Establishment and in vitro culture of porcine spermatogonial germ cells in low temperature culture conditions

Abstract

The objective of this study was to establish a porcine spermatogonial germ cell (pSGC) line and develop an in vitro culture system. Isolated total testicular cells (TTCs) from 5-day-old porcine testes were primary cultured at 31, 34, and 37 degrees C. Although the time of colony appearance was delayed at 31 degrees C, strong alkaline phosphatase staining, expressions of pluripotency marker genes such as OCT4, NANOG, and THY1, and the gene expressions of the undifferentiated germ cell markers PLZF and protein gene product 9.5 (PGP9.5) were identified compared to 34 and 37 degrees C. Cell cycle analysis for both pSGC and feeder cells at the three temperatures revealed that more pSGCs were in the G2/M phase at 31 degrees C than 37 degrees C at the subculture stage. In vitro, pSGCs could stably maintain undifferentiated germ cell and stem cell characteristics for over 60 days during culture at 31 degrees C. Xenotransplantation of pSGCs to immune deficient mice demonstrated a successful colonization and localization on the seminiferous tubule basement membrane in the recipient testes. In conclusion, pSGCs from neonatal porcine were successfully established and cultured for long periods under a low temperature culture environment in vitro. (C) 2013 Elsevier B.V. All rights reserved.