Azizi Hossein, Skutella Thomas, Shahverdi Abdolhossein
Faculty of Biotechnology, Amol University of Special Modern Technologies, Amol, Iran.
Institute for Anatomy and Cell Biology, Medical Faculty, University of Heidelberg, Heidelberg, Germany.
Cell J. 2017 Jul-Sep;19(2):238-249. doi: 10.22074/cellj.2016.4184. Epub 2017 Feb 22.
The properties of self-renewal and division in spermatogonial stem cells (SSCs) support spermatogenesis. There is a number of reported methods for SSC culture systems. The development of a culture system that effectively supports isolation and selfrenewal of germline stem cells (GSCs) is of tremendous benefit for clinical trials, experimental research, and as potential treatment for male infertility. The current study aims to consider the cultivation and behavior of GSCs in a non-adherent culture system.
In this experimental study, we cultured testicular cells from neonatal mice in agarose coated plates in the presence of Dulbecco's modified Eagle's medium (DMEM) medium (CTRL group), 10% fetal bovine serum (FBS)+DMEM (10% group), and growth factor (G group) that contained 2% FBS, glial cell-derived neurotrophic factor (GDNF), epidermal growth factor (EGF), and fibroblast growth factor (FGF). Mouse spermatogonial stem-like colonies were isolated approximately 3 weeks after digestion of the testis tissue. After passages 2-3, the identity of the mouse spermatogonial stem-like cells was confirmed by immunocytochemistry, reverse transcription-polymerase chain reaction (RT-PCR), and flow cytometry against the germ cell markers and . The statistical significance between mean values in different groups was determined by one-way analysis of variance (ANOVA).
We observed spermatogonial stem-like colonies in the G and 10% groups, but not the CTRL group. Immunocytochemistry, flow cytometry, and RT-PCR confirmed expressions of germ cell markers in these cells. In the spermatogonial stem-like cells, we observed a significant expression (P<0.05) of germ cell markers in the G and 10% groups versus the testis cells (T). Their proliferative and apoptotic activities were examined by Ki67 and PI/annexin V-FITC. Alkaline phosphatase assay showed that mouse spermato- gonial stem-like colonies were partially positive.
A non-adherent culture system could provide a favorable method for short-term culture of spermatogonial stem-like cell colonies.
精原干细胞（SSCs）的自我更新和分裂特性支持精子发生。目前已有多种关于SSC培养系统的报道方法。开发一种能有效支持生殖系干细胞（GSCs）分离和自我更新的培养系统，对临床试验、实验研究以及作为男性不育的潜在治疗方法具有巨大益处。本研究旨在探讨GSCs在非贴壁培养系统中的培养及行为。
在本实验研究中，我们将新生小鼠的睾丸细胞在涂有琼脂糖的培养板中培养，分别添加杜氏改良 Eagle 培养基（DMEM）（对照组）、10%胎牛血清（FBS）+DMEM（10%组）以及含有2% FBS、胶质细胞源性神经营养因子（GDNF）、表皮生长因子（EGF）和成纤维细胞生长因子（FGF）的生长因子（G组）。在睾丸组织消化后约3周分离出小鼠精原干细胞样集落。传代2 - 3次后，通过免疫细胞化学、逆转录 - 聚合酶链反应（RT-PCR）以及针对生殖细胞标志物的流式细胞术，确认小鼠精原干细胞样细胞的身份。不同组均值之间的统计学显著性通过单因素方差分析（ANOVA）确定。
我们在G组和10%组中观察到了精原干细胞样集落，而对照组未观察到。免疫细胞化学、流式细胞术和RT-PCR证实了这些细胞中生殖细胞标志物的表达。在精原干细胞样细胞中，我们观察到G组和10%组与睾丸细胞（T）相比，生殖细胞标志物有显著表达（P<0.05）。通过Ki67和PI/膜联蛋白V - FITC检测了它们的增殖和凋亡活性。碱性磷酸酶检测显示小鼠精原干细胞样集落部分呈阳性。
非贴壁培养系统可为精原干细胞样细胞集落的短期培养提供一种良好的方法。
