| Autor: |
Ahn Y; Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute for Agriculture and Life Science; College of Veterinary Medicine; Seoul National University, Seoul, South Korea., Jeong J; Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute for Agriculture and Life Science; College of Veterinary Medicine; Seoul National University, Seoul, South Korea., Choi KH; Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute for Agriculture and Life Science; College of Veterinary Medicine; Seoul National University, Seoul, South Korea.; Research and Development Center, Space F Corporation, Hwasung, South Korea., Lee DK; Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute for Agriculture and Life Science; College of Veterinary Medicine; Seoul National University, Seoul, South Korea.; Research and Development Center, Space F Corporation, Hwasung, South Korea., Lee M; Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute for Agriculture and Life Science; College of Veterinary Medicine; Seoul National University, Seoul, South Korea., Lee NY; Department of Veterinary Pathology, College of Veterinary Medicine; Seoul National University, Seoul, South Korea., Kim DY; Department of Veterinary Pathology, College of Veterinary Medicine; Seoul National University, Seoul, South Korea., Lee CK; Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute for Agriculture and Life Science; College of Veterinary Medicine; Seoul National University, Seoul, South Korea.; Institute of Green Bio Science and Technology, Seoul National University, Pyeongchang, South Korea. |
| Abstrakt: |
Feeder cells play a crucial role in maintaining the pluripotency of embryonic stem cells (ESCs) by secreting various extrinsic regulators, such as extracellular matrix (ECM) proteins and growth factors. Although primary mouse embryonic fibroblasts (MEFs) are the most widely used feeder cell type for the culture of ESCs, they have inevitable disadvantages such as batch-to-batch variation and labor-intensive isolation processes. Here, we revealed that the Sandoz inbred Swiss Mouse (SIM) thioguanine-resistant ouabain-resistant (STO) cell line, an immortalized cell line established from mouse SIM embryonic fibroblasts, can be used as a feeder layer for in vitro culture of authentic pig ESCs instead of primary MEFs. First, the expression of genes encoding ECM proteins and growth factors was analyzed to compare their secretory functions as feeder cells. Quantitative real-time polymerase chain reaction (qPCR) showed that the gene expression of these pluripotency-associated factors was downregulated in STO cells compared to primary MEFs of similar density. Therefore, subsequent optimization of the culture conditions was attempted using higher STO cell densities. Notably, pig ESCs cultured on STO cell density of 3 × (187,500 cells/cm 2 ) exhibited the most similar pluripotent state to pig ESCs cultured on primary MEF density of 1 × (62,500 cells/cm 2 ), as determined by alkaline phosphatase staining, qPCR, and immunocytochemistry. In addition, pig ESCs cultured on STO cell density of 3 × formed complex teratoma containing multiple types of tissues derived from all three germ layers. Our culture conditions using optimal STO cell density can be applied to fields requiring reproducible and scalable production of pig ESCs, such as preclinical research and cellular agriculture. |
