RESUMO
Monkey embryonic stem (ES) cells are a useful tool for studying early human development and evaluating the efficacy of stem cell therapy. Monkey ES cells show closer similarity to human ES cells than their mouse counterparts regarding morphology, cell surface markers, and the maintenance of pluripotency, including the leukemia inhibitory factor requirement. The generation of genetically modified monkey ES cells with a biomarker such as green fluorescent protein, which allows noninvasive monitoring of progeny ES cells, is invaluable for the development of cell transplantation therapy and the study of differentiation mechanisms in primates. Here, we describe the generation of green fluorescent protein-expressing monkey ES cells using a conventional electroporation method.
Assuntos
Embrião de Mamíferos/citologia , Proteínas de Fluorescência Verde/genética , Macaca fascicularis/embriologia , Células-Tronco Pluripotentes/metabolismo , Animais , Técnicas de Cultura de Células/métodos , Criopreservação , Eletroporação/métodos , Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Camundongos , Células-Tronco Pluripotentes/citologia , Proteínas Recombinantes/genética , TransfecçãoRESUMO
The major limitation of nonhuman primate (NHP) embryonic stem (ES) cell research is inefficient genetic modification and limited knowledge of differentiation mechanisms. A genetically modified NHP-ES cell with biomarkers, such as green fluorescent protein (GFP), that allow noninvasive monitoring of transgenic cells, is a useful tool to study cell differentiation control during preimplantation and fetal development, which also plays a crucial role in the development of cell transplantation medicine. Here we report the establishment of transgenic NHP-ES cell lines that express GFP without jeopardizing their pluripotency, which was confirmed by in vitro and in vivo differentiation. These GFP-expressing ES cells reproducibly differentiated into embryoid bodies, neural cells, and cardiac myocytes. They formed teratoma composed of tissues derived from the three embryonic germ layers when transplanted into severe combined immunodeficient disease (SCID) mice. GFP expression was maintained in these differentiated cells, suggesting that these cells were useful for cell transplantation experiments. Furthermore, we showed that these ES cells have the ability to form chimeric blastocysts by introducing into the early preimplantation stage NHP embryo.
