186 Improving Success Rate of Establishment and Maintenance of Porcine Induced Pluripotent Stem Cells by Investigation of Colony Morphology

Abstract

Induced pluripotent stem cells (iPS cells) are generated by reprogramming of somatic cells using ectopic introduction of 4 transcription factors, including OCT4, SOX2, KLF4, and c-MYC (OSKM). Fibroblast cells are the most commonly used as a primary cell source for iPS cells because they are easy to harvest and culture. However, reprogramming efficiency of porcine fibroblasts is poor (~0.1%). During reprogramming process, mixed populations of primary colonies become the major obstacle in iPS establishment. In this study, we characterised 2 different colony morphologies at colony pick-up (compact and loose iPS morphology). We hypothesised that compact colonies will proceed to long-term culture and pluripotency. The fibroblast cells were isolated from tails of piglets and transfected with retroviral vectors expressing OSKM. The primary colonies were counted on Day 7 after gene transduction. The iPS-like colonies were divided into compact (n = 10) and loose (n = 10) morphology at colony pick-up. The characteristics of iPS-like cell lines were analysed by morphology, alkaline phosphatase staining, G-banding karyotype, immunofluorescence staining (OCT4), pluripotent gene expression (RT-PCR), and embryoid body formation. A total of 1,697 iPS-like colonies (2.34%) were observed. The compact colonies contained with tightly packed cells with distinct border between iPS colony and feeder cells, while colonies with irregular shape and border were classified as loose colonies. These 2 types of iPS-like colonies expressed alkaline phosphatase and OCT4. A total 5 of 10 (50%) compact morphology cell lines could be maintained in the undifferentiated state for more than 50 passages. But only 3 of 10 (30%) loose morphology cell lines demonstrated pluripotent characteristics. G-Banding karyotype analysis revealed normal chromosome number (n = 38). All of the cell lines also expressed endogenous pluripotent genes, including OSKM and NANOG and formed three-dimensional aggregating masses. In this study, we found that the cell lines from compact morphology could be maintained for longer than those of the loose morphology. A high rate of differentiation of loose iPS colony may also indicate that this type of colony has different pluripotency signals or incomplete reprogramming compared with compact colony. In conclusion, selection of compact colony morphology at colony pick-up is simple and reflects long-term propagation of porcine iPS cell lines. This information is important for improving the success rate of establishment and maintenance of porcine iPS cells.