arrow, n = 10). (H and J) Representative immunofluorescence staining of iRPE patch (H) or iRPE cell suspension (J) implanted retina with ONL rescue (arrowheads) [red, human nuclear antigen (HuNu); green, human-specific anti-PMEL17]. Note that red arrowheads in (H) point to iRPE cells that likely dislodged from the scaffold during transplantation. (K) OKN tracking thresholds at p90. n = 10. *P < 0.05 and **P < 0.001, ANOVA.

  • Fig. 4 Development of a porcine iRPE patch efficacy model.

    (A) Schematics of micropulse laser injuring the pig RPE. Inset: Fluorescein angiogram depicting laser-induced outer blood-retinal barrier breakdown. (B and C) Representative OCT images at 24 and 48 hours after laser (arrowheads indicate RPE thinning). n = 3. (D) Heatmap of the P1 values of the visual streak region after 1% or 3% DC laser (laser areas outlined with dashed lines). White-red indicates the highest P1 values, and blue indicates the lowest P1 values. (E) Average mfERG waveform from healthy (black), 1% (light green), and 3% (dark green) DC laser areas. (F to I) Representative immunostaining for TUNEL (green), RPE65 (yellow), and PNA (magenta) (F and G) and H&E staining (H and I) at 48 hours (arrowheads indicate apoptotic RPE). n = 3.

  • Fig. 5 Efficacy assessment of clinical-grade AMD iRPE patch in a porcine retinal degeneration model.

    (A to C) Comparison of OCT from retina over a healthy region, retina transplanted with an empty PLGA scaffold, or retina transplanted with clinical-grade AMD iRPE patch (horizontal lines) (n = 3). (D to F) Immunostaining for STEM121 (green, arrowhead; F) and RPE65 (red) in the pig eye. PNA staining is shown in white; white arrowhead in (E) marks retinal tubulations (n = 3). (G) Immunostaining for red, blue, and green cone opsins (white; red arrowhead) and STEM121(green) in the pig eye after iRPE patch transplantation. (H and I) Rhodopsin (green) immunostaining shows phagocytosed (white arrowheads) POS by healthy pig RPE immunostained with RPE65 (red) and by human iRPE cells immunostained with STEM121 (red). Z sections show POS localization inside pig and human RPE cells (n = 3). (J to L) Heatmaps of N1P1 mfERG responses. (M and N) Average mfERG waveform (M) and mfERG data over 10 weeks of follow-up after surgery (N) (n = 3). LME was performed for data analysis, and ANOVA was used to determine statistical significance of the data. *P < 0.05.

  • Fig. 6 Integration of iRPE patch in a laser-induced retinal degeneration porcine model.

    (A to H) Representative OCT images of pig eyes 2 and 5 weeks after transplantation with empty scaffold, PLGA iRPE patch, transwell iRPE patch, and iRPE cell suspension. iRPE transplants are indicated by red horizontal line. Green arrowheads point to retinal tubulations. (I to L) Representative immunostaining for human antigen STEM121 (green) and RPE65 (red) in the pig eye [iRPE patch is indicated by horizontal white line in (J); white arrowheads in (J) and (L) mark iRPE transplants; green arrowheads in (I) marks retinal tubulations; and red arrowheads in (J) and (K) mark rat photoreceptors]. n = 3. (M and N) Individual and average mfERG responses from different transplant conditions (n = 3; *P < 0.05, ANOVA).

  • Table 1 Summary of preclinical rat and pig studies performed to demonstrate safety and efficacy of clinical-grade AMD iPSC-RPE patch.

    PR, photoreceptor; WT, wild-type; na, not available.

