184 Establishment of Porcine Induced Pluripotent Stem Cell Lines by Adding LIN 28 Transcription Factor

Abstract

The establishment of porcine induced pluripotent stem cells (piPSC) is important in the field of human biomedical regenerative medicine. The pig model is a more representative model than current rodent models because it better mimics human physiology in different organ systems. The piPSC can be traditionally generated by reprogramming somatic cells using 4 transcription factors (4TF: OCT4, SOX2, KLF4, and c-MYC), similarly in human. However, it is difficult to maintain the pluripotent state of reprogrammed cells and they exhibit poor differentiation capacity. Hence, the 4TF may be not enough to reprogram porcine somatic cells. This study aimed to establish piPSC by adding LIN28 (referred to as 5TF) to the traditional 4TF, via retroviral vector. Here, we report the successful establishment of 3 piPSC lines by using the 5TF. All 5TF-piPSC lines exhibited a normal karyotype (38, XY) and a typical mouse embryonic stem cell (ESC) morphology, including tightly packed and dome-like shape, even after they were propagated over 40 passages. All 5TF-piPSC lines were positive for alkaline phosphatase staining and expressed high levels of ESC-like markers (OCT4, SOX2, NANOG, and SSEA-1). Importantly, the 5TF-piPSC lines showed pluripotent capacity, as evidenced by differentiation into 3 germ layers in vitro following cystic embryoid body formation, as well as by efficiently forming teratomas containing all 3 embryonic germ layers in vivo. Moreover, the 5TF-piPSC lines showed spontaneously contractile cardiomyocytes and expressed cardiomyocyte markers (cardiac troponin T) during spontaneous cardiac differentiation using cell aggregation into spherical-like structures referred to as embryoid bodies. Thus, the addition of LIN28 TF promoted long-term pluripotency of piPSC and enhanced the ability to differentiate towards 3 embryonic germ layers and cardiac lineage. This research project is supported by grants from the Mahidol University, Thailand.