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.

Keywords: human embryonic stem cells; large-eye animal models; retinal organoids; subretinal transplantation; synaptic connectivity; vision restoration.

FIG. 1.

Differentiation of hESCs to retinal tissue (retinal organoids) and immunocytochemical characterization of retinal organoids before transplantation. (a) Schematic of three-dimensional retinal organoid differentiation protocol. (b–f) Representative bright-field images of retinal differentiation stages in culture. (g–n) Immunocytochemistry of hESC-derived retinal tissue (9–10 weeks) with antibodies specific to PAX6, NEUROD1, CALB2, CHX10, OTX2, ZO-1, BLIMP1, CRX, and BRN3A. Insets in panel j represent the magnification of the area marked with asterisks. hESC, human embryonic stem cell.

FIG. 2.

Transplantation of hESC-derived retinal tissue into the subretinal space of wild-type cats and imaging of grafts. (a) A routine two-port partial 23-gauge vitrectomy (following lateral canthotomy and conjunctival peritomy) is performed. (b) Creating subretinal bleb using Balanced Salt Solution delivered by a RetinaJect subretinal injection cannula. (c) Organoids were loaded into the glass cannula using a syringe attached to the cannula. (d) Retinal organoids can be seen in the subretinal space (RetCam II imaging). Black arrows indicate the extent of the retinal bleb that was formed before subretinal transplantation. (e–f) SD-OCT images showing presence of grafts in the subretinal space. SD-OCT, spectral-domain optical coherence tomography.

FIG. 3.

Infiltration of Iba1- and CD8-positive cells in the subretinal grafts maintained with prednisolone immunosuppression. (a–l) In subjects 1 and 2, we observed many Iba1-positive and CD8-positive cells in the grafts and surrounding host tissue. Insets represent the magnification of the area marked with asterisks. HNu staining (red) shows the poor survival of grafts. Scale bar: 50 μm. CH, choroid; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.

FIG. 4.

Mild immune response to the grafts maintained with prednisolone + cyclosporine A immunosuppression. (a–l) In subjects 4 and 5, we observed few Iba1- and CD8-positive cells in the subretinal grafts and surrounding host tissue. HNu staining shows good survival of graft.

FIG. 5.

Cytoplasmic projections connecting the graft and the host tissue. (a) Immunohistochemical staining shows presence of CALB2 in the graft and the host tissue. (b) STEM121 staining was restricted to the grafts. In addition, we observe STEM121-positive projections emanating from the graft to the host ONL (*), INL (**), and RGC layers (***). (c) Co-immunostaining of cat sections with CALB2 and STEM121 shows the cytoplasmic projections are not co-localized. (d–f) High magnification of the area marked with an asterisk (*) marked in (b) shows the cytoplasmic projections positive for CALB2 and STEM121 do not colocalize. (g–i) High magnification of the area marked with triple asterisks (***) shown in (b). Arrows indicate the STEM121-positive projections contacting the cat RGC layer. Scale bar: 50 μm. RGC, retinal ganglion cell.

FIG. 6.

Synaptic interaction between the graft and the host tissue. Low magnification images demonstrate presence of SYP staining in the graft and in cat ONL adjacent to the graft. (a-a’) Shows co-labeling of cat retinal section immunostained with SYP and CALB2. (b-b’) High magnification images of the area marked with asterisks (*) and (**) in (a’), showing SYP-positive boutons in the graft and host ONL. Arrows indicate SYP-positive boutons in the graft. The inset in (b) is a high magnification of area marked with asterisk (***). Scale bar: 20 μm (b); 50 μm (b’). SYP, synaptophysin.