RESUMO
Pluripotent stem cells can be established by various methods, but they share several cytological properties, including germ cell differentiation in vitro, independently of their origin. Although mouse induced pluripotent stem (iPS) cells can produce functional gametes in vivo, it is still unclear whether or not they have the ability to produce presumptive germ cells in vitro. Here, we show that mouse iPS cells derived from adult hepatocytes were able to differentiate into presumptive germ cells marked by mouse vasa homolog (Mvh) expression in feeder-free or suspension cultures. Embryoid body (EB) formation from iPS cells also induced the formation of round-shaped cells resembling immature oocytes. Mvh(+) cells formed clumps by co-aggregation with differentiation-supporting cells, and increased expression of germ cell markers was detected in these cell aggregates. Differentiation culture of presumptive germ cells from iPS cells could provide a conventional system for facilitating our understanding of the mechanisms underlying direct reprogramming and germline competency.
Assuntos
Células-Tronco Embrionárias/citologia , Hepatócitos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Diferenciação Celular , RNA Helicases DEAD-box/análise , RNA Helicases DEAD-box/genética , Corpos Embrioides/citologia , Células Germinativas/citologia , Masculino , Camundongos , Camundongos Transgênicos , Oócitos/citologia , RetroviridaeRESUMO
In a previous study, we established a system for visualizing the development of germ cells from mouse embryonic stem (ES) cells in culture using knock-in ES clones in which visual reporter genes were expressed from the mouse vasa homolog, Mvh. While assessing various culture conditions, we found that germ-cell formation was markedly depressed in low glucose medium. Using a repeated polymerase chain reaction (PCR) subtraction method, we identified genes that were differentially expressed in low versus high glucose media. Three genes that were predominantly expressed in high glucose medium, thioredoxin-interacting protein (Txnip), pituitary tumor-transforming gene 1 (Pttg), and RuvB-like protein 2 (RuvBl2), were further investigated. These genes were also found to be highly expressed in adult and embryonic gonads, and RuvBl2 in particular, which encodes an ATP-dependent DNA helicase, was specifically detected in the spermatocytes and spermatids of the adult testis as well as in primordial germ cells. Furthermore, using a green fluorescent protein (GFP) fusion construct, we found that RuvBl2 was expressed in both the nucleus and cytoplasm of testicular germ cells. These findings suggest a possible relationship between glucose metabolism and germ-cell development.
Assuntos
Proteínas de Transporte/genética , Diferenciação Celular/genética , DNA Helicases/genética , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Glucose/metabolismo , Óvulo/metabolismo , Espermatozoides/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Sequência de Aminoácidos , Animais , Proteínas de Transporte/biossíntese , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Linhagem Celular , RNA Helicases DEAD-box/biossíntese , RNA Helicases DEAD-box/genética , DNA Helicases/biossíntese , Células-Tronco Embrionárias/citologia , Glucose/fisiologia , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Óvulo/citologia , Securina , Espermatozoides/citologia , Testículo/metabolismo , Tiorredoxinas/biossíntese , Tiorredoxinas/genéticaRESUMO
Germ cell-specific ATP-dependent RNA helicase, the product of the mouse vasa homolog (Mvh), has been shown to play an essential role in the development of the male germ cell. In male Mvh knockout mice, premeiotic germ cells arrest at the zygotene stage. To investigate the role of MVH protein in the progression of meiosis, we searched for genes encoding partners that interact with MVH in testicular germ cells. Using the yeast two-hybrid system, we found that MVH interacts with mouse RanBPM, a Ran-GTP binding protein involved in microtubule nucleation. RanBPM is predominantly expressed in the testis, especially in maturating spermatocytes. Within the cell, RanBPM and MVH are closely associated with perinuclear RNA-protein complexes and chromatoid bodies. The interaction of MVH with RanBPM points to a functional relationship between translational regulation and the microtubule nucleation during meiosis. Mol. Reprod. Dev. 66: 1-7, 2004.