Piravar Zeinab, Jeddi-Tehrani Mahmood, Sadeghi Mohammad Reza, Mohazzab Arash, Eidi Akram, Akhondi Mohammad Mehdi
Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
J Reprod Infertil. 2013 Jan;14(1):17-22.
Spermatogonial stem cells are subpopulation of spermatogonial cells in testis tissue that support beginning and maintenance of spermatogenesis. Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) could be a specific marker for identification of spermatogonial stem cells including spermatogonial sperm cells (SSCs) in testis tissue and during the culture; therefore we undertook this study to culture these human testicular stem cells (hTSCs) in vitro and approved the presence of human testicular stem cells (hTSCs) by UCHL1, also known as PGP9.5.
Enzymatic digestion of human testicular biopsies was done by collagenase IV (4 mg/ml) and trypsin (0.25%). Differential plating of testicular cells in DMEM/F12 and 10% FBS was applied for 16 hr. Floating cells were collected and transferred onto laminin-coated plates with Stem-Pro 34 media supplemented with growth factors of GDNF, bFGF, EGF and LIF to support self-renewal divisions; testicular stem cell clusters were passaged every 14 days for two months. Spermatogonial cells propagation was studied through Expression of UCHL1 in testis tissue and the entire testicular stem cell culture.
Testicular stem cell clusters from 10 patients with obstructive azoospermia were cultured on laminin-coated plates and subsequently propagated for two months. The average of harvested viable cells was approximately 89.6%. UCHL1 was expressed as specific marker in testicular stem cells entire the culture.
Human testicular stem cells could be obtained from human testicular tissue by a simple digestion, culturing and propagation method for long-term in vitro conditions. Propagation of these cells approved by specific marker UCHL1, during the culture period.
精原干细胞是睾丸组织中精原细胞的一个亚群，支持精子发生的起始和维持。泛素羧基末端水解酶L1（UCHL1）可能是鉴定睾丸组织及培养过程中包括精原干细胞（SSCs）在内的精原干细胞的特异性标志物；因此，我们开展本研究以体外培养这些人睾丸干细胞（hTSCs），并通过UCHL1（也称为PGP9.5）证实人睾丸干细胞（hTSCs）的存在。
用IV型胶原酶（4mg/ml）和胰蛋白酶（0.25%）对人睾丸活检组织进行酶消化。将睾丸细胞接种于含10%胎牛血清的DMEM/F12培养基中进行差异铺板16小时。收集悬浮细胞并转移至包被层粘连蛋白的培养板上，使用添加了胶质细胞源性神经营养因子（GDNF）、碱性成纤维细胞生长因子（bFGF）、表皮生长因子（EGF）和白血病抑制因子（LIF）等生长因子的Stem-Pro 34培养基以支持自我更新分裂；睾丸干细胞簇每14天传代一次，共传代两个月。通过UCHL1在睾丸组织及整个睾丸干细胞培养物中的表达研究精原细胞的增殖情况。
来自10例梗阻性无精子症患者的睾丸干细胞簇在包被层粘连蛋白的培养板上培养，随后传代两个月。收获的活细胞平均比例约为89.6%。UCHL1在整个培养过程中作为睾丸干细胞的特异性标志物表达。
通过简单的消化、培养和传代方法，可在体外长期条件下从人睾丸组织中获取人睾丸干细胞。在培养期间，这些细胞的传代通过特异性标志物UCHL1得以证实。
