Delivery of Human iPSC-Derived RPE Cells in Healthy Minipig Retina Results in Interaction Between Photoreceptors and Transplanted Cells

Abstract

In late stages of inherited and acquired retinal diseases such as Stargardt disease (STGD) or dry age-related macular degeneration (AMD), loss of retinal pigment epithelia (RPE) cells and subsequently photoreceptors in the macular area result in a dramatic decline of central visual function. Repopulating this area with functional RPE cells may prevent or decline the progression of photoreceptor loss. In the present study, the viability, survival, and integration of human induced pluripotent stem cell (hiPSC)-derived RPE cells (hiPSC-RPE) is assessed generated using clinical-grade protocol and cultured on a clinically relevant scaffold (poly-L-lactide-co-D, L-lactide, PDLLA) after subretinal implantation in immunosuppressed minipigs for up to 6 weeks. It is shown that transplanted hiPSC-RPE cells maintain the RPE cell features such as cell polarity, hexagonal shape, and cell-cell contacts, and interact closely with photoreceptor outer segments without signs of gliosis or neuroinflammation throughout the entire period of examination. In addition, an efficient immunosuppressing strategy with a continuous supply of tacrolimus is applied. Continuous verification and improvement of existing protocols are crucial for its translation to the clinic. The results support the use of hiPSC-RPE on PDLLA scaffold as a cell replacement therapeutic approach for RPE degenerative diseases.

Keywords: Human induced pluripotent stem cells;minipigs; age‐related macular degeneration; cell therapy; retina; retinal degeneration; retinal pigment epithelium.

Figure 1

Differentiation of hiPSCs into RPE. A) Timeline of clinical‐grade RPE differentiation. Clinical‐grade RPE differentiation protocol begins using a hiPSC monolayer and uses clinical‐grade reagents. PM, plating medium; RIM, retinal induction medium; RDM, retinal differentiation medium; RMNA, retinal medium with Nicotinamide and Activin‐A; RPE‐MM, RPE‐maintenance medium. Lower images, Representative brightfield micrographs with hiPSC at the beginning of the protocol, the insert with the oval frame in the center, and higher magnification of the insert with highly pigmented cells at the time of transplantation B) Representative electron micrograph of cultured hiPSC‐RPE on porous scaffold. Apical microvilli (AmV), melanosomes (Mel), tight junction (white arrows), and basal infoldings (SBI) are detected. The nuclei (N) are located on the basal side of the cells. C) TEER values of hiPSC‐RPE patches during in vitro cell culture. D) qRT‐PCR analysis of mRNA expression for RPE markers (PMEL, RPE65, MITF, BEST1, CRALBP, MERTK) and the pluripotency marker NANOG. Data represents the fold‐change in mRNA expression of hiPSC‐ RPE relative to their parental hiPSCs. Each bar represents the average ± SEM of at least three independent replicates. ^** p ≤ 0.01, ^*** p ≤ 0.001, ^**** p ≤ 0.0001. E) Immunocytochemical analysis of RPE marker protein expression (MERTK, BEST1, CRALBP, EZRIN, RPE65 y ZO‐1) in hiPSC‐RPE patches. Vertical confocal sections showing apical localization of EZRIN and ZO1. Images were taken with Leica confocal microscope TCS SP8 using HCX PL APO lambda blue 63X/ 1.4 OIL objective. Scale bar = 25 µm.

Figure 2

Fundus imaging and OCT of implanted minipig retinas at 1, 2 or 6 weeks post‐implantation. A) A1‐A10, Fundus images of implanted minipig retinas. The ^* indicates the position of the implant in the eye fundus. B1‐B10, Fundus‐paired black‐white OCT scans (upper scans are horizontal, lower scans are vertical) of implanted retinas. White arrows show the location of the implant in the subretinal space. Magnifications with red borders demonstrate implanted areas of the retina, with green border non‐implanted adjacent areas. Images A9, B9, and A10, B10 demonstrate implanted acellular scaffolds at 6 weeks post‐implantation. Severe retinal atrophy was detected in the part of the neuroretina lying above the scaffolds (B9). The animal is identified by the number preceded by the B letter. Left eye (LE), right eye (RE). B) Tacrolimus concentration in vivo. Time course of intravenous Tacrolimus concentrations in treated animals. D0 – Cell seeded scaffold implantation + Depomedrol 80 mg/pig i.m. application, D5‐7–2nd TLPM s.c. application (TAC dose 0.3–0.8 mg kg⁻¹ BW), D14 – termination of animals for 2 weeks’ time point, D17‐19–3rd TLPM s.c. application (TAC dose 0.4–0.5 mg kg⁻¹ BW), D29‐30–4th TLPM s.c. application (TAC dose 0.5–0.6 mg kg⁻¹ BW), D42‐43–Experiment termination.

