Differentiation and characteristics of the enhanced green fluorescent protein gene transgenic goat neural stem cells cultured in attached and non‐attached plates

Abstract

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.

Isolation and propagation of neural stem cells in caprine (Capra hircus)

“…These NSCs have the capacity of self-renewal and differentiation (Zheng et al, 2010). Zheng et al (2011) succeeded in isolating stem cells from embryonic (day 98) foetal goat brain and transfecting EGFP gene into the cells. In vitro, piglet NSPs proliferated as neurospheres, expressed typical protein of neural progenitors, nestin, and a range of well-established neurodevelopmental markers.…”

“…Ara et al (2010) reported the characterisation of neural stem/progenitor cells (NSP) population from subventricular zone (SVZ) of neonatal piglet brain using in vivo and in vitro systems, and showed that the nestin and vimentin-positive neural progenitor cells are present in the SVZ of the lateral ventricles of neonatal piglet brain. Zheng et al (2011) showed that late stage foetus could be used for isolation of NSCs, which were characterised properly along with differentiation into cells of neural lineages. Zhao et al (2012) derived NSCs from enhanced green fluorescent protein (EGFP) transgenic porcine foetuses.…”

“…Zhao et al (2012) derived NSCs from enhanced green fluorescent protein (EGFP) transgenic porcine foetuses. Zheng et al (2011) succeeded in isolating stem cells from embryonic (day 98) foetal goat brain and transfecting EGFP gene into the cells. The transgenic NSCs could self-renewal, and grew as freefloating neurospheres with spindle-shaped cells.…”

“…The transgenic NSCs could self-renewal, and grew as freefloating neurospheres with spindle-shaped cells. Zheng et al (2011) showed that late stage foetus could be used for isolation of NSCs, which were characterised properly along with differentiation into cells of neural lineages. However, no transdifferentiation ability of these cells was tested.…”

Isolation and characterization of neural stem cells from ovine foetal cerebral cortex

“…Among domestic animals, isolation, propagation of these cells and their in vitro differentiation to neural cells were carried out by Zheng et al (2011) and Zhao et al . (2012) in porcine.…”