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In mammalian ovaries, many immature follicles remain after the dominant follicles undergo ovulation. Here we report the successful production of rabbit embryonic stem cells (ESCs) from oocytes produced by in vitro culture of immature follicles and subsequent in vitro maturation treatment. In total, we obtained 53 blastocysts from oocytes that received intracytoplasmic sperm injection followed by in vitro culture. Although only weak expression of POU5f1 was observed in the inner cell masses of in-vitro-cultured follicle-derived embryos, repeated careful cloning enabled establishment of 3 stable ESC lines. These ESC lines displayed the morphological characteristics of primed pluripotent stem cells. The ESC lines also expressed the pluripotent markers Nanog, POU5f1, and Sox2. Further, these ESCs could be differentiated into each of the 3 different germ layers both in vitro and in vivo. These results demonstrate that immature follicles from rabbits can be used to generate ESCs. Moreover, the use of rabbit oocytes as a cell source provides an experimental system that closely matches human reproductive and stem cell physiology.

Recently, an additional type of pluripotent stem cell-line derived from mouse embryos has been established and termed epiblast stem cell (EpiSC), and is expected to be an important tool for studying the mechanisms of maintenance of pluripotency since they depend on basic fibroblast growth factor-MAPK and Activin A-Smad2/3 signaling to maintain pluripotency, unlike mouse embryonic stem cells (ESCs). Further, because of the similarities between mouse EpiSCs and human ESCs, EpiSCs are expected to be effective experimental models for human stem cell therapy. Recently, study for conversion from ESC state to EpiSC state or reversion from EpiSC state to ESC state has attracted interest since these techniques may lead to increasing the potential of pluripotent stem cells and our knowledge about their developmental status. In the present study, we find that a low oxygen concentration in culture environment accelerated, improved, and stabilized the EpiSC state of the converted cells from the ESC state using Oct4ΔPE-GFP transgenic ESCs. Induced EpiSCs (iEpiSCs) in hypoxia possess closer gene expression patterns to native EpiSCs, and bisulfite sequences for the promoter regions of Stella and Oct4 genes have elucidated that the iEpiSC gain EpiSC-specific methylation patterns in hypoxia. Our data provide evidence that oxygen concentration is an important factor for establishment of the EpiSC-specific state.

Recently, several research groups have shown that germ cells can be produced in vitro from pluripotent embryonic stem cells (ESCs). In the mouse, live births of offspring using germ cells induced from ESCs in vitro have been reported. Furthermore, some efficient methods for inducing the useful number of germ cells from ESCs have also been developed. On the other hand, in primates, despite the appearances of germ cell-like cells including meiotic cells were observed by spontaneous differentiation or introducing transgenes, it has not been determined whether fully functional germ cells can be derived from ESCs. To elucidate the property for the germ cells induced from primate ESCs, specification of the promoting factors for the germ cell development and improving the efficiency of germ cell derivation are essential. Leukemia inhibitory factor (LIF) has been reported as one of the important factors for mouse primordial germ cell (PGC) survival in vitro. However, the effects of LIF on germ cell formation from pluripotent cells of primates have not been examined. The aim of this study is to determine whether LIF addition can improve in vitro germ cell production from cynomolgus monkey ESCs (cyESCs). After 8 days of differentiation, LIF added culture induced dome-shaped germ cell colonies as indicated by the intense expression of alkaline phosphatase activity (ALP). These cells also demonstrate high-level expression of the germ cell-marker VASA, OCT-4, and BLIMP-1, and show SSEA-1 expression that supports their early stage germ cell identity. Finally, we observed that adding LIF to differentiating cultures inhibited meiotic gene expressions and increased the percentage of ALP-positive cells, and demonstrate that the addition of LIF to differentiation media increases differentiation of early germ cells from the cyESCs.

Despite recent cell-engineering advances, treatment and repair of cartilage remains challenging. Although stem cell transplantation therapy using mesenchymal stem cells (MSCs) is considered a prominent strategy, the major problem of limited proliferative capacity of autologous cells has been unsolved. Recently, an induced pluripotent stem (iPS) cell line was suggested as an alternative way to cure various human diseases due to their potential proliferating infinitely while possessing the capacity to form all types of cells. However, the method to induce lineage-restricted differentiation has not been well examined or established. Here, we suggest a simple method to induce mesenchymal progenitors possessing chondrogenic property from mouse iPS cells. The MSC-like cells produced in our study expressed some MSC markers, and could also differentiate to osteoblast and adipocyte. The present study demonstrates the property of iPS cells as an alternative candidate for treatment of articular disorders, and suggests an effective approach for preparing chondrocyte from iPS cells.

Neural stem cells (NSCs) are tissue-specific stem cells with self-renewal potential in brain, and are committed cells of the central nervous system. Recently, some reports have suggested the possibility of the NSCs to differentiate into non-CNS mesodermal derivatives, such as blood cells and skeletal muscle cells. Here we isolated NSCs as neurospheres from a neonatal mouse brain using serum replacement medium, and demonstrated that the stem cell population expressing pluripotent-related genes such as Oct-4, Sox-2, and Nanog possess multiple differentiation potentials to ectodermal, mesodermal, and endodermal lineages, that is, some neural cells, beating cardiomyocytes, adipocytes, and insulin-producing cells. The results of the present study partly provide further evidence for multiple differentiation properties of NSCs and suggest common characteristics between NSCs and other pluripotent stem cells.

Embryonic stem cells (ESCs) are a good material for the study of mammalian development, production of genetically modified animals, and drug discovery because they proliferate infinitely while maintaining a multilineage differentiation potency and a normal karyotype. However, ethical considerations limit the use of human embryos for the establishment of ESCs. Recently, ESCs have been produced from blastomeres divided by biopsy in mice and humans. The method is expected to be less controversial because it does not destroy the embryo. However, no one has yet produced both a pup and an ESC from a single embryo. Here, we describe the production of individual/ESC pairs from each of three embryos out of 20 attempts, and is thus considered efficient. Blastomere-derived ESC could differentiate some types of tissues and contribute to chimera mouse. These results show that each blastomere at two-cell stage possesses pluripotency and separated blastomeres maintain viability to develop to a pup or pluripotent ESC.

Embryonic stem cells (ESCs) of nonhuman primates are important for research into human gametogenesis because of similarities between the embryos and fetuses of nonhuman primates and those of humans. Recently, the formation of germ cells from mouse ESCs in vitro has been reported. In this study, we established cynomolgus monkey ES cell lines (cyESCs) and attempted to induce their differentiation into germ cells to obtain further information on the development of primate germ cells by observing the markers specific to germ cells. Three cyESCs were newly established and confirmed to be pluripotent. When the cells are induced to differentiate, the transcripts of Vasa and some meiotic markers were expressed. VASA protein accumulated in differentiated cell clumps and VASA-positive cells gathered in clumps as the number of differentiation days increased. In the later stages, VASA-positive clumps coexpressed OCT-4, suggesting that these cells might correspond to early gonocytes at the postmigration stage. Furthermore, meiosis-specific gene expression was also observed. These results demonstrate that cyESCs can differentiate to developing germ cells such as primordial germ cells (PGCs) or more developed gonocytes in our differentiation systems, and may be a suitable model for studying the mechanisms of primate germ cell development.