Figure 3

Hematoxylin & eosin staining of the retinal area containing the transplanted cells on nanofibrous carrier membrane surrounded by PET frame structure (arrows). Implanted hiPSC‐derived RPE cells are heavily pigmented and in close proximity to photoreceptor inner and outer segments. Observations are presented at 1 (C), 2 (D), and 6 (E) weeks post‐implantation as well as an untreated control eye (A). The level of the implanted RPE cell layer is marked with a bar indicating the heights of the RPE cells (marked by a medium sized arrow in C–E). Retina with implanted cell‐free membrane shows a disorganized outer nuclear layer after six weeks (B). Big arrows in B, C & E point to the PET frame (ring) of the PDLLA nanofibrous membrane. The retina adjacent to the implanted area is unaffected by the implantation F). Carrier dimensions, 5.2 mm × 2.1 mm. ONL = outer nuclear layer; INL = inner nuclear layer; IPL = inner plexiform layer; GCL = ganglion cell layer; NFL = nerve fiber layer. All scale bars represent 200 µm.

Figure 4

Expression of Human Nuclear Antigen (HNAA, green) and STEM121 (red) on hiPSC‐RPEs implanted on nanofibrous carriers, followed up to 6 weeks. Tissue was stained with HNAA A–C) and STEM121 D–F) antibodies. The third panel (D’‐F’) shows bright field images of the STEM121 staining. Observations are presented at 1 (A, D, D’), 2 (B, E, E’), and 6 (C, F, F’) weeks. Arrows in E and E’ point to heavily pigmented RPE cells partially masking immunohistochemical staining in implanted hiPSC‐RPE cells. They appear as a monolayered or a multi‐layered cell structure after 1, 2, or 6 weeks showing a high cell density., i.e., the distance between the nuclei is very small which is unusual for RPE cells. Nuclear staining was performed by DAPI (purple and light blue). ONL = outer nuclear layer; nfm = nanofibrous membrane; hiPSC‐RPE = human iPSC derived RPE cells; RPE = retinal pigment epithelium.

Figure 5

Immunohistochemical detection of tight junction marker ZO‐1 (red). Vertical and flat sections through the posterior part of the minipig eye holding implanted hiPSC‐RPE cells on nanofibrous carrier membrane after 1 (B) 2 (C) and 6 weeks (D–G). In flat views, the typical hexagonal shape of RPE cells is nicely visible (E, E’, F) due to the ZO‐1 immunoreactive staining of the tight junctions. In vertical views, the ZO‐1 staining appears rather dot‐like or like a dashed line (arrows in A–D, G, G’). Nuclear staining by DAPI is shown in magenta. OLM = outer limiting membrane. The scale bar in D holds for A–D, in G‘ for G, scale bar in E‐F, 10 µm.

Figure 6

Hematoxylin & eosin staining of the retinal area containing the implanted nanofibrous carrier membrane with and without hiPSC‐RPE cells A–D). Massive rosette formation underneath the cell‐free nanofibrous membrane is visible (C, D, I). In contrast, no rosette formation was detected in the retina underneath the implanted carrier membrane with the human RPE cells (A, B, E). Immunohistochemistry of reactive gliosis marker GFAP in minpig retina showed massive reactive gliosis after implantation of cell‐free nanofibrous carrier membrane after six weeks (H, I). No signs of reactive gliosis or rosette formation were detected in the retina underneath the implanted carrier membrane with the human RPE cells (A, B, E–G). Massive growth of Müller glia cell processes is visibly filling the gap of the dying photoreceptors (H‐I’). (F’, G’, I’) represent the respective bright field images of (F, G, I). Nuclear staining by DAPI is shown in blue. Arrows mark the PET ring structure surrounding the nanofibrous carrier membrane (A–D, E, H). IS = inner segment, OLM = outer limiting membrane, ONL = outer nuclear layer, OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer, GCL = ganglion cell layer, NFL = nerve fiber layer. Scale bar in G, 50 µm.

Figure 7

Expression of peanut agglutinin (PNA, green), MERTK (blue), and STEM121 (red) in the upper row B–D) and PNA and rhodopsin (red) (E’‐H’, E” – H”) in treated and untreated minipig eyes (the two lower rows). The second row (E–H) represents the bright field images of the respective time points and images in the two lower rows. Nuclear staining was performed by DAPI (light blue or dark blue). Observations are presented at 1 (B, F, F’, F”), 2 (C, G, G’, G”), and 6 (D, H, H’, H”) weeks post‐implantation as well as an untreated control eye (A, E, E’, E”). PNA brightly labels the inner and outer segments of cone photoreceptors in treated eyes. Cone pedicles are labeled by PNA too but less intensely (arrowheads). MERTK is localized in the microvilli of RPE cells. Especially at 6 weeks p.i. Close connections are visible between PNA‐positive cone photoreceptor outer segments and MERTK‐immunoreactive microvilli of hiPSC‐RPE cells (D). In one animal each at week 1 p.i. and week 2 p.i. We observed a close connection between cone outer segments and hiPSC‐RPE microvilli. Rod outer segments labeled by the rhodopsin antibody are present to some extent at all time points shown here (F’‐H’, F”–H”). Bright‐field images of the retinal implant region reveal heavy pigmentation of the hiPSC derived RPE cells and their close proximity to photoreceptor outer segments (F–H). hiPSC‐RPE = human iPSC derived RPE cells; RPE = retinal pigment epithelium, OS = outer segment, IS = inner segment, ONL = outer nuclear layer, OPL = outer plexiform layer. The scale bar in A, E”‐H” is 50 µm. Scale bars in B‐D, E‐H, E’‐H’ are 20 µm.