Lai Dongmei, Wang Yongwei, Sun Jian, Chen Yifei, Li Ting, Wu Yi, Guo Lihe, Wei Chunsheng
The International Peace Maternity and Child Health Hospital, School of medicine, Shanghai Jiaotong University, Shanghai, China.
Eye and ENT Hospital, Fudan University, Shanghai, China.
Sci Rep. 2015 May 7;5:10014. doi: 10.1038/srep10014.
Culture conditions that support the growth of undifferentiated human embryonic stem cells (hESCs) have already been established using primary human amnion epithelial cells (hAECs) as an alternative to traditional mitotically inactivated mouse embryonic fibroblasts (MEFs). In the present work, inner cell masses (ICM) were isolated from frozen embryos obtained as donations from couples undergoing in vitro fertilization (IVF) treatment and four new hESC lines were derived using hAECs as feeder cells. This feeder system was able to support continuous growth of what were, according to their domed shape and markers, undifferentiated naïve-like hESCs. Their pluripotent potential were also demonstrated by embryoid bodies developing to the expected three germ layers in vitro and the productions of teratoma in vivo. The cell lines retained their karyotypic integrity for over 35 passages. Transmission electron microscopy (TEM) indicated that these newly derived hESCs consisted mostly of undifferentiated cells with large nuclei and scanty cytoplasm. The new hESCs cultured on hAECs showed distinct undifferentiated characteristics in comparison to hESCs of the same passage maintained on MEFs. This type of optimized culture system may provide a useful platform for establishing clinical-grade hESCs and assessing the undifferentiated potential of hESCs.
已经建立了使用原代人羊膜上皮细胞（hAECs）替代传统的经有丝分裂灭活的小鼠胚胎成纤维细胞（MEFs）来支持未分化的人胚胎干细胞（hESCs）生长的培养条件。在本研究中，从接受体外受精（IVF）治疗的夫妇捐赠的冷冻胚胎中分离出内细胞团（ICM），并使用hAECs作为饲养层细胞获得了4个新的hESC系。这种饲养层系统能够支持根据其圆顶形状和标志物判断为未分化的幼稚样hESCs的持续生长。通过体外形成预期的三个胚层的胚状体以及体内产生畸胎瘤，也证明了它们的多能潜力。这些细胞系在超过35代时保持了其核型完整性。透射电子显微镜（TEM）表明，这些新获得的hESCs主要由细胞核大、细胞质少的未分化细胞组成。与在MEFs上培养的同一代hESCs相比，在hAECs上培养的新hESCs表现出明显的未分化特征。这种优化的培养系统可能为建立临床级hESCs和评估hESCs的未分化潜力提供一个有用的平台。
