Factors supporting long-term culture of bovine male germ cells.
Factors supporting long-term culture of bovine male germ cells.
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DOI：
10.1071/rd15003
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发表时间：
2016-11
期刊：
影响因子：
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通讯作者：
Mahesh Sahare;Sung-min Kim;Ayagi Otomo;Kana Komatsu;N. Minami;M. Yamada;H. Imai
中科院分区：
文献类型：
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作者：
Mahesh Sahare;Sung-min Kim;Ayagi Otomo;Kana Komatsu;N. Minami;M. Yamada;H. Imai
精原干细胞（SSCs）本质上是单能性的，但小鼠SSCs在适当的培养条件下具有多能性。虽然培养系统可用于啮齿动物和人类生殖细胞系，但尚未证实可用于牲畜物种的培养系统。在这里，我们研究了生长因子、基质底物和无血清补充剂，以开发一种用于培养新生牛睾丸原始生殖细胞（性腺细胞）的确定系统。聚l -赖氨酸是一种合适的选择性抑制体细胞生长的底物，与明胶、胶原蛋白或双花凝集素（DBA）底物相比，它可以维持较高的性腺细胞：体细胞比例。在我们的培养基中测试的无血清补品中，在连续传代菌落后，敲除血清替代（KSR）比补品B-27和StemPro-SFM更能支持gonocytes的增殖和存活。在我们优化的培养条件下，在聚l -赖氨酸包被的培养皿上添加15% KSR，培养的性腺细胞的干细胞和生殖细胞潜能保持正常核型超过2个月（超过13代）。该培养系统能够维持牛生殖干细胞的增殖，可用于家畜SSC生物学和种系修饰的研究。
Spermatogonial stem cells (SSCs) are unipotent in nature, but mouse SSCs acquire pluripotency under the appropriate culture conditions. Although culture systems are available for rodent and human germ-cell lines, no proven culture system is yet available for livestock species. Here, we examined growth factors, matrix substrates and serum-free supplements to develop a defined system for culturing primitive germ cells (gonocytes) from neonatal bovine testis. Poly-L-lysine was a suitable substrate for selective inhibition of the growth of somatic cells and made it possible to maintain a higher gonocyte:somatic cell ratio than those maintained with gelatin, collagen or Dolichos biflorus agglutinin (DBA) substrates. Among the serum-free supplements tested in our culture medium, knockout serum replacement (KSR) supported the proliferation and survival of gonocytes better than the supplements B-27 and StemPro-SFM after sequential passages of colonies. Under our optimised culture conditions consisting of 15% KSR supplement on poly-L-lysine-coated dishes, the stem-cell and germ-cell potentials of the cultured gonocytes were maintained with normal karyotype for more than 2 months (over 13 passages). The proposed culture system, which can maintain a population of proliferating bovine germ stem cells, could be useful for studying SSC biology and germline modifications in livestock animals.