Hum. Reprod. Advance Access originally published online on October 6, 2005
Human Reproduction 2006 21(2):471-476; doi:10.1093/humrep/dei319
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Isolation of male germ stem cell-like cells from testicular tissue of non-obstructive azoospermic patients and differentiation into haploid male germ cells in vitro
1 Fertility Center of CHA General Hospital, CHA Research Institute, Pochon CHA University, 606-5 Yeoksam-dong, Gangnam-gu, Seoul, 135-081 and 2 Department of Anatomy and Cell Biology, College of Medicine, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul, 133–791, Korea
3 To whom correspondence should be addressed at: Fertility Center of CHA General Hospital, CHA Research Institute, Pochon CHA University, 606-5 Yeoksam-dong, Gangnam-gu, Seoul, 135-081, Korea. E-mail: drleedr{at}cha.ac.kr
* D.R.Lee and K.-S.Kim contributed equally to this work.
BACKGROUND: The purpose of this study was to establish the culture conditions required to isolate, identify and expand male germ stem cell-like cells (GSC-LC) from the testicular tissue of patients with non-obstructive azoospermia (NOA). METHODS AND RESULTS: Testicular tissues obtained from patients (two with maturation arrest (MA, n = 2) and Sertoli cell-only syndrome (SCOS, n = 11) were dissociated and plated into gelatin-coated dishes. After 2–4 weeks, cultures from both MA patients (100%) and four SCOS patients (36.3%) exhibited multicellular colonies, which proliferated successfully until passage 10. GSC-LC in the colonies displayed alkaline phosphatase activity, as well as Oct-4 and integrin b1 expression after every passage. After the fifth passage, GSC-LC were differentiated by encapsulation in calcium alginate and further cultivation. At 2 and 6 weeks, cells expressed c-Kit, Scp3, testis-specific histone protein 2B (TH2B), and transition protein (TP)-1. Fluorescence in situ hybridization additionally disclosed a few tetraploid and haploid cells at 6 weeks. Human oocytes were activated in the absence of artificial activation and cleaved after the injection of presumptive spermatids. CONCLUSIONS: Our novel culture system may be useful for diagnosing the existence of germ cells and facilitating the treatment of NOA patients.
Key words: embryo production/germ stem cell-like cells/haploid male germ cells/non-obstructive azoospermic patients/spermatogenesis in vitro
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