    PhaseGroupNo. of eyesDuration
    (weeks)
    Observations
    1iPSC-RPE patch safety study* in RNU (Crl:NIH-Foxn1rnu) rats (0.5-mm-diameter iPSC-RPE patch)
    1aSham surgery1070/8 STEM121 positive (2 unscheduled deaths)
    1biPSC-RPE sheet2076/15 eyes showed integrated iPSC-RPE patch (one
    unscheduled death)
    2Cell suspension safety study* in RNU (Crl:NIH-Foxn1rnu) rats (100,000 cells)
    2aVehicle control550/5 STEM121 positive
    2bPure iPSCs1053/10 teratoma
    2cScaffold pieces cut from 8-mm2 PLGA punch1050/10 STEM121 positive
    2diPSC-RPE cell suspension1051/10 endo-ophthalmitis; 9/10 STEM121 and PMEL17 positive;
    9/10 Ki67 negative; no teratoma
    3Cell suspension and patch efficacy study in RCS rats (1 mm iPSC-RPE patch or 100,000 cells)
    3aSham surgery185 /10PR degeneration
    3bSuspension (100,000 cells)115 /10PR rescue in all eyes
    3cSheet (1-mm scaffold)75 /108/10 eyes showed integrated iPSC-RPE patch and PR rescue
    4Empty PLGA scaffold safety study in WT pig eye (4 × 2 mm PLGA piece)
    4aSham surgery310na
    4bEmpty PLGA scaffold310No inflammation, slow degradation of PLGA, and recovery of
    mfERG signals
    5iPSC-RPE patch efficacy study in laser-injured pig eye
    5aEmpty PLGA scaffold (4 × 2 mm)38Slow degeneration of PRs
    5biPSC-RPE patch (4 × 2 mm)38Integration of iPSC-RPE patch in pig eye and no degeneration of PRs
    5ciPSC-RPE cell suspension (100,000 cells)38
    5diPSC-RPE transwell (4 × 2 mm)38No degeneration of PRs, but no integration of iPSC-RPE
    transwell in pig eye

    *Body weight and food consumption were checked weekly for immunocompromised rats in the safety study. There was no apparent effect of the patch or cell suspension on weight and food consumption of these animals.

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/11/475/eaat5580/DC1

      Materials and Methods

      Fig. S1. Optimization of iRPE differentiation.

      Fig. S2. Degradation kinetics of PLGA scaffold.

      Fig. S3. Evaluation of functionally mature iRPE patch.

      Fig. S4. Assessment of iPSC survival in iRPE cultures.

      Fig. S5. Safety and efficacy assessment of iRPE patch in rodents.

      Fig. S6. Optimization of laser-induced RPE injury in pig eyes.

      Fig. S7. Optimization of subretinal transplantation procedure in pigs.

      Fig. S8. Analysis of iRPE patch in pig model of laser-induced retinal degeneration.

      Fig. S9. Comparative efficacy analysis of iRPE patch and iRPE suspension in the pig model.

      Table S1. Validation of clinical (GMP)–grade iPSC Working Bank derived from CD34+ cells.

      Table S2. Oncogene exome analysis of iPSC versus donor PBMCs.

      Table S3. Detailed list of clinical-grade reagents used in iPSC generation and RPE differentiation.

      Table S4. Summary of body weight change in rats transplanted with iPSC-derived RPE.

      Table S5. Lactic acid release analysis of in vitro degraded PLGA scaffolds.

      File S1. Images for all replicates described in Figs. 2 to 6 (provided as separate Word file).

      File S2. Raw data for the main figures (provided as separate Excel file).

      File S3. Raw data for the supplementary figures (provided as separate Excel file).

      References (53, 54)

    • The PDF file includes:

      • Materials and Methods
      • Fig. S1. Optimization of iRPE differentiation.
      • Fig. S2. Degradation kinetics of PLGA scaffold.
      • Fig. S3. Evaluation of functionally mature iRPE patch.
      • Fig. S4. Assessment of iPSC survival in iRPE cultures.
      • Fig. S5. Safety and efficacy assessment of iRPE patch in rodents.
      • Fig. S6. Optimization of laser-induced RPE injury in pig eyes.
      • Fig. S7. Optimization of subretinal transplantation procedure in pigs.
      • Fig. S8. Analysis of iRPE patch in pig model of laser-induced retinal degeneration.
      • Fig. S9. Comparative efficacy analysis of iRPE patch and iRPE suspension in the pig model.
      • Table S1. Validation of clinical (GMP)–grade iPSC Working Bank derived from CD34+ cells.
      • Table S2. Oncogene exome analysis of iPSC versus donor PBMCs.
      • Table S3. Detailed list of clinical-grade reagents used in iPSC generation and RPE differentiation.
      • Table S4. Summary of body weight change in rats transplanted with iPSC-derived RPE.
      • Table S5. Lactic acid release analysis of in vitro degraded PLGA scaffolds.
      • Legends for files S1 to S3
      • References (53, 54)

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

      • File S1 (Microsoft Word format). Images for all replicates described in Figs. 2 to 6 (provided as separate Word file).
      • File S2 (Microsoft Excel format). Raw data for the main figures (provided as separate Excel file).
      • File S3 (Microsoft Excel format). Raw data for the supplementary figures (provided as separate Excel file).

    Stay Connected to Science Translational Medicine

    Navigate This Article