Nuclear receptor subfamily 0, group B, member 1 (Nr0b1, also known as Dax1) is regarded as an important component of the transcription factor network that governs pluripotency in mouse embryonic stem (ES) cells. Here we generated inducible knockout ES cells for Nr0b1 using the Cre-loxP system and analyzed its precise function. We succeeded in establishing the Nr0b1-null ES cells and confirmed their pluripotency by showing their contribution to chimeric embryos. However, they proliferated slowly with over-expression of 2-cell stage specific transcripts including Zscan4c, which is known to be involved in telomere elongation in ES cells. We revealed that over-expression of Zscan4c prevents normal self-renewal by inducing arrest at G2 phase followed by cell death and that Nr0b1 directly represses the Zscan4c promoter. These data indicated that Nr0b1 is not essential to maintain pluripotency but is involved in the proper activation of 2-cell specific transcripts for self-renewal.

Recently, we successfully transplanted an autograft, or major histocompatibility complex (MHC)-matched allografts, from induced-pluripotent-stem-cell-derived retinal pigment epithelial (iPSC-RPE) cells in patients with age-related macular degeneration. However, there was an issue regarding immune rejection after transplantation. In this study, we established a preoperational in vitro “drug–lymphocytes–grafts immune reaction (Drug-LGIR)” test to determine the medication for immune rejection using host immunocompetent cells (lymphocytes) and transplant cells (target iPSC-RPE cells) together with different medications. The adequacy of the test was assessed by in vivo transplantation in monkey models together with medication based on in vitro data. In the results of Drug-LGIR tests, some drugs exhibited significant suppression of RPE cell-related allogeneic reactions, while other drugs did not, and the efficacy of each drug differed among the recipient monkeys. Based on the results of Drug-LGIR, we applied cyclosporine A or local steroid (triamcinolone) therapy to two monkeys, and successfully suppressed RPE-related immune rejections with RPE grafts, which survived without any signs of rejection under drug administration. We propose that our new preoperational in vitro Drug-LGIR test, which specifies the most efficacious medication for each recipient, is useful for controlling immune attacks with personalized treatment for each patient after retinal transplantation.

Currently, retinal pigment epithelium (RPE) transplantation includes sheet and single-cell transplantation, the latter of which includes cell death and may be highly immunogenic, and there are some issues to be improved in single-cell transplantation. Y-27632 is an inhibitor of Rho-associated protein kinase (ROCK), the downstream kinase of Rho. We herein investigated the effect of Y-27632 in vitro on retinal pigment epithelium derived from induced pluripotent stem cells (iPS-RPE cells), and also its effects in vivo on the transplantation of iPS-RPE cell suspensions. As a result, the addition of Y-27632 in vitro showed suppression of apoptosis, promotion of cell adhesion, and higher proliferation and pigmentation of iPS-RPE cells. Y-27632 also increased the viability of the transplant without showing obvious retinal toxicity in human iPS-RPE transplantation into monkey subretinal space in vivo. Therefore, it is possible that ROCK inhibitors can improve the engraftment of iPS-RPE cell suspensions after transplantation.

In patients with retinitis pigmentosa (RP), color fundus photography and fundus autofluorescence (FAF) have been used to estimate the disease progression. To understand the origin and the diagnostic interpretation of the fundus color and FAF, we performed in vivo imaging of fundus color and FAF together with histological analyses of the retinal degeneration process using the RP model mice, rd10. FAF partly represented the accumulation of microglia in the photoreceptor outer segments. Fundus whitening suggested the presence of apoptotic cells, which spatiotemporally preceded increase in FAF. We observed two patterns of FAF localization, arcuate and diffuse, each indicating different pattern of apoptosis, wavy and diffuse, respectively. Diffuse pattern of apoptosis was suppressed in dark-raised rd10 mice, in which outer nuclear layer (ONL) loss was significantly suppressed. The occupancy of FAF correlated with the thinning rate of the ONL. Fractalkine, a microglia chemotactic factor, was detected in apoptotic photoreceptors, suggesting chemokine-induced recruitment of microglia into the ONL, which paralleled with accelerated ONL loss and increased FAF occupancy. Thus, we propose that the degree of photoreceptor apoptosis and the rate of ONL thinning in RP patients might be read from the fundus color and the FAF.

Retinal ganglion cells (RGCs) are impaired in patients such as those with glaucoma and optic neuritis, resulting in permanent vision loss. To restore visual function, development of RGC transplantation therapy is now underway. Induced pluripotent stem cells (iPSCs) are an important source of RGCs for human allogeneic transplantation. We therefore analyzed the immunological characteristics of iPSC-derived RGCs (iPSC-RGCs) to evaluate the possibility of rejection after RGC transplantation. We first assessed the expression of human leukocyte antigen (HLA) molecules on iPSC-RGCs using immunostaining, and then evaluated the effects of iPSC-RGCs to activate lymphocytes using the mixed lymphocyte reaction (MLR) and iPSC-RGC co-cultures. We observed low expression of HLA class I and no expression of HLA class II molecules on iPSC-RGCs. We also found that iPSC-RGCs strongly suppressed various inflammatory immune cells including activated T-cells in the MLR assay and that transforming growth factor-β2 produced by iPSC-RGCs played a critical role in suppression of inflammatory cells in vitro. Our data suggest that iPSC-RGCs have low immunogenicity, and immunosuppressive capacity on lymphocytes. Our study will contribute to predicting immune attacks after RGC transplantation.