217 Induction and differentiation of porcine induced pluripotent stem cells into neuronal precursor cell-like cells

Abstract

The invitro cellular induction into pluripotency [induced pluripotent stem cell (iPSC) generation] has much evolved since its first report and may play a central role in veterinary regenerative medicine. In particular, the generation of invitro neural precursor cells (NPCs) enabled the creation of models for human genetic diseases, such as autism, schizophrenia, and Parkinson disease. The swine model is known to present many advantages over others, including the physiological similarities of pigs and humans. This project aimed to reprogram porcine embryonic fibroblasts (pEF) into iPSCs, and further differentiate them into NPCs. The iPSCs were generated through lentiviral transduction of OCT4, SOX2, c-MYC, and KLF4 genes and cultured with Dulbecco's modified Eagle's medium (DMEM)/F12 KO, 20% knockout serum replacement (KSR), nonessential amino acids, glutamine, β-mercaptoethanol, 10ngmL−1 basic fibroblast growth factor (bFGF), and antibiotics. Three clonal iPSCs lines with typical pluripotent morphology (high nuclear/cytoplasm ratio and well-defined colony borders) were alkaline phosphatase (AP) positive and were further analysed. All three lines [porcine (p)iPSCs lines 1, 2, and 3] expressed exogenous factors for at least 10 passages. Quantitative real-time (qRT)-PCR showed that line 1 expressed endogenous OCT4 and all three lines expressed NANOG. The pEFs were used as controls for 2−ΔΔCt analysis. piPSC line 1 was positive for OCT4, SOX2, NANOG, SSEA-1, TRA-1-60 through immunofluorescence; line 2 was negative for NANOG; and line 3 only presented SOX2 expression. piPSCs lines 2 and 3 differentiated into NPC-like cells. NPC induction was accomplished by 14 days of culture in commercial extracellular matrix (Geltrex) and neural induction media supplemented by SMAD inhibitors LDN193189 (0.2 μM) and SB431542 (10 μM), and then maintained at 20ngmL−1 of epidermal growth factor and basic fibroblast growth factor. They expressed NESTIN and GFAP by qRT-PCR and were positive for NESTIN, β-tubulin III and vimentin by immunofluorescence, confirming the NPC-like cell status. Interestingly, during all stages (pEF, iPSCs, and NPC-like cells), cells were positive to some degree for the neural markers tested by immunofluorescence. Flow cytometry analyses showed a 30% higher intensity of those markers at the NPC-like stage than the pEF and pluripotent stage. These results suggest preliminary evidence for neural differentiation. This will contribute to the use of the porcine model in future regenerative and translational medicine research. This study was funded by FAPESP (grant nos. 2015/26818-5, 2017/02159-8) and CAPES.