Proliferation of small number of human spermatogonial stem cells obtained from azoospermic patients

• DOI: 10.1007/s10815-012-9817-8
• Publication Date: 2012-06-27T12:57:33Z
• Authors: Morteza Koruji; Abdulhossein Shah...; Arghavan Janan; Abbas Piryaei; Mohammad Reza Lak...; Mohammad Ali Gila...
• Keywords: Male; 0301 basic medicine; Analysis of Variance; Epidermal Growth Factor; Biopsy; Cell Culture Techniques; Cell Count; Leukemia Inhibitory Factor; Culture Media; 3. Good health; Mice; 03 medical and health sciences; Animals; Humans; Fibroblast Growth Factor 2; Glial Cell Line-Derived Neurotrophic Factor; Laminin; Busulfan; Biomarkers; Cells, Cultured; Infertility, Male; Azoospermia; Cell Proliferation

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.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.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.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.