Identification, culture and characterization of adult human spermatogonial stem cells in vitro

Abstract

OBJECTIVE: Recurring efforts have been devoted to developing germ cell cultures for basic and clinical research over recent decades. However, there remains much confusion, particularly concerning the fate of human germ cells in culture. The purpose of this study was to establish the culture conditions required to isolate, identify and expand adult human spermatogonial stem cells (SSCs). DESIGN: We developed a serum-free defined culture system for SSCs and identified potential markers of undifferentiated SSCs by immunofluorescence staining and RT-PCR assay. MATERIALS AND METHODS: Fourteen specimens of testis tissue were obtained from the donors aged 28-35 years. We examined the expression of c-Kit, CD90, GFRα-1, oct3/4 and MIS in cryosections of 6 samples of testicular tissue by immunohistochemical staining. For developing a model SSC culture system, we obtained enriched populations of SSCs by two-step enzymatic digestion and concentrated by Ficoll fractionation. This enriched population of SSCs was used to develop a culture system that consisted of serum-free defined medium and mouse embryonic fibroblasts (MEF) feeders, which routinely maintained stem cell activity for 2 weeks. Co-labelling for a germ cell marker (c-Kit), and a marker used to detect dividing cells (bromodeoxyuridine, BrdU), showed proliferation of germ cells. We evaluated the stem cell activity of cultured SSCs by RT-PCR and immunohistochemistry analysis for oct3/4, DAZL, VASA, STRA8, c-Kit, SCP3. RESULTS: Oct3/4+/c-Kit+/CD90- cells were found in the cords surrounding the periphery of the testis. Based on morphology and germ cell marker expression of c-Kit we suggest these cells are supposed to be SSCs. We analyzed the effect of in vitro environment on the maintenance of adult SSCs in a 15-day culture system. Although the number of adult SSCs decreased in a time-dependent manner, nearly two in three stem cells (62%) could be maintained in vitro for 2 weeks, and 24% of them were BrdU-labelled. The undifferentiation of SSCs was confirmed by the expression of oct3/4, DAZL, VASA and c-Kit. CONCLUSIONS: For the first time we have shown that SSCs cultures could be established from adult human testis by using a modified system composed of serum-free defined medium and MEF feeders. We also propose that oct3/4, DAZL, VASA and c-Kit may be the specific markers for undifferentiated SSCs.