RESUMEN
We have isolated stem cells from amniotic fluid of goat at terminal gestational age and transferred the EGFP (enhanced green fluorescent protein) gene into the stem cells previously. The aim of this study was to determine whether the transgenic stem cells have the capability of multipotent differentiation. The transgenic stem cells were induced to differentiate into neurogenic, adipogenic, osteogenic and endothelial cells in vitro. Markers associated with AFS (amniotic fluid-derived stem) cells and the differentiated cells were tested by RT-PCR (reverse transcription-PCR). The results demonstrated that the transgenic AFS cells were capable of self-renewal, a defining property of stem cells. AFS cells were positive for the undifferentiated cell markers, Oct4, Nanog, Sox2 and Hes1, while following differentiation cells expressed markers for neurogenic cells such as astrocyte [GFAP (glial fibrillary acidic protein)] and NSE (neuron-specific enolase), adipogenic cells [LPL+ (lipoprotein lipase+)], osteogenic cells (osteocalcin+ and osteonectin+) and endothelium [CD34+ and eNOS+ (endothelial nitric oxide synthase)]. The results demonstrated that the EGFP gene transgenic AFS cells have the capability of multipotent differentiation, which means that the transgenic AFS cells may be useful in cell-transplantation studies in future.
Asunto(s)
Líquido Amniótico/citología , Diferenciación Celular , Proteínas Fluorescentes Verdes/genética , Células Madre/citología , Líquido Amniótico/metabolismo , Animales , Animales Modificados Genéticamente , Biomarcadores/metabolismo , Femenino , Cabras , Células Madre Multipotentes/citología , Embarazo , Células Madre/metabolismoRESUMEN
The aims of this study were (i) to determine whether NSCs (neural stem cells) could be isolated from the brain of embryonic day 98 fetal goat, (ii) to determine if these stem cells have the capability of multipotent differentiation following transfection with a reporter gene, EGFP (enhanced green fluorescent protein) and (iii) to study the characteristics of the stem cells cultured in attached and non-attached plates. NSCs were isolated from embryonic day 98 fetal goat brain, transfected with EGFP gene using lipofection, and subcultured in attached and non-attached plates respectively. The transgenic stem cells were induced to differentiate into osteogenic and endothelial cells in vitro respectively. Markers associated with undifferentiated NSCs and their differentiated cells were tested by RT-PCR (reverse transcription-PCR). The results demonstrated that stem cells could be isolated from embryonic day 98 fetal goat brain, and EGFP gene could be transfected into the cells. The transgenic NSCs were capable of self-renewal, a defining property of stem cells, and were grown as free-floating neurospheres in non-attached plates. When the neurospheres were transferred and cultured in attached plates, cells migrate from the neurospheres and are grown as spindle cells. The stem cells were grown as quasi-circular cells when the single stem cells were cultured in attached plates. Both the NSCs cultured in non-attached and attached plates could express Hes1 (hairy and enhancer of split 1), Oct4 (octamer-binding protein 4), Nanog, Sox2 [SRY (sex-determining region Y)-box 2] and Nestin, while following differentiation cells expressed markers for osteogenic cells (Osteocalcin+ and Osteonectin+) and endothelium (CD34+ and eNOS+). The results demonstrated that the goat EGFP gene transgenic NSCs have the capability of multipotent differentiation, which means that the transgenic NSCs may be useful in cell transplantation studies in future.
Asunto(s)
Diferenciación Celular/genética , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Proteínas Fluorescentes Verdes/genética , Células-Madre Neurales/citología , Animales , Antígenos CD34/biosíntesis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Técnicas de Cultivo de Célula , Línea Celular , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Cabras , Proteínas de Homeodominio/biosíntesis , Proteínas de Filamentos Intermediarios/biosíntesis , Liposomas , Proteínas del Tejido Nervioso/biosíntesis , Nestina , Células-Madre Neurales/metabolismo , Óxido Nítrico Sintasa de Tipo III/biosíntesis , Factor 3 de Transcripción de Unión a Octámeros/biosíntesis , Osteocalcina/biosíntesis , Osteonectina/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción SOXB1/biosíntesis , Transfección , TransgenesRESUMEN
We have obtained the EGFP (enhanced green fluorescence protein) gene transgenic porcine fetuses before. The aims of this study were (i) to determine whether stem cells could be isolated from amniotic fluid of the transgenic porcine fetuses, and (ii) to determine if these stem cells could express EGFP and differentiate in vitro. The results demonstrated that stem cells could be isolated from amniotic fluid of the EGFP gene transgenic porcine fetuses and could express EGFP and differentiate in vitro. Undifferentiated AFSs (amniotic fluid-derived stem cells) expressed POU5F1, THY1 and SOX2, while the following differentiation cells expressed markers for chondrogenic (COL2A1), osteogenic (osteocalcin and osteonectin) and neurogenic cells such as astrocyte (GFAP), oligodendrocyte (GALC) and neuron (NF, ENO2 and MAP).