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    • OCT NANOG and THY are referred to as stem

    2018-10-22

    OCT4, NANOG, and THY1 are referred to as stem cell pluripotency markers. NANOG is expressed in porcine male germ cells, but OCT4 is not expressed in gonocytes of neonatal testes (Goel et al., 2008). However, OCT4 expression is detected in 2week cultures of PGP9.5 positive porcine spermatogonia from 10-week-old testes (Luo et al., 2006). Nevertheless, OCT4 expression in mouse gonocytes and undifferentiated SSCs has been reported (Pesce et al., 1998; Tadokoro et al., 2002), and the expression of OCT4 is essential to maintain mice spermatogonial stem Sulfo-NHS-Biotin cost in vitro and retain their self-renewal property (Dann et al., 2008). OCT4 and NANOG expression has also been detected in in vitro cultured bovine male germ cells, and transplantation of these cells results in stable colonization in the testes of recipient immune deficient mice (Fujihara et al., 2011). THY1 expression has been reported in undifferentiated spermatogonia and SSCs in rodent and non-human primates. In addition, THY1 positive human male germ cells that express OCT4 and NANOG, and stained with AP are maintained in vitro for 1week, and transplanting these cells into immune deficient nude mice does not cause tumor formation over at least 6months (Kobayashi et al., 2009). THY1 positive cells reportedly express VASA and the spermatogonial specific transcription factor PLZF in bovine male germ cells (Reding et al., 2010). Taken together, these results suggest that the expression of the pluripotency markers OCT4, NANOG, and THY1 is required to maintain spermatogonial germ cells in vitro, and that the porcine male germ cell markers are very conserved with other species but that the expression patterns of these markers need to be elucidated at specific spermatogenesis developmental stages. In the present study, pSGCs cultured at 31°C expressed PGP9.5, PLZF, OCT4 and NANOG; however, pSGCs cultured at 34 and 37°C expressed only PGP9.5 and PLZF, suggesting that the 31°C culture condition may be an optimal environment to maintain porcine spermatogonial germ cells. Unlike mice SSC cultured in vitro, long-term culture of porcine male germ cells has not been successfully conducted (Goel et al., 2008; Luo et al., 2006), and in vitro cultured porcine male germ cells have been maintained for only up to 1–3weeks. However, the present study showed stable maintenance of colonized pSGCs expressing both SSC and pluripotency marker genes over 60days at 31°C. Furthermore, long-term cultured pSGCs bound lectin DBA (Fig. 6C). Lectin DBA binding to germ cells is an indicator of the presence of germ cells (Goel et al., 2007) and germ cells with strong DBA binding are more primitive than those that weakly bind. Therefore, our results suggest that the low temperature culture condition may not only be suitable for the expression of pSGC molecular markers but is also important to maintain stem cell characteristics. Stem cells are typically cultured on feeder cells. We tried to derive and culture our SCCs on MEFs; however, no germ cell colonies were detected on MEFs at the three temperature conditions. pSGC colonies appeared only on porcine testicular somatic cells in Stempro 34 medium, suggesting that pSGCs may require signals from CD34 positive testicular cells for proliferation and maintenance of stem cell characteristics. Supporting this hypothesis, CD34 positive testicular stromal cells support long-term expansion of adult spermatogonial and progenitor cells (Kim et al., 2008) and spermatogonial stem cells can be proliferated and remain undifferentiated on bone marrow stromal cells (Xu et al., 2006). Additionally, bovine male germ cells have also been successfully cultured in vitro for 1.5months on bovine testicular somatic cells as a feeder, based on an unknown factor from the testicular somatic cells (Fujihara et al., 2011). In the present study, pSGCs were cultured over 60days with maintaining of both pluripotency and undifferentiated germ cell marker gene expressions. However detachment of pSGC colonies from the feeder cells and reduced colony formation after subculture was observed over 70days, and cultures were eventually limited. This may be due to the aging of feeder cells, and the aged feeder cells could not provide sufficient survival environment for pSGCs. Although the biochemical and cellular characteristics in between freshly isolated feeder cells and aged feeder cells have not been compared in this study, aged niches inhibited cell proliferation and initiated the loss of stemness marker gene expression in human embryonic and muscle stem cells (Carlson and Conboy, 2007). Therefore, identification and development of suitable feeder cell characteristics for pSGCs will be important to maintain undifferentiated spermatogonial germ cells for long-term culture.