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Hum. Reprod. Advance Access published online on October 6, 2005
Human Reproduction, doi:10.1093/humrep/dei319
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© The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Received May 25, 2005
Revised August 26, 2005
Accepted September 1, 2005

Article

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

Dong Ryul Lee 1*, Kye-Seong Kim 2, Yun Hee Yang 1, Hwa Soon Oh 1, Sook Hwan Lee 1, Tae Gyu Chung 1, Jung Hyun Cho 1, Hyun Joo Kim 1, Tae Ki Yoon 1, and Kwang Yul Cha 1

1 Fertility Center of CHA General Hospital, CHA Research Institute, Pochon CHA University, 606-5 Yeoksam-dong, Gangnam-gu, Seoul, 135-081
2 Department of Anatomy and Cell Biology, College of Medicine, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul, 133-791, Korea

* To whom correspondence should be addressed.
Dong Ryul Lee, E-mail: drleedr{at}cha.ac.kr


  Abstract

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.

Keywords: embryo production/germ stem cell-like cells/haploid male germ cells/non-obstructive azoospermic patients/spermatogenesis in vitro.
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