earticle

영어

Jeju Black Cattle (JBC, endangered native Korean cattle), a species characterized by their pitch-black coat color, live in Jeju Special Self-Governing Province. Pluripotent stem cells (PSCs) derived from farm animals have potential for various applications in transgenic breeding, basic research and agriculture. However, there were a few reports for establishment of bovine induced PSC (iPSC) and their culture conditions were not optimized yet. In this study, JBC bull #5293 (date of birth, October 31, 2013) ear cells were transfected by retroviral vectors that include 5 pluripotent reprogramming factors, hOCT4, hSOX2, hKLF4, hc-MYC and hNANOG. The JBC cells were exposed to retrovirus in suspended state in a fibroblast culture medium (10% FBS+DMEM) for 3 h and then plated on culture dish (3×104 cells/60mm-dish). After three days, infected cells were dissociated and replated to a new feeder-free culture dish or mouse embryonic fibroblast (MEF) feeder cell dish (3×104 cells/60 mm-dish). And these cells were cultured in two different ES media (15% FBS+DMEM; 15% FBS or 20% SR+DMEM/F12; 20% SR) commonly supplemented with 5 ng/mL bFGF and 1,000 U LIF. Produced iPSC-like colony was separated by mechanical dissociation and transferred to a new MEF cell dish. In the result, cell aggregation before colony formation was predominantly appeared in 20% SR group compared with 15% FBS group, while the JBC cell growth was faster in 15% FBS group. In comparison of feeder-dependent or -free culture, differentiation was more frequently appeared in feeder-free condition. However, a iPSC-like colony was recovered in 15% FBS group under feeder-free culture. These iPSC-like colonies were maintained up to 3 passages. When we checked the pluripotent cell marker alkaline phosphatase (AP), the activity was strongly detected in 2nd passage JBC iPS-like cells. Further experiments were needed to find the better stable culture condition able to more passages culture (>15) for establishment of JBC-iPSCs.