Department of Fisheries Biology, Pukyong National University, Busan 48513, Korea.
Department of Marine-Biomaterials and Aquaculture, Pukyong National University, Busan 48513, Korea.
Biomolecules. 2020 Oct 24;10(11):1477. doi: 10.3390/biom10111477.
Fish ovarian germline stem cells (OGSCs) have great potential in various biological fields due to their ability to generate large numbers of mature eggs. Therefore, selective enrichment of OGSCs is a prerequisite for successful applications. To determine the optimal conditions for the enrichment of OGSCs from Japanese medaka (), we evaluated the effects of Percoll density gradient centrifugation (PDGC), differential plating (DP), and a combination of both methods. Based on cell morphology and gene expression of germ cell-specific and OGSC-specific , we demonstrated that of seven density fractions obtained following PDGC, the 30-35% density fraction contained the highest proportion of OGSCs, and that Matrigel was the most effective biomolecule for the enrichment of OGSCs by DP in comparison to laminin, fibronectin, gelatin, and poly-l-lysine. Furthermore, we confirmed that PDGC and DP in combination significantly enhanced the efficiency of OGSC enrichment. The enriched cells were able to localize in the gonadal region at a higher efficiency compared to non-enriched ovarian cells when transplanted into the developing larvae. Our approach provides an efficient way to enrich OGSCs without using OGSC-specific surface markers or transgenic strains expressing OGSC-specific reporter proteins.
鱼类卵原生殖细胞（OGSCs）因其能够产生大量成熟卵子的能力，在各个生物领域具有巨大的潜力。因此，OGSCs 的选择性富集是成功应用的前提。为了确定从日本青鳉（）中富集 OGSCs 的最佳条件，我们评估了 Percoll 密度梯度离心（PDGC）、差速贴壁（DP）和这两种方法的组合对 OGSCs 富集的影响。基于细胞形态和生殖细胞特异性基因表达和 OGSC 特异性，我们证明在 PDGC 获得的七个密度级分中，30-35%的密度级分包含最高比例的 OGSCs，并且与层粘连蛋白、纤连蛋白、明胶和多聚赖氨酸相比，Matrigel 是 DP 富集 OGSCs 的最有效生物分子。此外，我们证实 PDGC 和 DP 的组合显著提高了 OGSC 富集的效率。与非富集的卵巢细胞相比，当移植到发育中的幼虫中时，富集的细胞能够以更高的效率定位于生殖区域。我们的方法提供了一种无需使用 OGSC 特异性表面标记或表达 OGSC 特异性报告蛋白的转基因株系来富集 OGSCs 的有效方法。
