Agricultural College, Guangdong Ocean University, Zhanjiang, Guangdong, China.
Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
Reprod Domest Anim. 2024 Jul;59(7):e14661. doi: 10.1111/rda.14661.
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.
精原干细胞（SSC）是精子发生的基础，因此在保存稀有或濒危物种的生育能力和开发转基因动物和鸟类方面具有巨大的潜力。然而，开发最佳条件以在体外分离、培养和维持 SSC 仍然具有挑战性，特别是对于鸡而言。本研究的目的是：（1）确定在华美鸡中分离 SSC 的最佳年龄；（2）开发有效的分离、（3）富集和（4）培养分离的 SSC 方法。在本研究中，我们首先比较了使用 11 个不同年龄组（8-79 日龄）的华美鸡分离 SSC 的效率。我们发现 21 日龄鸡的睾丸产生的细胞活力最高。接下来，我们比较了两种不同的酶解组合用于分离 SSC，发现 0.125%胰蛋白酶和 0.02g/L EDTA 支持最高数量和活力的 SSC。接下来，我们研究了富集 SSC 的最佳条件，发现与 Percoll 梯度和磁性激活细胞分选方法相比，差速贴壁法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% FBS 与包括 GDNF（20ng/mL）、bFGF（30ng/mL）或 LIF（5ng/mL）在内的单个生长因子的组合最能支持 SSC 的增殖和集落形成。总之，通过酶消化从 21 日龄鸡的睾丸中最佳地分离 SSC，然后通过差速贴壁法进行富集。此外，在培养物中添加 2% FBS 和优化浓度的 GFNF、bFGF 或 LIF 可促进鸡 SSC 的增殖。
