Subretinal Implantation of Human Primary RPE Cells Cultured on Nanofibrous Membranes in Minipigs

Abstract

Purpose: The development of primary human retinal pigmented epithelium (hRPE) for clinical transplantation purposes on biodegradable scaffolds is indispensable. We hereby report the results of the subretinal implantation of hRPE cells on nanofibrous membranes in minipigs.

Methods: The hRPEs were collected from human cadaver donor eyes and cultivated on ultrathin nanofibrous carriers prepared via the electrospinning of poly(L-lactide-co-DL-lactide) (PDLLA). "Libechov" minipigs (12-36 months old) were used in the study, supported by preoperative tacrolimus immunosuppressive therapy. The subretinal implantation of the hRPE-nanofibrous carrier was conducted using general anesthesia via a custom-made injector during standard three-port 23-gauge vitrectomy, followed by silicone oil endotamponade. The observational period lasted 1, 2, 6 and 8 weeks, and included in vivo optical coherence tomography (OCT) of the retina, as well as post mortem immunohistochemistry using the following antibodies: HNAA and STEM121 (human cell markers); Bestrophin and CRALBP (hRPE cell markers); peanut agglutining (PNA) (cone photoreceptor marker); PKCα (rod bipolar marker); Vimentin, GFAP (macroglial markers); and Iba1 (microglial marker).

Results: The hRPEs assumed cobblestone morphology, persistent pigmentation and measurable trans-epithelial electrical resistance on the nanofibrous PDLLA carrier. The surgical delivery of the implants in the subretinal space of the immunosuppressed minipigs was successfully achieved and monitored by fundus imaging and OCT. The implanted hRPEs were positive for HNAA and STEM121 and were located between the minipig's neuroretina and RPE layers at week 2 post-implantation, which was gradually attenuated until week 8. The neuroretina over the implants showed rosette or hypertrophic reaction at week 6. The implanted cells expressed the typical RPE marker bestrophin throughout the whole observation period, and a gradual diminishing of the CRALBP expression in the area of implantation at week 8 post-implantation was observed. The transplanted hRPEs appeared not to form a confluent layer and were less capable of keeping the inner and outer retinal segments intact. The cone photoreceptors adjacent to the implant scaffold were unchanged initially, but underwent a gradual change in structure after hRPE implantation; the retina above and below the implant appeared relatively healthy. The glial reaction of the transplanted and host retina showed Vimentin and GFAP positivity from week 1 onward. Microglial activation appeared in the retinal area of the transplant early after the surgery, which seemed to move into the transplant area over time.

Conclusions: The differentiated hRPEs can serve as an alternative cell source for RPE replacement in animal studies. These cells can be cultivated on nanofibrous PDLLA and implanted subretinally into minipigs using standard 23-gauge vitrectomy and implantation injector. The hRPE-laden scaffolds demonstrated relatively good incorporation into the host retina over an eight-week observation period, with some indication of a gliotic scar formation, and a likely neuroinflammatory response in the transplanted area despite the use of immunosuppression.

Keywords: human primary RPE; minipigs; nanofibrous PDLLA membranes; subretinal implantation.

Figure 1

Nanofibrous carrier with embedded supporting PET frame. The carrier was cut from a cell culture insert with a modified punch. Three visible marks on the frame allow to control a side orientation of the carrier (A). Electron microscopy of the nanofibrous membrane (B).

Figure 2

Tacrolimus blood levels measured throughout the experimental studies in minipigs. The concentration of tacrolimus in the blood was examined on Days 5, 14, and 23 after initial application.

Figure 3

Morphology of the cultured hRPEs on the nanofibrous carrier. (A) Typical morphology of the cells between Days 15–44 (20× magnification, scale bar = 200 µm), (B) at Day 74 (20× magnification, scale bar = 200 µm), and (C) at Day 74 (4× magnification, scale bar = 1 mm). (D) Typical morphology of a cross-section of cultured hRPEs (10× magnification, scale bar = 400 µm), also found to be stained by DAPI (E).

Figure 4

Fundus (A) and Optical Coherence Tomography (B) imaging of the implanted hRPEs on a nanofibrous carrier into minipigs. (A,A¹,A²,A³) The star indicates the position of the implant in the superior nasal area of the retina. (B,B¹,B²,B³) White arrow highlights the location of the implant underneath the retina in two cross-sectional images of the retina. The strong red line indicates the reflection from the RPE, which seems to be thicker in the area of the transplant.

Figure 5

Hematoxylin & eosin staining of the retinal area containing the transplanted cells. Expression of Human Nuclear Antigen (HNAA, green) was observed in hRPE cells at week 1 and 2 post implantation and only occasionally at 6 weeks. Observations are presented at 1 (A,A’), 2 (B,B’), 6 (C,C’,D,D’), and 8 (E,E’) weeks as well as a control eye (F,F’). Nuclear staining was performed by DAPI (purple).

Figure 6

Expression of the human cell markers HNAA (green) and STEM121 (red) on hRPEs implanted on nanofibrous carriers and followed up to 8 weeks. Tissue was stained with HNAA (A–D) and STEM121 (E–H). Nuclear staining was performed by DAPI (purple and blue). ONL = outer nuclear layer.

Figure 7

Expression of the RPE cell markers bestropin (B–E) and cellular retinaldehyde binding protein (CRALBP, red) (G–J) in the treated minipigs continued for up to 8 weeks. (A,F) are images of the control eyes to demonstrate specificity of the staining. Immunofluorescence analysis of the given marker and its appropriate color is shown. Nuclear staining was performed by DAPI (purple in (A–E) and blue in (F–J)). ONL = outer nuclear layer, INL = inner nuclear layer.

Figure 8

Expression of the cone photoreceptor (peanut agglutinin, PNA) (B–E) and the rod bipolar (PKCα) (G–J) markers in the neuroretina underlying the hRPE cell implant followed up to 8 weeks. (A,F) are images of control eyes to demonstrate specificity of the staining. Immunofluorescence analysis of the given marker and its appropriate color is shown. Nuclear staining was performed by DAPI (purple in (A–E) and blue in (F–J)). Asterisks in B mark cone cell bodies that were slightly dislocated in the INL due to retinal detachment. Cp = cone pedicles, ONL = outer nuclear layer, INL = inner nuclear layer, OPL = outer plexiform layer, IPL = inner plexiform layer, GCL = ganglion cell layer.

Figure 9

Expression of macroglial (Vimentin, B–H, GFAP, J–M) and microglial (Iba1, Q–W) markers in the neuroretina underlying the hRPE cells implant followed up to 8 weeks. (A,I,P) are images of control eyes to demonstrate specificity of the staining. Immunofluorescence analysis of the given marker and its appropriate color is shown. Nuclear staining was performed by DAPI (purple in (A–H,P–W) and blue in (I–M)). OLM = outer limiting membrane, ONL = outer nuclear layer, INL = inner nuclear layer, OPL = outer plexiform layer, IPL = inner plexiform layer, GCL = ganglion cell layer.