RESUMEN
Fluorescent live imaging of cells and embryos at subcellular resolution poses significant challenges for biologists due to morbidity and mortality ensuing from phototoxicity. Here we report the use of a spinning-disk confocal microscope to image mouse and bovine preimplantation embryos without impairing their developmental potential. We also present data indicating that this imaging technique does not affect the functionality of subcellular components as assessed by reactive oxygen species (ROS) production, caspase activity, and DNA integrity. Spinning-disk confocal microscopy was also useful in determining cell number and allocation in transgenic bovine blastocysts. We conclude that this imaging method is suitable for monitoring preimplantation embryos.
Asunto(s)
Blastocisto/fisiología , Blastocisto/ultraestructura , Desarrollo Embrionario , Microscopía Confocal/métodos , Animales , Bovinos , Núcleo Celular/ultraestructura , Femenino , Ratones , Especies Reactivas de OxígenoRESUMEN
Induced pluripotent stem cells (iPSCs) have radically advanced the field of regenerative medicine by making possible the production of patient-specific pluripotent stem cells from adult individuals. While cell differentiation protocols have been successfully developed, and animal models of human disease have proved that these cells have the potential to treat human diseases and conditions produced as a consequence of aging, degeneration, injury, and birth defects, logistical issues still remain unsolved and hamper the possibility of testing these cells in human clinical trials. Among them is the widely spread use of animal products for the generation and culture of iPSCs. We report here a xeno-free iPSC generation system that addresses all the steps of iPSCs production including the isolation and culture of adult skin fibroblasts, and iPSCs generation, expansion, and maintenance. iPSCs generated with a polycistronic lentiviral vector under xeno-free conditions displayed markers of pluripotency and gave rise to embryoid bodies (EBs) displaying indicators of the 3 primary germ layers. Xeno-free iPSCs injected into nude mice produced classic teratomas, and teratoma explants cultured under conditions favoring fibroblastic cells gave rise to cells morphologically indistinguishable from input cells. Protocols here described will facilitate the implementation of new cellular therapies for preclinical and clinical studies, potentially reducing the regulatory burden without compromising the differentiation potential of the cells.
Asunto(s)
Fibroblastos/citología , Células Madre Pluripotentes Inducidas/citología , Animales , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Desdiferenciación Celular/genética , Diferenciación Celular/fisiología , Técnicas Citológicas/métodos , Cuerpos Embrioides/citología , Cuerpos Embrioides/metabolismo , Fibroblastos/metabolismo , Expresión Génica/genética , Vectores Genéticos/biosíntesis , Vectores Genéticos/genética , Estratos Germinativos/patología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/trasplante , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Lentivirus/genética , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Factor 5 Regulador Miogénico/metabolismo , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Antígenos Embrionarios Específico de Estadio/metabolismo , Teratoma/patología , Transducción GenéticaRESUMEN
The identification of genes and deduced pathways from the mature human oocyte can help us better understand oogenesis, folliculogenesis, fertilization, and embryonic development. Human metaphase II oocytes were used within minutes after removal from the ovary, and its transcriptome was compared with a reference sample consisting of a mixture of total RNA from 10 different normal human tissues not including the ovary. RNA amplification was performed by using a unique protocol. Affymetrix Human Genome U133 Plus 2.0 GeneChip arrays were used for hybridizations. Compared with reference samples, there were 5,331 transcripts significantly up-regulated and 7,074 transcripts significantly down-regulated in the oocyte. Of the oocyte up-regulated probe sets, 1,430 have unknown function. A core group of 66 transcripts was identified by intersecting significantly up-regulated genes of the human oocyte with those from the mouse oocyte and from human and mouse embryonic stem cells. GeneChip array results were validated using RT-PCR in a selected set of oocyte-specific genes. Within the up-regulated probe sets, the top overrepresented categories were related to RNA and protein metabolism, followed by DNA metabolism and chromatin modification. This report provides a comprehensive expression baseline of genes expressed in in vivo matured human oocytes. Further understanding of the biological role of these genes may expand our knowledge on meiotic cell cycle, fertilization, chromatin remodeling, lineage commitment, pluripotency, tissue regeneration, and morphogenesis.