Novel autogenic feeders derived from human embryonic stem cells (hESCs) support an undifferentiated status of hESCs in xeno-free culture conditions
Novel autogenic feeders derived from human embryonic stem cells (hESCs) support an undifferentiated status of hESCs in xeno-free culture conditions
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DOI：
10.1093/humrep/dep003
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发表时间：
2009-05-01
影响因子：
6.1
通讯作者：
Kuo, Hung-Chih
中科院分区：
文献类型：
--
作者：
Chen, Hsin-Fu;Chuang, Ching-Yu;Kuo, Hung-Chih
理想情况下，临床级人类胚胎干细胞（hESCs）应该使用特定的化学物质或人类来源的材料在无异种培养条件下获得和维持。这将减少临床应用中外源致病性感染和/或不良免疫反应的可能性。因此，本研究旨在从hESCs中获得自体喂食器，并评估其在无异种培养条件下支持hESCs多能性的能力。H9 hESCs在含有人血清（HS）、血清替代（SR）或KFM组合的培养基中培养，生成自源饲料（分别命名为HSdF、SRdF和KFMdF）。利用多能干细胞标记物、早期细胞系标记物和表面标记物进行逆转录聚合酶链反应、流式细胞术和免疫荧光分析显示，HSdF、SRdF和KFMdF可能属于不同的细胞亚群。用含SR、胎牛血清、HS或1% HS加各种生长因子组合的培养基，通过流式细胞术分析Oct4的表达，评价自源性饲养者维持H9 hESCs多能性的效率。在含sr培养基中长期培养，这三种自源喂食器均能维持H9 hESCs的未分化状态。在含有1% HS的培养基中，当添加bFGF、激活素A和noggin时，hESCs在KFMdF上也能保持良好的生长，而不失去其体外和体内的多能性。在含低浓度HS的无xeno培养基中，hESCs可以在无xeno条件下获得新的自生供体，并能稳定地保持hESCs的多能性。
Clinical-grade human embryonic stem cells (hESCs) ideally should be derived and maintained in xeno-free culture conditions using defined chemicals or materials of human origin. This will reduce the possibility of xeno-derived pathogenic infection and/or unfavorable immune reaction in clinical application. The present study therefore aimed to derive autogenic feeders from hESCs and evaluate their capability to support the pluripotency of hESCs in xeno-free culture conditions.H9 hESCs were cultured in media containing human serum (HS), serum replacement (SR) or KFM combination, to generate autogenic feeders (named HSdF, SRdF and KFMdF, respectively). Reverse transcription polymerase chain reaction, flow cytometry and immunofluorescence analysis using pluripotent stem cell markers, markers of early cell lineages and surface markers revealed that HSdF, SRdF and KFMdF likely belonged to different cellular subpopulations. The efficiency of the autogenic feeders in maintaining pluripotency of H9 hESCs using media containing SR, fetal bovine serum, HS or 1% HS plus various combinations of growth factors was evaluated by flow cytometric analysis of Oct4 expression. All three autogenic feeders were shown to be capable of maintaining the undifferentiated status of H9 hESCs in SR-containing media in long-term culture. When supplemented with bFGF, activin A and noggin, hESCs could also be maintained favorably on KFMdF in a medium containing 1% HS without losing their pluripotent potentials both in vitro and in vivo.Novel autogenic feeders can be derived from hESCs under xeno-free conditions and they can robustly maintain the pluripotent identity of hESCs in xeno-free media containing a low concentration of HS.