Establishment of alternative culture method for spermatogonial stem cells using knockout serum replacement.
Establishment of alternative culture method for spermatogonial stem cells using knockout serum replacement.
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DOI：
10.1371/journal.pone.0077715
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Okada Y
中科院分区：
文献类型：
--
作者：
Aoshima K;Baba A;Makino Y;Okada Y
由于精原干细胞具有自我更新和向子代细胞分化的能力，因此建立高效的体外培养体系对精子发生相关的研究具有重要意义。尽管目前可用的系统是无血清的，并且只包含化学定义的成分，但它高度依赖于牛血清白蛋白(BSA)，这是一种与胎儿牛血清类似的、具有批次到批次质量变化的成分。因此，我们寻找了另一种保存SSCs特性的无BSA培养体系。在本研究中，我们使用SSC培养液中的敲除血清替代(KSR)作为BSA的替代品。结果表明，在饲养层细胞与小鼠胚胎成纤维细胞相结合的情况下，KSR支持SSC在体外的持续生长和体内无BSA的SSC活性。在KSR中添加BSA进一步促进了细胞周期的进展，而移植实验表明，添加BSA并不影响体内SSCs的数量。KSR和BSA联合使用还可以消除GFRA1和FGF2，并将GDNF浓度从20 ng/ml降至5 ng/ml，同时保持生长速度和SSC标志物的表达。此外，KSR也适用于非DBA/2菌株的SSCs，如C57BL/6和ICR。提示KSR是长期培养SSCs的一种有效的BSA替代物。因此，该方法适用于与SSCs相关的各种研究，包括精子发生和生殖干细胞生物学。
Since spermatogonial stem cells (SSCs) are capable of both self-renewal and differentiation to daughter cells for subsequent spermatogenesis, the development of an efficient in vitro culture system is essential for studies related to spermatogenesis. Although the currently available system is serum-free and contains only chemically-defined components, it highly relies upon bovine serum albumin (BSA), a component with batch-to-batch quality variations similar to those of fetal bovine serum. Thus, we searched for an alternative BSA-free culture system that preserved the properties of SSCs. In this study, we utilized Knockout Serum Replacement (KSR) in the SSC culture medium, as a substitute for BSA. The results demonstrated that KSR supported the continuous growth of SSCs in vitro and the SSC activity in vivo without BSA, in a feeder-cell combination with mouse embryonic fibroblasts. The addition of BSA to KSR further facilitated cell cycle progression, whereas a transplantation assay revealed that the addition of BSA did not affect the number of SSCs in vivo. The combination of KSR with BSA also allowed the elimination of GFRA1 and FGF2, and the reduction of the GDNF concentration from 20 ng/ml to 5 ng/ml, while maintaining the growth rate and the expression of SSC markers. Furthermore, KSR was also useful with SSCs from non-DBA/2 strains, such as C57BL/6 and ICR. These results suggested that KSR is an effective substitute for BSA for long-term in vitro cultures of SSCs. Therefore, this method is practical for various studies related to SSCs, including spermatogenesis and germ stem cell biology.
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影响因子：
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