REPROGRAMMING OF PORCINE EPIBLAST-DERIVED NEURAL PROGENITOR CELLS TO PLURIPOTENCY

Abstract

Human induced pluripotent stem cells (iPSC) and neural progenitor cells (NPC) are envisioned to play a vital role in future cell replacement therapy. In this context, porcine iPSC and NPC would be highly useful for pre-clinical safety testing by autologous transplantation in a porcine biomedical model. 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 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 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 sub-cultured 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 latter resulted in establishment of new NPC lines. The system can be used to study mechanisms involved in the early transition from pluripotency to multipotency in the pig and the reversal of the process caused by reprogramming.

The Danish Agency for Science, Technology and Innovation, the Danish National Advanced Technology Foundation as well as the EU projects, EU FP7 Stem Cell Project “PartnErS” (218205; 204, 523) and EU FP7 Stem Cell Project “PluriSys” (223485).