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.
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).


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.
Lee
Dong Ryul
DR
Fertility Center of CHA General Hospital, CHA Research Institute, Pochon CHA University, 606-5 Yeoksam-dong, Gangnam-gu, Seoul, Korea.
Kim
Kye-Seong
KS
Yang
Yun Hee
YH
Oh
Hwa Soon
HS
Lee
Sook Hwan
SH
Chung
Tae Gyu
TG
Cho
Jung Hyun
JH
Kim
Hyun Joo
HJ
Yoon
Tae Ki
TK
Cha
Kwang Yul
KY
D018929:Cell Culture Techniques; D002454:Cell Differentiation; D049109:Cell Proliferation; D002469:Cell Separation; D006238:Haploidy; D006801:Humans; D019012:Integrin beta1 / Q000378:metabolism; D008297:Male; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D009845:Oligospermia / Q000473:pathology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013091:Spermatogenesis; D013094:Spermatozoa / Q000166:cytology* / Q000254:growth & development / Q000473:pathology; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism / Q000473:pathology; D013737:Testis / Q000473:pathology*
D019012:Integrin beta1; D050814:Octamer Transcription Factor-3