397 DERIVATION OF NEURAL STEM CELLS FROM PORCINE EPIBLAST CELLS

Abstract

The use of neural stem cells (NSC) has gained increased attention as a means of treating neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. However, before regenerative treatment of humans can be undertaken, safety studies of NSC using animal models are required. The mouse has been the model of choice so far; however, testing in larger mammals such as the pig is essential. The aim of this study was to derive NSC from porcine epiblast cells and to analyze these cells using neural stem cell markers. A total of 47 epiblasts were isolated from E9 porcine embryos and grown on mouse embryonic fibroblast cells in a porcine embryonic stem cell medium. After 5 days, 23 outgrowth colonies had formed (49%). Based on morphology, 8 outgrowth colonies were selected and cut into 63 smaller pieces, which were transferred to MS5 stromal cells in a serum replacement medium, and after an additional 12 days, rosette structures had formed. These structures were transferred to Matrigel-coated dishes in a neural stem cell medium containing EGF and FGF. Under such conditions, bipolar cells containing large nuclei and several nucleoli grew out from the rosettes. The bipolar cells have been expanded for more than 8 passages without any change in morphology or growth rate, and upon high-density culture, the cells spontaneously form floating neurospheres. Stainings revealed that the cells expressed the neural stem cell markers Nestin (100%), Sox2 (100%), Pax6 (100%), and Vimentin (100%), as well as the proliferation marker Ki67 (54%). The same markers were found to be expressed in the lateral ventricles of the developing porcine brain, a location known to have high neurogenic activity. When growth factors were withdrawn from the culture medium, a higher proportion of TujI expressing cells were observed, especially when cells were cultured as neurospheres. We conclude that it is possible to derive presumptive NSC from porcine epiblast cells and that these express the same markers as reported for human NSC. Further studies are required to determine if the cells can be cultured long term and differentiate into various neuronal and glial cell types.