Nguyen Huy-Hoang, Nhu Bui Le Quynh, Uyen Nguyen Nhat Phuong, Nguyen Van-Thuan, Bui Hong-Thuy
Cellular Reprogramming Laboratory, School of Biotechnology, International University, Vietnam National University, Ho Chi Minh City 700000, Vietnam.
J Reprod Dev. 2019 Oct 23;65(5):423-432. doi: 10.1262/jrd.2019-050. Epub 2019 Aug 4.
Historically, it had been widely accepted that the female mammalian ovary contained a limited number of oocytes that would reduce over time, without the possibility of replenishment. However, recent studies have suggested that female germline stem cells (FGSCs) could replenish the oocyte-pool in adults. The aim of this study was to isolate FGSCs from porcine ovaries and differentiate them into oocyte-like cells (OLCs). The FGSCs were successfully isolated from porcine ovarian tissue and cultured in vitro, in DMEM/F-12 medium supplemented with growth factors (EGF, FGF, GDNF, etc.) and a supplement (N21). These cells possessed spherical morphology and expressed specific germline characteristics (Vasa, Stella, Oct4, c-kit). By evaluating different conditions for in vitro differentiation of FGSCs, co-culturing the isolated FGSCs with MEF cells, under three-dimensional (3D) cell cultures, were shown to be optimal. FGSCs could successfully be differentiated into OLCs and reached about 70 µm in diameter, with a large number of surrounding somatic cells. Importantly, OLCs contained large nuclei, about 25-30 µm, with filamentous chromatin, similar to oocyte morphology, and expressed oocyte-specific markers (Gdf9, Zp2, SCP3, etc.) at the same level as oocytes. In conclusion, we successfully isolated FGSCs from porcine ovarian tissue and differentiated them into oocyte-like cells. This will provide a valuable model for studying a new, alternative source of oocytes.
从历史上看，人们普遍认为雌性哺乳动物卵巢中的卵母细胞数量有限，且会随着时间减少，无法补充。然而，最近的研究表明，雌性生殖系干细胞（FGSCs）可以补充成年动物的卵母细胞库。本研究的目的是从猪卵巢中分离FGSCs，并将其分化为卵母细胞样细胞（OLCs）。FGSCs成功地从猪卵巢组织中分离出来，并在添加了生长因子（EGF、FGF、GDNF等）和添加剂（N21）的DMEM/F-12培养基中进行体外培养。这些细胞具有球形形态，并表达特定的生殖系特征（Vasa、Stella、Oct4、c-kit）。通过评估FGSCs体外分化的不同条件，结果表明，在三维（3D）细胞培养下，将分离的FGSCs与MEF细胞共培养是最佳的。FGSCs能够成功分化为OLCs，直径约达70 µm，周围有大量体细胞。重要的是，OLCs含有大细胞核，约25-30 µm，有丝状染色质，类似于卵母细胞形态，并表达与卵母细胞相同水平的卵母细胞特异性标记物（Gdf9、Zp2、SCP3等）。总之，我们成功地从猪卵巢组织中分离出FGSCs，并将其分化为卵母细胞样细胞。这将为研究一种新的、替代性的卵母细胞来源提供有价值的模型。
