Abstract:

【Objective】 Despite the increasing importance of the pig as a large animal model, little is known about the porcine neural stem cells (NSCs). 【Method】 To evaluate the markers expressed by NSCs, expression of EGFP by the cells and in vitro differentiation of the cells, brains were dissected from 30-day fetuses, enzymatically dissociated, and grown in the presence of epidermal growth factor, basic fibroblast growth factor etc. Porcine NSCs could be grown as suspended spheres. Plasmid containing EGFP gene was transfected into NSCs by lipofection and selected by G418. The positive NSCs were abtained. NSCs were induced to differentiate into cell types representing each embryonic germ layer, including cells of adipogenic, osteogenic, myogenic, endothelial, neuronal and chondrogenic lineages. 【Result】 Expanded NSCs and differentiated cells were banked or harvested for analysis using reverse transcription–polymerase chain reaction (RT-PCR). Cultured porcine NSCs widely expressed Nestin, NogoA, DCX, CyclinD2, CD133, Hes1, Oct4, CD-90, Nanog and Sox2. NSCs were differentiated into astrocyte (GFAP+), oligodendrocyte (GalC+), neuron (NF+、NSE+ and MAP2+), adipocyte (LPL+ and PPARγ-D+), osteoblast (Osteonectin+ and Osteocalcin+), myocyte (myf-5+, myf-6+ and myoD+), endothelium (CD31+, CD34+, CD144+ and eNOS+) and chondrogenic cells (COL2A1+). 【Conclusion】This study shows that porcine NSCs are broadly multipotent, thus, may be useful in porcine cell transplantation studies potentially leading to the application of this strategy in the setting of nervous system disease and injury.

Key words: neural stem cells')">neural stem cells, EGFP gene, markers, differentiation, porcine fetus