Department of Biomedical Sciences, College of Medicine, Chang Gung University, Linkou, Taiwan.
Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital, Linkou, Taiwan.
FEBS J. 2018 May;285(9):1667-1683. doi: 10.1111/febs.14436. Epub 2018 Apr 11.
A reprogrammable transgenic mouse strain, called Col1a1 4F2A-Oct4-GFP, was bred for the present study. Because the somatic cells of this mouse strain contain only two copies of each Yamanaka factor, these animals are inefficient at producing induced pluripotent stem cells (iPSCs; approx. 0.005%) under traditional culture conditions. With an optimized culture condition, the iPSC production rate of mouse embryonic fibroblasts (MEFs) of Col1a1 4F2A-Oct4-GFP mice (MEF ) was increased to approximately 8%. Further, promotion of cell proliferation by serum supplementation did not enhance iPSC production. Inhibition of transforming growth factor β (TGF-β) in the serum by SB431542 neither affected the growth rate of MEF nor promoted iPSC production. However, the use of the gamma-irradiated STO-NEO-LIF (γSNL) cells to serve as feeders for iPSC production resulted in a 5-fold higher rate of iPSC production than the use of γMEF feeders. Interestingly, the use of SB431542 with the γMEF -adopted system could eliminate this difference. RT-PCR-based comparative analysis further demonstrated that TGF-β expression was 10-fold higher in γMEF than in γSNL cells. Consistent with previous reports, mesenchymal to epithelial transition was found to participate in the initial steps of reprogramming in the specific context of MEF . Moreover, we found that the initial seeding density is one of the pivotal factors for determining a high efficiency of iPSC generation. The iPSCs efficiently generated from our MEF resembled mouse embryonic stem cells (mESCs) in aspects of teratoma formation and germline transmission. Depending on the culture conditions, our Col1a1 4F2A-Oct4-GFP mouse system can generate bona fide iPSCs with variable efficiencies, which can serve as a tool for interrogating the route taken by cells during somatic reprogramming.
本研究培育了一种可重编程的转基因小鼠品系，称为 Col1a1 4F2A-Oct4-GFP。由于该小鼠品系的体细胞仅含有每个 Yamanaka 因子的两个拷贝，因此在传统培养条件下，这些动物产生诱导多能干细胞（iPSC；约 0.005%）的效率很低。通过优化培养条件，Col1a1 4F2A-Oct4-GFP 小鼠的胚胎成纤维细胞（MEF）的 iPSC 产生率提高到约 8%。此外，血清补充促进细胞增殖并没有提高 iPSC 的产生。血清中转化生长因子-β（TGF-β）的抑制作用 SB431542 既不影响 MEF 的生长速度，也不促进 iPSC 的产生。然而，使用γ辐射的 STO-NEO-LIF（γSNL）细胞作为 iPSC 生产的饲养细胞可使 iPSC 的产生率提高 5 倍，而使用γMEF 饲养细胞则不行。有趣的是，在采用γMEF 系统的情况下，使用 SB431542 可以消除这种差异。基于 RT-PCR 的比较分析进一步表明，TGF-β在γMEF 中的表达比在γSNL 细胞中高 10 倍。与先前的报道一致，在特定的 MEF 背景下，间充质到上皮的转变被发现参与了重编程的初始步骤。此外，我们发现初始接种密度是决定 iPSC 高效产生的关键因素之一。从我们的 MEF 中高效产生的 iPSC 在畸胎瘤形成和种系传递方面与小鼠胚胎干细胞（mESC）相似。根据培养条件的不同，我们的 Col1a1 4F2A-Oct4-GFP 小鼠系统可以以不同的效率产生真正的 iPSC，可作为研究体细胞重编程过程中细胞所走途径的工具。
