Lai Dongmei, Xu Minhua, Zhang Qiuwan, Chen Yifei, Li Ting, Wang Qian, Gao Yimeng, Wei Chunsheng
The Center of Research Laboratory, The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, 200030, China.
State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China.
Stem Cell Res Ther. 2015 Feb 20;6(1):13. doi: 10.1186/s13287-015-0004-6.
Follicular fluid is important for follicular development and oocyte maturation. Evidence suggests that follicular fluid is not only rich in proteins but cells. Besides oocytes, the follicular fluid contains granulosa, thecal, and ovarian surface epithelial cells, and both granulosa and thecal cells are well-characterized. However, epithelial cells in follicular fluid are poorly studied. This study aims to isolate and characterize in vitro epithelial cells that originate from human ovarian follicular fluid retrieved in the assisted fertilization program.
Follicular fluid samples were collected from 20 women in the assisted reproduction program. Epithelial cells were characterized by flow cytometry assay, immunofluorescence staining, real-time PCR, and time lapse photography.
Epithelial cell cultures were established from 18 samples. A small population of epithelial cells expresses germ-line stem cell markers, such as octamer-binding transcription factor 4 (OCT4), NANOG, and DEAD box polypeptide 4 (DDX4). In the epithelial cell culture system, oocyte-like cells formed spontaneously in vitro and expressed the following transcription markers: deleted in azoospermia-like (DAZL), developmental pluripotency associated protein 3 stella-related protein (STELLA), zona pellucida gene family C (ZPC), Syntaptonemal complex protein (SCP), and growth and differentiation factor 9 (GDF9). Some of the oocyte-like cells developed a zona pellucida-like structure. Both the symmetric and asymmetric division split of epithelial cells and early developing oocytes were observed using time lapse photography. Cell colonies were formed during epithelial culturing, which maintained and proliferated in an undifferentiated way on the feeder layer and expressed some pluripotency markers. These colonies differentiated in vitro into various somatic cell types in all three germ layers, but did not form teratoma when injected into immunodeficient mice. Furthermore, these epithelial cells could be differentiated directly to functional hepatocyte-like cells, which do not exist in ovarian tissues.
The epithelial cells derived from follicular fluid are a potential stem cell source with a pluripotent/multipotent character for safe application in oogenesis and regenerative medicine.
卵泡液对卵泡发育和卵母细胞成熟至关重要。有证据表明，卵泡液不仅富含蛋白质，还含有细胞。除了卵母细胞外，卵泡液还包含颗粒细胞、卵泡膜细胞和卵巢表面上皮细胞，其中颗粒细胞和卵泡膜细胞已得到充分表征。然而，卵泡液中的上皮细胞研究较少。本研究旨在分离并在体外鉴定源自辅助生殖项目中获取的人卵巢卵泡液的上皮细胞。
从20名参与辅助生殖项目的女性中收集卵泡液样本。通过流式细胞术检测、免疫荧光染色、实时聚合酶链反应和延时摄影对上皮细胞进行表征。
从18个样本中建立了上皮细胞培养体系。一小部分上皮细胞表达生殖系干细胞标志物，如八聚体结合转录因子4（OCT4）、NANOG和DEAD盒多肽4（DDX4）。在上皮细胞培养体系中，卵母细胞样细胞在体外自发形成，并表达以下转录标志物：无精子症缺失样蛋白（DAZL）、发育多能性相关蛋白3（STELLA）、透明带基因家族C（ZPC）、联会复合体蛋白（SCP）和生长分化因子9（GDF9）。一些卵母细胞样细胞形成了类似透明带的结构。使用延时摄影观察到上皮细胞和早期发育的卵母细胞的对称和不对称分裂。上皮细胞培养过程中形成了细胞集落，这些集落在饲养层上以未分化的方式维持和增殖，并表达一些多能性标志物。这些集落在体外分化为所有三个胚层的各种体细胞类型，但注射到免疫缺陷小鼠体内时未形成畸胎瘤。此外，这些上皮细胞可直接分化为功能性肝细胞样细胞，而肝细胞样细胞在卵巢组织中并不存在。
源自卵泡液的上皮细胞是一种具有多能/多向分化特性的潜在干细胞来源，可安全应用于卵子发生和再生医学。
