Transplantation of human embryonic stem cell-derived photoreceptors restores some visual function in Crx-deficient mice

Abstract

Some of the most common causes of blindness involve the degeneration of photoreceptors in the neural retina; photoreceptor replacement therapy might restore some vision in these individuals. Embryonic stem cells (ESCs) could, in principle, provide a source of photoreceptors to repair the retina. We have previously shown that retinal progenitors can be efficiently derived from human ESCs. We now show that retinal cells derived from human ESCs will migrate into mouse retinas following intraocular injection, settle into the appropriate layers, and express markers for differentiated cells, including both rod and cone photoreceptor cells. After transplantation of the cells into the subretinal space of adult Crx(-/-) mice (a model of Leber's Congenital Amaurosis), the hESC-derived retinal cells differentiate into functional photoreceptors and restore light responses to the animals. These results demonstrate that hESCs can, in principle, be used for photoreceptor replacement therapies.

Fig.1

Intra-vitreal transplantation in neonatal mice. A shows the retina stained with recoverin (red) following transplantation of GFP-expressing human ES-derived retinal cells. The cells have migrated into all the layers of the retina (11μm confocal stack). Other sections labeled with HuC/D (red) (B,B′, 1μm confocal slice) indicate some transplanted cells develop amacrine cell characteristics, recoverin (red) (D,D′, 1μm confocal slice), and S-opsin (red)(E-E″′, 0.9μm confocal slice) consistent with photoreceptor differentiation. (E′-E″′) are zoomed in view of baxed region in (E). The * in D indicates a GFP expressing cell in the inner nuclear layer which is not recoverin positive. (C, C′) shows fluorescence microscope view of synaptophysin (red) expression in the synaptic terminals (arrowhead) of the transplanted photoreceptors (green). DAPI (blue) in C′ shows that typical condensed nuclear deposits in the transplanted cell nucleus (green in C, arrow) characteristic of photoreceptors. (GCL – ganglion cell layer, INL – inner nuclear layer, ONL – outer nuclear layer).

Fig.2

Subretinal transplantation in adult wild-type retina. A. GFP expressing human photoreceptors that have migrated into the ONL from the subretinal space. Cells show highly differentiated photoreceptor morphology with outer segments. The section is co-stained for Pax6 in red (1μm confocal slice). A′ shows a high-power view of the human photoreceptors. B, B′ show a similar image of transplanted mouse GFP+ retinal cells into wild-type mouse. Arrows in A′ and B′ indicate outer segments of the transplanted human and mouse photoreceptors respectively. (C-F) Additional examples of human ES-derived retinal cells transplanted to the subretinal space. Transplanted human cells expressed recoverin (C-C″″, red, 6 μm confocal stack for C) and rhodopsin (D (5 μm confocal stack), E-E″, F-F″, red). (C′-C″) show magnified view of the cell body with co-localization between GFP and recoverin in a 1μm confocal slice and (C″′, C″″) shows similar co-localization in the outer segment in a 1μm confocal. (E-E″) show magnified view for co-localization between rhodopsin and GFP for the cell marked by arrow in D in a 1μm confocal slice and (F-F″) show similar co-localization for the outer segments for the cell marked by arrowhead in a in a 1μm confocal slice.

Fig.3

Subretinal transplantation in adult Crx-/- retina. (A) Low-magnification view of transplanted human cells (green) in a Crx-/- retina. (B) Higher magnification image of the boxed region in A co-stained for recoverin (red) and GFP/NCAM-FITC (green). (C, C′, C″, 1μm confocal slice) Higher magnification view of the boxed region in B. (D,D′, D″, 1μm confocal slice) showing another example of a high magnification view of double-labeled cells for recoverin and GFP/NCAM-FITC. (E-E″) Coexpression of rhodopsin (red, E′, E″) and GFP and NCAM-FITC (green, E, E′) by the transplanted retinal cells (1.1μm confocal slice). (E-E″) Presence of human rods in the transplant site using of human-specific Nrl antibody (red, F′, F″). Note that a large number of human rod photoreceptors have downregulated their GFP (green, F, F′) upon differentiation (1μm confocal slice).

Fig.4

Functional integration of human cells in Crx-/- retina. (A-A′″)Synaptophysin expression in the synaptic terminal of the transplanted human cells. (A) is a low-power view stained for GFP (green), NCAM-FITC (green) and synaptophysin (red). A′-A′″ show high power view for separate and merged view of the boxed region in (A) showing co-localization (arrow, 0.9μm confocal slice). (B-B″) PSD95 expression in the terminals of transplanted cells. There is co-localization between PSD95 (red, Figure B, B′, B″) and GFP and NCAM-FITC (green, Figure B′, B″, 1μm confocal slice). (B″) shows a high power view of the boxed region in B′. (C) is a representative ERGs from a control non-transplanted eye having no response to light stimulus and (D) is averaged traces from the eye of the same animal which had received the transplanted human ES-derived retinal cells. (E) shows the B-wave amplitudes following light flash in control un-injected, sham surgery with no-subretinal transplant, subretinal transplanted human cells, subretinal transplanted wild-type mouse cells and finally traces from a normal wild-type mouse. Eyes that received subretinal human and mouse retinal cells were light-responsive (*** =p<0.0001). (F) shows a correlation analysis between size of transplanted area and corresponding B-wave response (r=0.8139, p<0.0001). (G, H) shows a similar strong correlation between number of Nrl cells per section (K, r=0.8537, p=0.0070) or total Nrl cells per eye (L, r=0.8977, p=0.0025) and the corresponding B-wave response.