Abstract：
Spermatogonial stem cells (SSCs) comprise the foundation of spermatogenesis and hence have great potential for fertility preservation of rare or endangered species and the development of transgenic animals and birds. Yet, developing optimal conditions for the isolation, culture, and maintenance of SSCs in vitro remains challenging, especially for chicken. The objectives of this study were to (1) find the optimal age for SSC isolation in Huaixiang chicken, (2) develop efficient protocols for the isolation, (3) enrichment, and (4) culture of isolated SSCs. In the present study, we first compared the efficiency of SSC isolation using 11 different age groups (8-79 days of age) of Huaixiang chicken. We found that the testes of 21-day-old chicken yielded the highest cell viability. Next, we compared two different enzymatic combinations for isolating SSCs and found that 0.125% trypsin and 0.02 g/L EDTA supported the highest number and viability of SSCs. This was followed by investigating optimal conditions for the enrichment of SSCs, where we observed that differential plating had the highest enrichment efficiency compared to the Percoll gradient and magnetic-activated cell sorting methods. Lastly, to find the optimal culture conditions of SSCs, we compared adding different concentrations of foetal bovine serum (FBS; 2%, 5%, 7%, and 10%) and different concentrations of GDNF, bFGF, or LIF (5, 10, 20, or 30 ng/mL). We found that a combination of 2% FBS and individual growth factors, including GDNF (20 ng/mL), bFGF (30 ng/mL), or LIF (5 ng/mL), best supported the proliferation and colony formation of SSCs. In conclusion, SSCs can be optimally isolated through enzymatic digestion from testes of 21-day-old chicken, followed by enrichment using differential plating. Furthermore, adding 2% FBS and optimized concentrations of GFNF, bFGF, or LIF in the culture promotes the proliferation of chicken SSCs.
摘要：
精原干细胞（SSC）是精子发生的基础，因此在保护稀有或濒危物种的生育力以及开发转基因动物和鸟类方面具有巨大的潜力。然而，开发隔离的最佳条件，文化,在体外维持SSC仍然具有挑战性，尤其是鸡肉。本研究的目的是（1）找到怀乡鸡SSC分离的最佳年龄，(2)制定高效的隔离协议,(3)富集,和(4)分离的SSC的培养。在本研究中,我们首先比较了11个不同年龄组（8-79日龄）淮乡鸡的SSC分离效率。我们发现21日龄鸡的睾丸产生最高的细胞活力。接下来,我们比较了两种不同的酶组合来分离SSC，发现0.125％胰蛋白酶和0.02g/LEDTA支持最高的SSC数量和活力。随后是研究富集SSC的最佳条件，其中我们观察到，与Percoll梯度和磁激活细胞分选方法相比，差异铺板具有最高的富集效率。最后,为了找到SSC的最佳培养条件，我们比较了添加不同浓度的胎牛血清(FBS；2%，5%,7%,和10%)和不同浓度的GDNF，bFGF,或LIF（5、10、20或30ng/mL）。我们发现，2%的FBS和个体生长因子的组合，包括GDNF（20ng/mL），bFGF（30ng/mL），或LIF（5ng/mL），最好地支持了SSC的增殖和集落形成。总之,SSC可以通过酶消化从21天大的鸡的睾丸中最佳地分离，然后使用差异电镀进行富集。此外，添加2%FBS和优化浓度的GFNF，bFGF,或培养物中的LIF促进鸡SSC的增殖。
