Abstract
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.
Highlights
Spermatogonial stem cells (SSCs) are the first precursors to spermatogenic cells and are essential for continued spermatogenesis throughout the life span [1]
When cells prepared from germ cell-derived colonies (GDCs) in passage 0 were cultured with old feeder cells (OFCs), some colonies formed in passage 1 were smaller than those in passage 0 (Figure 2(B))
When the cells of colonies in passages 1, 3, 7, and were cultured with fresh feeder cells (FFCs), colony formation was observed in passages 2, 4, 8, and (Figures 2(G)–2(J)); OFCs were used in subculture for all the passages
Summary
Spermatogonial stem cells (SSCs) are the first precursors to spermatogenic cells (spermatocytes, spermatids, and spermatozoa) and are essential for continued spermatogenesis throughout the life span [1]. Owing to their small population, long-term culture-mediated enrichment of SSCs is important for investigating their self-renewal and differentiation. A 5-month culture of mice SSCs has been reported, where mouse embryonic fibroblasts (MEFs) were used as feeder cells [3]. Long-term culture of SSCs is difficult in domestic animals because of the unavailability of feeder cells (like MEFs or STO cells) for subculture, shortterm culture (
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