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Human-induced pluripotent stem cells (iPSC) are envisioned to play a vital role in future cell replacement therapy. In this context, porcine iPSC would be highly useful for pre-clinical safety testing by autologous transplantation in a porcine biomedical model. However, a major impediment is that currently, continuous expression of reprogramming factors is required to maintain the pluripotent state of porcine iPSC. In the mouse, neural progenitor cells (NPC) have proved to be highly amenable to reprogramming due to their partly stem-like epigenetic state and expression of pluripotency-related genes such as SOX2. The objective of this study was to establish iPSC from porcine epiblast-derived NPC by use of a tetracycline-inducible Tet-ON approach. A total of 1.5 × 105 porcine NPC at passage 6 (Rasmussen et al. 2011) were transduced O/N with 0.5 mL of active virus containing the following porcine pluripotency genes: pOCT4 (pO); pOCT4 and pKLF4 (pOK); pOCT4 and pC-MYC (pOM); pOCT4, pC-MYC and pKLF4 (pOMK) or polycistronic pOCT4, pSOX2, pC-MYC and pKLF4 (pOSMK); all including 0.25 mL of transactivator (rtTA). After 3 days, the cells were trypsinized and passaged to MEF feeder cells and cultured in iPSC medium containing DMEM/F12, 20% KSR, 1% NEAA, 10 μM β-Me, 20 ng mL-1 human bFGF and 2 μg mL-1 doxycycline. On Day 8, tightly packed colonies of cells presenting an embryonic stem cell-like morphology were visible in the pOM, pOMK and pOSMK combinations. In contrast, colonies were not observed with the pO and pOK combination. On Day 14, several iPSC-like colonies were manually picked and subcultured on MEF feeder cells in iPSC medium. Two lines from the pOSMK combination were capable of prolonged clonal propagation while maintaining an ESC-like morphology. However, when doxycycline was removed from the culture medium, growth arrest and spontaneous differentiation occurred. The iPSC-like lines expressed OCT4, SOX2, C-MYC and KLF4, as evaluated by immunocytochemistry and expression of NANOG, SSEA-1 and SSEA-4 was also confirmed, demonstrating activation of endogenous pluripotency genes. The iPSC-like lines were capable of forming embryoid bodies (EB) without addition of doxycycline and in vitro differentiation of EB in medium containing DMEM and 15% FCS confirmed the presence of meso- (SMA) and endodermal (AFP) derivatives by immunocytochemistry. Furthermore, co-culture experiments with MS5 stromal cells in medium containing DMEM, 15% KSR and 150 ng mL-1 human Noggin resulted in differentiation into neuroectoderm (NESTIN and SOX2), as well as more mature neurons (TUJI and GFAP). The porcine iPSC-like lines could serve as an excellent platform for optimizing culture conditions, which may sustain the pluripotency network in the pig and could be applied for autologous stem cell transplantation in a porcine model for evaluation of safety and efficacy. The Danish Agency for Science, Technology and Innovation, the Danish National Advanced Technology Foundation as well as the EU projects, EU FP7 (PartnErS, PIAP-GA-2008-218205; PluriSys, HEALTH-2007-B-223485). [ABSTRACT FROM AUTHOR]

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 vitromodels 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 4h 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 20ngmL-1human recombinant basic fibroblast growth factor (Prospec) and 20ngmL-1human 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 BETAIIITUBULINand 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 vitrostudy 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. [ABSTRACT FROM AUTHOR]