Establishment of Immunodeficient Retinal Degeneration Model Mice and Functional Maturation of Human ESC-Derived Retinal Sheets after Transplantation

Increasing demand for clinical retinal degeneration therapies featuring human ESC/iPSC-derived retinal tissue and cells warrants proof-of-concept studies. Here, we established two mouse models of end-stage retinal degeneration with immunodeficiency, NOG-rd1-2J and NOG-rd10, and characterized disease progress and immunodeficient status. We also transplanted human ESC-derived retinal sheets into NOG-rd1-2J and confirmed their long-term survival and maturation of the structured graft photoreceptor layer, without rejection or tumorigenesis. We recorded light responses from the host ganglion cells using a multi-electrode array system; this result was consistent with whole-mount immunostaining suggestive of host-graft synapse formation at the responding sites. This study demonstrates an application of our mouse models and provides a proof of concept for the clinical use of human ESC-derived retinal sheets

Proliferation potential of Müller glia after retinal damage varies between mouse strains.

Retinal Müller glia can serve as a source for regeneration of damaged retinal neurons in fish, birds and mammals. However, the proliferation rate of Müller glia has been reported to be low in the mammalian retina. To overcome this problem, growth factors and morphogens have been studied as potent promoters of Müller glial proliferation, but the molecular mechanisms that limit the proliferation of Müller glia in the mammalian retina remain unknown. In the present study, we found that the degree of damage-induced Müller glia proliferation varies across mouse strains. In mouse line 129×1/SvJ (129), there was a significantly larger proliferative response compared with that observed in C57BL/6 (B6) after photoreceptor cell death. Treatment with a Glycogen synthase kinase 3 (GSK3) inhibitor enhanced the proliferation of Müller glia in 129 but not in B6 mouse retinas. We therefore focused on the different gene expression patterns during retinal degeneration between B6 and 129. Expression levels of Cyclin D1 and Nestin correlated with the degree of Müller glial proliferation. A comparison of genome-wide gene expression between B6 and 129 showed that distinct sets of genes were upregulated in the retinas after damage, including immune response genes and chromatin remodeling factors

Expression of <i>Cyclin D1</i> was higher in 129 compared with B6 explants.

<p>(A–D) Immunostatining of Cyclin D1 (magenta) and GS (green) in retinal explants. Cyclin D1 was not detected in the control normal retina from B6 and 129 (A, B), but it accumulated in the nucleus of Müller glia in the retinal explants at 4DIV (arrows) (C, D). Several Cyclin D1 positive Müller glial nuclei were located in the OPL side (arrowheads). (E, F) Ki67 (magenta) was detected in the GS (green)-positive Müller glia only in the retinal explant from 129. (G, H) Cells expressing Ki67 (green) and Cyclin D1 (magenta) in a retinal explant from B6 in the ONL (arrowheads) (G) and 129 (arrow) (H). (I) Ki67 and Iba1 positive microglia in the ONL (open arrowheads). (J) Ki67 positive and Iba1 negative cells in the INL (arrow), and Ki67 positive and Iba1 positive cells in the ONL (open arrowhead). Brackets indicate OPL. Scale bar: 100 µm. (K) Number of cells positive for Cyclin D1 in the ONL and cells positive for Cyclin D1 and GS in the INL (OPL side) and the inner INL of the retinal explants. n.s.: not significant. (L) Relative expression levels of <i>Cyclin D1</i> and <i>Cyclin D3</i> transcripts in the retinal explants from B6 (black rectangle) and 129 (white square) at indicated time points.</p

Temporal expression of <i>Hmga2</i> and <i>Mbd1</i> transcripts in the retinal explants from B6 and 129.

<p>(A, D) Hmga2 was not detected in the normal retina of B6 and 129. (B, E) Hmga2 was detected in the INL of the retinal explants at 1.5 DIV from both B6 and 129 (arrows). (C, F) Hmga2 was maintained only in the INL of the mouse retinal explant from 129 at 3DIV (arrows). (G–L) Mbd1 was not detected in the normal retina of B6 and 129 (G, J) or in the retinal explant at 1.5 DIV (H, K), but it was detected at 3DIV in the INL of B6 retinal explants (arrows) (I). (L) Mbd1 expression was not detected in the retinal explant from 129. Scale bar: 100 µm.</p

Candidate proliferation repressors.

<p>Candidate proliferation repressors.</p

Retinal progenitor-like cells were generated in retinal explants from B6 and 129.

<p>Retinal explants from adult B6 (A, C, E,) and 129 (B, D, F,) mice were stained for indicated markers. (A, B) A portion of GS-positive Müller glia (green) expressed Nestin (magenta) in the retinal explants from B6 and 129 (arrows) at 4DIV. (C, D) Pax6 (magenta) was detected in the GS-positive Müller glia (green) (arrows) in the retinal explants from B6 and 129 at 4DIV. A number of Pax6-positive Müller glia were located in the ONL (arrowheads). (E, F) Cells in the INL co-expressed Pax6 (magenta) and Chx10 (green, arrows) in the retinal explants from B6 and 129 at 4DIV. An example of double-positive cells located in the ONL (arrowheads). (G) The number of Pax6 and Chx10 positive cells in the ONL and the INL. ***: p<0.005, n.s.: not significant. (H) Relative expression levels of <i>Nestin</i> transcripts at indicated time points. Brackets indicate OPL. Scale bar: 100 µm.</p

Gene expression profiles comparing normal retinas and retinal explants from B6 and 129.

<p>(A) Schematic diagram of the preparation of the retinal samples. Two retinas were prepared for each condition (1 to 6). (B) Expression profile of the 1000 most differentially expressed probe sets between the retinal explants from B6 and 129 at 3DIV. (C) Schematic diagram of the classification of the genes selected in (B). (D) Strategies to narrow down the candidate proliferation promoters and repressors.</p