We assessed the feasibility of transplanting a sheet of retinal pigment epithelial (RPE) cells differentiated from induced pluripotent stem cells (iPSCs) in a patient with neovascular age-related macular degeneration. The iPSCs were generated from skin fibroblasts obtained from two patients with advanced neovascular age-related macular degeneration and were differentiated into RPE cells. The RPE cells and the iPSCs from which they were derived were subject to extensive testing. A surgery that included the removal of the neovascular membrane and transplantation of the autologous iPSC-derived RPE cell sheet under the retina was performed in one of the patients. At 1 year after surgery, the transplanted sheet remained intact, best corrected visual acuity had not improved or worsened, and cystoid macular edema was present. (Funded by Highway Program for Realization of Regenerative Medicine and others; University Hospital Medical Information Network Clinical Trials Registry [UMIN-CTR] number, UMIN000011929 .).

We previously performed mutant screens in the medaka for defects in gonadal development and identified a mutant of interest in this regard, which was designated as hotei (hot). This mutant manifests a number of remarkable phenotypic abnormalities including: (i) excessive proliferation of germ cells that initiates at around the hatching stage regardless of the genetic sex of the fish; (ii) initiation of premature meiosis in phenotypically male hot homozygotes; (iii) one-half of the hot-homozygous XY fish undergo sex reversal, which accompanies the expression of the femalecharacteristic aromatase gene in the somatic cells of the gonad; and (iv) in phenotypically female homozygotes, follicular development is arrested at an early stage. We have also performed genetic mapping, chromosome walking, and candidate gene sequencing analysis of hot and demonstrate that the underlying mutation occurs in the recently identified medaka anti-Mü llerian hormone (Amh) receptor type II (amhrII) gene. Moreover, this gene was found to be responsible for each of the hot phenotypes, as an amhrII transgene rescues these abnormalities. In addition, the amhrII gene is expressed in the somatic cells of the gonads of both sexes. The phenotypes of the hot homozygotes indicate that there are multiple regulatory functions of the AMH/AMHRII signaling system in the development of the gonad, including the sexdependent regulation of germ cell proliferation and follicular development. These presumably represent the basic roles of Amh, which precede Mü llerian duct evolution during phylogeny.gonad ͉ sex differentiation ͉ hotei mutant

Basic studies of human pluripotential stem cells have advanced rapidly and stem cell products are now seeing therapeutic applications. However, questions remain regarding the tumorigenic potential of such cells. Here, we report the tumorigenic potential of induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) for the treatment of wet-type, age-related macular degeneration (AMD). First, immunodeficient mouse strains (nude, SCID, NOD-SCID and NOG) were tested for HeLa cells’ tumor-forming capacity by transplanting various cell doses subcutaneously with or without Matrigel. The 50% Tumor Producing Dose (TPD50 value) is the minimal dose of transplanted cells that generated tumors in 50% of animals. For HeLa cells, the TPD50 was the lowest when cells were embedded in Matrigel and transplanted into NOG mice (TPD50 = 101.1, n = 75). The TPD50 for undifferentiated iPSCs transplanted subcutaneously to NOG mice in Matrigel was 102.12; (n = 30). Based on these experiments, 1×106 iPSC-derived RPE were transplanted subcutaneously with Matrigel, and no tumor was found during 15 months of monitoring (n = 65). Next, to model clinical application, we assessed the tumor-forming potential of HeLa cells and iPSC 201B7 cells following subretinal transplantation of nude rats. The TPD50 for iPSCs was 104.73 (n = 20) and for HeLa cells 101.32 (n = 37) respectively. Next, the tumorigenicity of iPSC-derived RPE was tested in the subretinal space of nude rats by transplanting 0.8–1.5×104 iPSC-derived RPE in a collagen-lined (1 mm×1 mm) sheet. No tumor was found with iPSC-derived RPE sheets during 6–12 months of monitoring (n = 26). Considering the number of rodents used, the monitoring period, the sensitivity of detecting tumors via subcutaneous and subretinal administration routes and the incidence of tumor formation from the iPSC-derived RPE, we conclude that the tumorigenic potential of the iPSC-derived RPE was negligible.

The proliferation of germ cells becomes sexually dimorphic during gonadal sex differentiation, although the underlying dynamics of this are not well understood in vertebrates. By tracing GFP-labeled germ cells in vivo and analyzing the germ cell-depleted mutant, zenzai, we show that the proliferation and differentiation of germ cells are regulated in a sexually dimorphic manner in the teleost fish medaka. In the undifferentiated gonads, germ cells resume proliferation by slow intermittent division (type I), producing isolated daughter cells. While germ cells in the male gonads continue this mode of proliferation, some germ cell fractions in the female gonads initiate two to four rounds of continuous division (type II), forming cysts of four, eight, or sixteen cells, which subsequently enter meiosis synchronously. Thus, female germ cells become differentiated much earlier than do male germ cells. In the zenzai mutant, a defect in slow intermittent division eventually leads to the depletion of germ cells in the adult gonads in both sexes, despite the fact that cyst-forming division is unaffected. This argues that slow intermittent division is essential for the maintenance of germ cells. The proliferation and differentiation of germ cells are thus important components of gonadal sex differentiation in vertebrates.

A large-scale mutagenesis screen was performed in Medaka to identify genes acting in diverse developmental processes. Mutations were identified in homozygous F3 progeny derived from ENU-treated founder males. In addition to the morphological inspection of live embryos, other approaches were used to detect abnormalities in organogenesis and in specific cellular processes, including germ cell migration, nerve tract formation, sensory organ differentiation and DNA repair. Among 2031 embryonic lethal mutations identified, 312 causing defects in organogenesis were selected for further analyses. From these, 126 mutations were characterized genetically and assigned to 105 genes. The similarity of the development of Medaka and zebrafish facilitated the comparison of mutant phenotypes, which indicated that many mutations in Medaka cause unique phenotypes so far unrecorded in zebrafish. Even when mutations of the two fish species cause a similar phenotype such as one-eyed-pinhead or parachute, more genes were found in Medaka than in zebrafish that produced the same phenotype when mutated. These observations suggest that many Medaka mutants represent new genes and, therefore, are important complements to the collection of zebrafish mutants that have proven so valuable for exploring genomic function in development.