Morphological and Functional Evaluation of hESC-RPE Cell Suspension Injection in RCS Rats

Abstract

Purpose: : To evaluate whether transplantation of retinal pigment epithelial (RPE) cells derived from H9 human embryonic stem cells (hESC-RPE) delivered as cell suspension can maintain the RPE functionality and prevent photoreceptor degeneration in the dystrophic Royal College of Surgeons (RCS) rat. The study will also evaluate the immunological reactions of the host retina to non-polarized hESC-RPE.

Methods: : HESC-RPE cells were cultured for 4 weeks and digested into cell suspension at the cell density of 5.0x10⁷/ml. Subretinal injections of hESC-RPE cells (10⁵, 2µl) were performed in 28 to 31 day old RCS rats. Prednisolone was administered to all rats through drinking water (0.002mg/liter) for the entire period of study. Animals were sacrificed and eyes enucleated at 10 days (n=3), and 2 months (n=9) after surgery; tissue sections underwent histological evaluation using hemotoxylin and eosin (H&E) staining and immunostaining for RPE markers (RPE65), human marker (TRA-1-85), and immune response markers (CD3, CD68, GFAP). Visual functional evaluation was performed using optokinetic testing. Aperio Scanscope was used for measurement of outer nuclear layer thickness and performing cell counts.

Results: : Based on H&E staining, presence of pigmented cells was observed in all transplanted rats. In the 10 days post-transplantation group, hESC-RPE cells showed co-localized expression of TRA-1-85 and RPE-65 indicative of viable donor cells. In this group, transplanted cell clumps were surrounded by CD68 positive cells indicating macrophage activity. In the 2 months post-transplantation group, CD68 expression was observed mostly over the cell clumps, but TRA-1-85 and RPE-65 markers were not identified. Optokinetic testing suggested some improvement in the implanted eye; however the difference with the non-transplanted eye was not significant. Based on histological assays, no appreciable rescue of the outer retinal layer was observed in the transplanted area.

Conclusions: : Dissociated hESC-RPE cells do not survive long-term and do not function in the rat’s subretinal space. One possible mechanism for the accelerated loss of the transplanted hESC-RPE cells can be due to the increased host immune reaction to non-polarized hESC-RPE.

Keywords: retinal pigment epithelium • transplantation • age-related macular degeneration