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Stem Cells Int. 2016;2016:6029271. doi: 10.1155/2016/6029271. Epub 2016 Jan 6.

Subculture of Germ Cell-Derived Colonies with GATA4-Positive Feeder Cells from Neonatal Pig Testes.

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Department of Animal Biotechnology, College of Animal Bioscience & Technology, Konkuk University, Seoul 143-701, Republic of Korea.
Department of Food Bioscience, College of Biomedical & Health Science, Konkuk University, Chungju 380-701, Republic of Korea.
Department of Biomedical Science, College of Life Science, CHA University, Seongnam 463-836, Republic of Korea.
Department of Animal Science, College of Agriculture, Chungbuk National University, Cheongju 361-763, Republic of Korea.


Enrichment of spermatogonial stem cells is important for studying their self-renewal and differentiation. Although germ cell-derived colonies (GDCs) have been successfully cultured from neonatal pig testicular cells under 31°C conditions, the short period of in vitro maintenance (<2 months) limited their application to further investigations. To develop a culture method that allows for in vitro maintenance of GDCs for long periods, we subcultured the GDCs with freshly prepared somatic cells from neonatal pig testes as feeder cells. The subcultured GDCs were maintained up to passage 13 with the fresh feeder cells (FFCs) and then frozen. Eight months later, the frozen GDCs could again form the colonies on FFCs as shown in passages 1 to 13. Immunocytochemistry data revealed that the FFCs expressed GATA-binding protein 4 (GATA4), which is also detected in the cells of neonatal testes and total testicular cells, and that the expression of GATA4 was decreased in used old feeder cells. The subcultured GDCs in each passage had germ and stem cell characteristics, and flow cytometric analyses revealed that ~60% of these cells were GFRα-1 positive. In conclusion, neonatal pig testes-derived GDCs can be maintained for long periods with GATA4-expressing testicular somatic cells.

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