Rasmussen M A, Hall V J, Carter T F, Hyttel P
Department of Basic Animal and Veterinary Sciences, Faculty of Life Sciences, University of Copenhagen, Groennegaardsvej 7, DK-1870 Frederiksberg C, Denmark.
Stem Cell Res. 2011 Sep;7(2):124-36. doi: 10.1016/j.scr.2011.04.004. Epub 2011 May 7.
Neural progenitor cells (NPCs) are promising candidates for cell-based therapy of neurodegenerative diseases; however, safety concerns must be addressed through transplantation studies in large animal models, such as the pig. The aim of this study was to derive NPCs from porcine blastocysts and evaluate their in-vitro differentiation potential. Epiblasts were manually isolated from expanded hatched blastocysts and cultured on MEF feeder cells. Outgrowth colonies were passaged to MS5 cells and rosettes were further passaged to Matrigel-coated dishes containing bFGF and EGF. Three NPC lines were established which showed expression of SOX2, NESTIN and VIMENTIN. One line was characterised in more detail, retaining a normal karyotype and proliferating for more than three months in culture. Following differentiation, TUJI was significantly up-regulated in protocol 2 (RA and SHH; 58% positive cells) as were NF and TH. In contrast, MBP was significantly up-regulated in protocol 3 (FGF8 and SHH; 63% positive cells), whereas, GFAP was significantly up-regulated in protocols 1-4 (33%, 25%, 43% and 22%). The present study provides the first report of a porcine blastocyst-derived NPC line capable of differentiating into both neurons and glia, which may be of paramount importance for future transplantation studies in large animal models of neurodegenerative diseases.
神经祖细胞（NPCs）是神经退行性疾病细胞治疗中很有前景的候选细胞；然而，必须通过在大型动物模型（如猪）中的移植研究来解决安全问题。本研究的目的是从猪囊胚中获取NPCs，并评估它们的体外分化潜能。从扩张孵化的囊胚中手动分离出上胚层，并在MEF饲养细胞上培养。生长的集落被传代到MS5细胞上，玫瑰花结进一步传代到含有bFGF和EGF的基质胶包被的培养皿中。建立了三个NPC系，它们表现出SOX2、NESTIN和波形蛋白的表达。对其中一个系进行了更详细的表征，其保持正常核型并在培养中增殖超过三个月。分化后，在方案2（视黄酸和音猬因子；58%阳性细胞）中TUJI显著上调，NF和TH也是如此。相比之下，在方案3（FGF8和音猬因子；63%阳性细胞）中MBP显著上调，而在方案1 - 4（33%、25%、43%和22%）中GFAP显著上调。本研究首次报道了一种能够分化为神经元和神经胶质细胞的猪囊胚来源的NPC系，这对于未来神经退行性疾病大型动物模型的移植研究可能至关重要。
