Effective culture conditions for the induction of pluripotent stem cells
Effective culture conditions for the induction of pluripotent stem cells
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DOI：
10.1016/j.bbagen.2010.04.004
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发表时间：
2010-09-01
影响因子：
3
通讯作者：
Yoneda, Yoshihiro
中科院分区：
文献类型：
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作者：
Okada, Minoru;Oka, Masahiro;Yoneda, Yoshihiro
背景：诱导多能干细胞在功能上与胚胎干细胞相似，可通过表达一组转录因子Oct4、Sox2、Klf4和c-Myc从小鼠成纤维细胞中获得。由于iPS细胞是从体细胞产生的，它们为细胞移植治疗提供了宝贵的多潜能干细胞来源，而不存在伦理问题。然而，重编程效率极低，iPS细胞分化的最佳培养条件尚未明确。方法：为了高效地获得iPS细胞，我们测试了10种不同的培养条件：DMEM+15%胎牛血清(FBS)，敲除DMEM+15%Invitgen FBS。Eguitech或HyClone。使用15%的敲除血清替代(KSR)的DMEM，以及使用10%、15%、20%、25%或35%KSR的敲除DMEM。这些培养基均含有2 mM的L-谷氨酰胺、100mM的非必需氨基酸、100 mM的β-巯基乙醇、1000单位毫升(-1)的白血病抑制因子(LIF)、50单位毫升(-1)的青霉素和50毫升(-1)的链霉素。结果：含有20%KSR的敲除DMEM的培养液可以有效地诱导小鼠胚胎成纤维细胞(MEF)和成人尾尖成纤维细胞(TTF)的iPS细胞。在此条件下产生的小鼠iPS细胞表达的ES细胞标志基因如Oct4、Sox2、Rex1和Nanog的水平与ES细胞相当。结论：我们的iPS细胞诱导效率高于其他报道。一般意义：这些发现为检测不同培养环境下iPS细胞的产生提供了重要的催化剂。(C)2010爱思唯尔B.V.保留所有权利。
Background: Induced pluripotent stem (iPS) cells, which are functionally comparable to embryonic stem (ES) cells, can be generated from mouse fibroblasts by expression of a defined set of transcription factors Oct4, Sox2, Klf4, and c-Myc. Since iPS cells are generated from somatic cells, they provide an invaluable source of pluripotent stem cells for cell transplantation therapy that does not present ethical problems. However, the reprogramming efficiency is extremely low, and optimal culture conditions for iPS cell derivation have not been clearly defined.Methods: To generate iPS cells efficiently, we tested 10 different culture conditions: DMEM supplemented with 15% fetal bovine serum (FBS), Knockout DMEM with 15% FBS from Invitrogen. Eguitech, or HyClone. DMEM with 15% Knockout Serum Replacement (KSR), and Knockout DMEM with 10%, 15%, 20%, 25%, or 35% KSR. These media all contain 2 mM L-glutamine, 100 mu M nonessential amino acids, 100 AM beta-mercaptoethanol, 1000 units ml(-1) leukemia inhibitory factor (LIF), 50 units ml(-1) penicillin, and 50 mu g ml(-1) streptomycin.Results: Medium containing Knockout DMEM with 20% KSR permits efficient induction of iPS cells from both mouse embryonic fibroblasts (MEFs) and adult tail tip fibroblasts (TTFs). Mouse iPS cells generated in the condition express ES cell marker genes such as Oct4, Sox2, Rex1, and Nanog at levels comparable to those of ES cells. Furthermore, iPS cells derived form MEFs and adult TTFs can contribute to adult chimeras.Conclusion: Our iPS cell induction efficiency is greater than that described in other reports.General significance: These findings provide an important catalyst for examining different culture environments for the generation of iPS cells. (C) 2010 Elsevier B.V. All rights reserved.