Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in the Subretinal Space of the Cat Eye

Abstract

To develop biological approaches to restore vision, we developed a method of transplanting stem cell-derived retinal tissue into the subretinal space of a large-eye animal model (cat). Human embryonic stem cells (hESC) were differentiated to retinal organoids in a dish. hESC-derived retinal tissue was introduced into the subretinal space of wild-type cats following a pars plana vitrectomy. The cats were systemically immunosuppressed with either prednisolone or prednisolone plus cyclosporine A. The eyes were examined by fundoscopy and spectral-domain optical coherence tomography imaging for adverse effects due to the presence of the subretinal grafts. Immunohistochemistry was done with antibodies to retinal and human markers to delineate graft survival, differentiation, and integration into cat retina. We successfully delivered hESC-derived retinal tissue into the subretinal space of the cat eye. We observed strong infiltration of immune cells in the graft and surrounding tissue in the cats treated with prednisolone. In contrast, we showed better survival and low immune response to the graft in cats treated with prednisolone plus cyclosporine A. Immunohistochemistry with antibodies (STEM121, CALB2, DCX, and SMI-312) revealed large number of graft-derived fibers connecting the graft and the host. We also show presence of human-specific synaptophysin puncta in the cat retina. This work demonstrates feasibility of engrafting hESC-derived retinal tissue into the subretinal space of large-eye animal models. Transplanting retinal tissue in degenerating cat retina will enable rapid development of preclinical in vivo work focused on vision restoration.

Development of a protocol for maintaining viability while shipping organoid‐derived retinal tissue

“… Saturate 15 ml of retinal organoid medium (Singh et al, ) in a 5% CO 2 incubator for 1 hr using a 100‐mm tissue culture dish (rather than 15‐ml tube) to maximize saturation of media in the minimum amount of time (normoxia conditions were used, but percentage of oxygen inside the tissue culture incubator may vary; therefore, it is best to saturate media with percentage of oxygen and CO 2 used for the organoid culture). Transfer saturated media into 15‐ml tissue culture tube, add organoids, add basic fibroblast growth factor (bFGF) and brain‐derived neurotrophic factor (BDNF; 20 ng/ml each, Peprotech, Rocky Hills, NJ, USA, or R&D Systems, Minneapolis, MN, USA), close the cap, seal with parafilm (with the sterile side facing the tube), put a layer of prewarmed heat‐absorbing pouches (Cameron's Hot/Cold Gel Pak TM , Figure f‐3), and place the 15‐ml tube with organoids horizontally into the shipper (Figure f‐4). Place the SpotBot tracker (Figure f‐5) and one of the TempTale Ultra trackers (Figure f‐6) inside and activate them according to manufacturer's instructions. Place the other TempTale Ultra tracker outside the shipper (Figure f‐6) in the blue bin and activate. Cover with a cloth/insulation material, enabling easy temperature circulation within the shipper and preventing the tube from bouncing in the shipper. Insulate the shipper inside the crate (Figure f‐7) and especially the housing with robust layer of insulation (Figure f‐8) to reduce mechanical damage and battery drain. Remove retinal organoids (shaking slowly inside 37°C tissue culture incubator) at about 1 hr before the nearby FedEx shipping facility closes and ship by “priority overnight” service for delivery by 10:30 a.m. the next day (which is usually 9:00–9:30 a.m. the next day local time). We experimented with 7:30 a.m. delivery the next day (which is an option too) but found it unnecessary.…”

“…Developing preclinical vision restoration protocols based on transplanting stem cell–derived retinal tissue (Lin et al, ; Singh et al, ) or cells (Hambright et al, ) into the ocular space (subretinal or epiretinal) of experimental animals requires close coordination of work between the team generating the organoids and the team doing surgical grafting. Precise and reliable methods are needed, because (a) biological material is expensive and takes time to generate, (b) the surgical team, equipment, and surgical rooms are scheduled in advance and availability of all three takes time to plan, and (c) critically, the animals, especially those models with progressive retinal atrophy (Petersen‐Jones & Komaromy, ; Seiler et al, ), are scheduled for surgeries on specific days depending on the dynamics of retina degeneration and multiple experiments are usually done before each study is completed (with each cohort of animals being implanted at the same age, to be able to pool the data from multiple experiments).…”

“…1. Saturate 15 ml of retinal organoid medium (Singh et al, 2019) in a 5% CO 2 incubator for 1 hr using a 100-mm tissue culture dish (rather than 15-ml tube) to maximize saturation of media in the minimum amount of time (normoxia conditions were used, but percentage of oxygen inside the tissue culture incubator may vary; therefore, it is best to saturate media with percentage of oxygen and CO 2 used for the organoid culture).…”

“…Developing preclinical vision restoration protocols based on transplanting stem cell-derived retinal tissue (Lin et al, 2018;Singh et al, 2019) or cells (Hambright et al, 2012) are scheduled for surgeries on specific days depending on the dynamics of retina degeneration and multiple experiments are usually done before each study is completed (with each cohort of animals being implanted at the same age, to be able to pool the data from multiple experiments). Developing neural retinal tissue is sensitive to shipping conditions and needs to be transported quickly and in the optimal conditions to maintain viability ahead of transplantation.…”

“…We remove retinal organoids from a stem cell incubator at about 4:00–4:30 p.m. local time, place in a 15‐ml tube filled with culture medium (Singh, Occelli, Binette, Petersen‐Jones, & Nasonkin, ) pre‐saturated (for 1–2 hr, in the same incubator) with optimized gas concentrations (CO 2 and oxygen), place the tube in the shipping container (preequilibrated to reach 37°C), which is then placed in a shipping crate recognized by FedEx, with signs “do not X‐ray” and “keep in the upright position,” and deliver the crate to a nearby FedEx shipping facility about 30–45 min before the designated cutoff pickup time, which may vary between the locations and also depends on the shipping destination (see protocol below). We use three tracking devices: two inside the shipper—each reporting temperature conditions inside the shipper, and one additionally reporting the Global Positioning System (GPS) location and the g‐forces inside the shipper; one tracker is placed outside the shipper to monitor temperature conditions experienced by the 37°C shipping container inside the shipping crate.…”