296 PRODUCTION OF HEMIZYGOUS AND HOMOZYGOUS EMBRYONIC STEM CELL-DERIVED NEURAL PROGENITOR CELLS FROM THE TRANSGENIC ALZHEIMER GÖTTINGEN MINIPIG

Alzheimer’s disease is the most prevalent cause of dementia and afflicts ∼26 million people worldwide. There are currently no cures for this disease. Production of in vitro models of the disease would be extremely useful for studying disease mechanisms and for potential screening of novel drugs. In this study we produced 2 hemizygote and 2 homozygote embryonic stem cell-derived neural progenitor cell lines from Day 8 transgenic blastocysts carrying a human gene linked to early-onset Alzheimer’s disease [Swedish mutation of the amyloid precursor protein (hAPPsw)]. Following onset of spontaneous oestrus, a mating of hAPPsw± × hAPPsw± Göttingen transgenic progeny was performed. Eight days after the first of 2 matings, embryos were flushed from the tip of both cornuas of the gilt under surgical anaesthesia. A total of 6 blastocysts were obtained and 7 corpora lutei recorded. Blastocysts were transported for 4 h in porcine zygote medium 3 (PZM-3) in hypoxic, humidified conditions at 39°C to the cell laboratory. Compact epiblasts were mechanically isolated from the embryo using insulin needles and cultured on inactivated mouse embryonic fibroblasts in embryonic stem cell medium, supplemented with 20 ng mL–1 human recombinant basic fibroblast growth factor (Prospec) and 20 ng mL–1 human recombinant Activin A (Prospec), for a period of 5 days in hypoxic conditions at 39°C. Five of the 6 epiblasts expanded to form embryonic stem-cell-like outgrowth colonies. These were cut into small colonies and plated on MS5 murine stromal cells to induce spontaneous neural differentiation in DMEM medium containing 15% knockout serum replacement. Neuronal rosette-like structures were identified from Day 10 of differentiation onward. Six rosette structures were mechanically isolated from 4 outgrowths and plated in serum-free conditions on Matrigel-coated dishes. Two of the 6 lines failed to proliferate beyond passage 2. The 4 remaining cell lines have currently been cultured to passage 7. These lines were analysed at passage 5 by comparative real-time PCR and found to be positive for the neural progenitor markers VIMENTIN, SOX2, NESTIN PAX6, MUSASHI; other neural markers BETAIIITUBULIN and NCAM; and the astrocyte marker, GFAP. These lines were also subjected to analysis by immunocytochemistry and found to express SOX2, VIMENTIN, and NESTIN. Further genotyping by comparative real-time PCR using primers designed to target the hAPPsw gene revealed that 2 lines carried a single copy of hAPPsw and 2 lines carried 2 copies of hAPPsw. The expression levels of the hAPPsw transgene in these cell lines were determined using quantitative PCR. These cell lines are currently being investigated for their ability to differentiate into cholinergic neurons and for their expression of hyperphosphorylated TAU and β-Amyloid secretion. These cell lines will be potentially relevant for the in vitro study of amyloid precursor protein accumulation in neural cells and its role in cell death, as well as for potential screening of novel drugs for Alzheimer’s disease.