Helical Fasciculation of Bipolar and Horizontal Cell Neurites for Wiring With Photoreceptors in Macaque and Mouse Retinas.

Purpose: The three-dimensional configurations of rod and cone bipolar cell (BC) dendrites and horizontal cell (HC) processes outside rod and cone synaptic terminals have not been fully elucidated. We reveal how these neurites are mutually arranged to coordinate formation and maintenance of the postsynaptic complex of ribbon synapses in mouse and monkey retinas. Methods: Serial section transmission electron microscopy was utilized to reconstruct BC and HC neurites in macaque monkey and mouse, including metabotropic glutamate receptor 6 (mGluR6)-knockout mice. Results: Starting from sporadically distributed branching points, rod BC and HC neurites (B and H, respectively) took specific paths to rod spherules by gradually adjusting their mutual positions, which resulted in a closed alternating pattern of H‒B‒H‒B neurites at the rod spherule aperture. This order corresponded to the array of elements constituting the postsynaptic complex of ribbon synapses. We identified novel helical coils of HC processes surrounding the rod BC dendrite in both mouse and macaque retinas, and these structures occurred more frequently in mGluR6-knockout than wild-type mouse retinas. Horizontal cell processes also formed hook-like protrusions that encircled cone BC and HC neurites below the cone pedicles in the macaque retina. Conclusions: Bipolar and horizontal cell neurites take specific paths to adjust their mutual positions at the rod spherule aperture. Some HC processes are helically coiled around rod BC dendrites or form hook-like protrusions around cone BC dendrites and HC processes. Loss of mGluR6 signaling may be one factor promoting unbalanced neurite growth and compensatory neurite coiling.

HLA-Matched Allogeneic iPS Cells-Derived RPE Transplantation for Macular Degeneration.

Immune attacks are key issues for cell transplantation. To assess the safety and the immune reactions after iPS cells-derived retinal pigment epithelium (iPS-RPE) transplantation, we transplanted HLA homozygote iPS-RPE cells established at an iPS bank in HLA-matched patients with exudative age-related macular degeneration. In addition, local steroids without immunosuppressive medications were administered. We monitored immune rejections by routine ocular examinations as well as by lymphocytes-graft cells immune reaction (LGIR) tests using graft RPE and the patient's blood cells. In all five of the cases that underwent iPS-RPE transplantation, the presence of graft cells was indicated by clumps or an area of increased pigmentation at 6 months, which became stable with no further abnormal growth in the graft during the 1-year observation period. Adverse events observed included corneal erosion, epiretinal membrane, retinal edema due to epiretinal membrane, elevated intraocular pressure, endophthalmitis, and mild immune rejection in the eye. In the one case exhibiting positive LGIR tests along with a slight fluid recurrence, we administrated local steroid therapy that subsequently resolved the suspected immune attacks. Although the cell delivery strategy must be further optimized, the present results suggest that it is possible to achieve stable survival and safety of iPS-RPE cell transplantation for a year.

Inhibition of T-cell activation by retinal pigment epithelial cells derived from induced pluripotent stem cells.

PURPOSE: The purpose of this study was to determine whether human retinal pigment epithelial (RPE) cells from induced pluripotent stem (iPS) cells could inhibit T-cell activation in vitro. METHODS: Cultured iPS-derived RPE (iPS-RPE) cells were established from fresh skin tissues or dental pulp cells obtained from healthy donors or a retinal patient after informed consent was obtained. To confirm expression of the specific markers on iPS and iPS-RPE cells, immunohistochemistry, quantitative RT-PCR (qRT-PCR), and flow cytometry were performed. Target T cells were obtained from peripheral blood mononuclear cells of healthy donors. Target T cells were assessed for proliferation by incorporation of bromodeoxyuridine or carboxyfluorescein succinimidyl ester for production of cytokines such as IFN-γ. Expression of TGFß and other candidate molecules by iPS-RPE cells was evaluated with flow cytometry, ELISA, multiplex cytokine array, immunohistochemistry, and qRT-PCR. RESULTS: The RPE cells we established from iPS cells had many characteristics of mature RPE cells but no characteristics of pluripotent stem cells. Cultured iPS-RPE cells inhibited cell proliferation and production of IFN-γ by activated CD4(+) T cells. In some bystander T cells, iPS-derived RPE cells induced CD25(+)Foxp3(+) regulatory T cells in vitro. Induced pluripotent stem-RPE cells constitutively expressed TGFß and suppressed activation of T cells via soluble TGFß, because TGFß-downregulated iPS-RPE cells did not inhibit this T-cell activation. CONCLUSIONS: Cultured iPS-derived retinal cells fully suppress T-cell activation. Transplantation of iPS-RPE cells into the eye might be a therapy for retinal disorders.

Use of lectins to enrich mouse ES-derived retinal progenitor cells for the purpose of transplantation therapy.

Using the mouse ES cell line with green fluorescent protein knocked-in at the Rx locus (Rx-KI ES cell), we previously showed that photoreceptors can be efficiently obtained in defined culture conditions by enriching Rx-positive retinal progenitor cells. We aimed to explore a protocol applicable for non-Rx-labeled stem cell lines for subsequent enrichment of retinal photoreceptor precursors for transplantation. The Rx-KI ES cell line was differentiated according to the serum-free suspension conditions with serum-free suspension/Dkk1/LeftyA/serum/activin method (SFEB/DLFA) described previously. Enrichment efficacy by negative selection was compared among 20 different lectins and the lectin combination that effectively enriched the Rx-positive cells by selecting the lectin low-binding population was determined. Subsequent differentiation efficiency to photoreceptor precursors and the contamination of Nanog or Oct3/4(+) cells in the culture were evaluated between the cell cultures using negative selection with lectins and Rx positive selection. The effect of cytarabine (Ara-C) for minimizing the contamination of undifferentiated cells after the selection was also studied. The combination of the lectins, wheat germ agglutinin (WGA), and Erythrina crista-galli agglutinin (ECA) enabled us to enrich the Rx-positive population by approximately twice the original Rx percentage. The selection also minimized the percentage of Oct3/4(+) cells. The lectin-selected cells produced a comparable percentage of Crx/rhodopsin-positive colonies with Rx-positive selection and were differentiated into photoreceptors. The Ara-C treatment on differentiating days 24-26 decreased Nanog and Oct3/4 expression in subsequent cultures. Enrichment of Rx-positive cells using WGA and ECA was comparable to Rx-positive selection, and the method could be applied to achieve efficient photoreceptor differentiation from other ES or iPS cell lines in which the Rx gene is not marked.