Proliferation of small number of human spermatogonial stem cells obtained from azoospermic patients.
OBJECTIVE
This study aims to proliferate spermatogonial stem cells (SSCs) and compare the in-vitro effects of laminin and growth factors on the proliferation of adult human SSC.


METHODS
Isolated testicular cells were cultured in DMEM supplemented with 5 % fetal calf serum (FCS). During the culture, enriched spermatogonial cells were treated with a combination of glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and mouse leukemia inhibitory factor (LIF) in the presence or absence of human placental laminin-coated dishes. Cluster assay was performed during culture. Presence of spermatogonia was determined by an ultrastructural study of the cell clusters, reverse transcription polymerase chain reaction (RT-PCR) for spermatogonial markers and xenotransplantation to the testes of busulfan-treated recipient mice. Statistical significance between mean values was determined using statistical ANOVA tests.


RESULTS
The findings indicated that the addition of GDNF, bFGF, EGF and LIF on laminin-coated dishes significantly increased in-vitro spermatogonial cell cluster formation in comparison with the control group (p ≤ 0.001). The expression of spermatogonial markers was maintained throughout the culture period. Furthermore, a transplantation experiment showed the presence of SSC among the cultured cells. In addition, a transmission electron microscopy (TEM) study suggested the presence of spermatogonial cells of typical morphology among the cluster cells.


CONCLUSIONS
It can be concluded that human SSCs obtained from non-obstructive azoospermic (NOA) patients had the ability to self-renew in the culture system. This system can be used for the propagation of a small number of these cells from small biopsies.
Cellular and Molecular Research Center and Department of Anatomical Sciences, School of Medicine, Tehran University of Medical Sciences, Hemmat Highway, Tehran, Iran. skoruji@tums.ac.ir
Morteza
M
Koruji
Abdulhossein
A
Shahverdi
Arghavan
A
Janan
Abbas
A
Piryaei
Mohammad Reza
MR
Lakpour
Mohammad Ali
MA
Gilani Sedighi
D000704:Analysis of Variance; D000818:Animals; D053713:Azoospermia; D015415:Biomarkers / Q000737:chemistry; D001706:Biopsy; D002066:Busulfan / Q000494:pharmacology; D002452:Cell Count; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002478:Cells, Cultured; D003470:Culture Media / Q000737:chemistry; D004815:Epidermal Growth Factor / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000494:pharmacology; D006801:Humans; D007248:Infertility, Male / Q000473:pathology; D007797:Laminin / Q000494:pharmacology; D053605:Leukemia Inhibitory Factor / Q000494:pharmacology; D008297:Male; D051379:Mice; D046529:Microscopy, Electron, Transmission; D020133:Reverse Transcriptase Polymerase Chain Reaction; D044967:Serum / Q000737:chemistry; D013093:Spermatogonia / Q000166:cytology / Q000187:drug effects / Q000648:ultrastructure*; D033581:Stem Cell Transplantation / Q000379:methods; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000648:ultrastructure*; D013737:Testis / Q000737:chemistry / Q000166:cytology; D014183:Transplantation, Heterologous / Q000379:methods*
D015415:Biomarkers; D003470:Culture Media; D051100:Glial Cell Line-Derived Neurotrophic Factor; D007797:Laminin; D053605:Leukemia Inhibitory Factor; C507838:Lif protein, mouse; D016222:Fibroblast Growth Factor 2; D004815:Epidermal Growth Factor; D002066:Busulfan