Yoo Seung Jun, Yoon Byung Sun, Kim Jin Mee, Song Ji Min, Roh Sung ll, You Seungkwon, Yoon Hyun Soo
Laboratory of Stem Cell Research, Medical Research Center, MizMedi Hospital, 701-4 Naebalsan-dong, Kangseo-gu, Seoul 157-280, Korea.
Exp Mol Med. 2005 Oct 31;37(5):399-407. doi: 10.1038/emm.2005.50.
Human embryonic stem cells (hESCs) need feeder cells for their maintenance in an undifferentiated state. In conventional culture systems, mouse embryonic fibroblasts (MEFs) serve as feeder cells to maintain hESCs. However, the use of MEFs elevates the risk of transmitting mouse pathogens and thus limits the potential of hESCs in cell replacement therapy. Consequently, the use of human feeder cells would be an important step forward in this in vitro technology. To address this issue, we used fibroblast-like cells differentiated from the Miz-hES6 hESC line (DiffMiz-hES6) as feeder cells to support the in vitro growth of three hESC lines. Immunofluorescence microscopy and reverse transcription-PCR assessing the expression of undifferentiated hESC markers revealed all three hESC lines were maintained in an undifferentiated state. In vitro proliferation proceeded as efficiently as when the hESCs were cultured on MEFS. Moreover, karyotype analysis revealed the chromosomal normality of the hESC lines and the DiffMiz-hES6 feeders themselves after even 50 passages. Furthermore, the hESC lines maintained their pluripotency since they remained capable of forming embryoid bodies (EBs) in vitro. Thus, hESC-derived fibroblast-like cells successfully support in vitro hESC propagation.
人类胚胎干细胞（hESCs）需要饲养层细胞来维持其未分化状态。在传统培养系统中，小鼠胚胎成纤维细胞（MEFs）用作饲养层细胞来维持hESCs。然而，使用MEFs会增加传播小鼠病原体的风险，从而限制了hESCs在细胞替代疗法中的潜力。因此，使用人类饲养层细胞将是这项体外技术向前迈出的重要一步。为了解决这个问题，我们使用从Miz-hES6 hESC系分化而来的成纤维样细胞（DiffMiz-hES6）作为饲养层细胞，以支持三种hESC系的体外生长。免疫荧光显微镜和逆转录PCR评估未分化hESC标志物的表达显示，所有三种hESC系均维持在未分化状态。体外增殖与hESCs在MEFs上培养时一样高效。此外，核型分析显示，即使经过50代传代，hESC系和DiffMiz-hES6饲养层细胞本身的染色体仍保持正常。此外，hESC系保持了它们的多能性，因为它们在体外仍能够形成胚状体（EBs）。因此，hESC衍生的成纤维样细胞成功地支持了hESC的体外增殖。
