Subretinal transplantation and survival of hESC-derived photoreceptor precursor cells in normal canine eyes

Abstract

Purpose : To inject fluorescently-labeled human embryonic stem cell (hESC)-derived photoreceptor precursor cells (PRPCs) in the subretinal space and assess their survival in normal dogs maintained under systemic immunosuppression.

Methods : Optic vesicle-like structures originating from a WA09 CRX^+/tdTomato hESC line were cultured for 108 to 132 days. Following mechanical dissociation or enzymatic digestion, aggregated or dissociated cells highly enriched in PRPCs expressing tdTomato were subretinally-delivered in 4 normal dogs (8 eyes) using a modified subretinal injector.
A Spectralis HRA/OCT unit and a modified Topcon fundus camera with specific exciter/emission filters were used to confirm the subretinal localization of tdTomato+ PRPCs, and monitor their survival over time. All dogs received a systemic triple immunosuppression regimen (Prednisone, Cyclosporine A, Mycophenolate Mofetil), which in one dog was interrupted 10 weeks before termination. Eyes were processed for immunohistochemistry at 11 days, 12 weeks, 22 weeks, or 32 weeks post-transplantation.

Results : WA09-hESC-PRPCs were successfully injected in the subretinal space of non-vitrectomized and vitrectomized normal eyes (6 and 2, respectively); however, some reflux into the vitreous was observed. Retinas reattached in 1-2 days but cell distribution within the injected area was primarily limited to the inferior border of the bleb. In all dogs maintained under immunosuppression, no clinical signs of transplant rejection were seen. Cells that had refluxed into the vitreous did not cause formation of epiretinal membranes nor tractional retinal detachments, and gradually disappeared with time. Cells in the subretinal space and tdTomato fluorescence persisted until termination with rare features of cell migration into the ONL and inner retina in one dog. PRPC survival was reduced in the one dog that was taken off immunosuppression, which also showed massive infiltration of the xenotransplant with CD4, CD8, and CD20 positive cells by IHC.

Conclusions : Successful subretinal delivery of WA09-hESC-PRPCs was achieved in normal dogs. Cell survival was seen in vivo for up to 22 weeks post-injection in immunosuppressed dogs. These results pave the way for evaluating delivery, survival, and integration of hESC-PRPCs in canine models with ongoing retinal degeneration.

This is a 2020 ARVO Annual Meeting abstract.