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Optimization of reprogramming culture conditions for the generation of induced pluripotent stem cells from Col1a1 4F2A‐Oct4‐ GFP mice with high efficiency
Author(s) -
Lin PoYing,
Hsu ShengChieh,
Chen HungChi,
Len WenBin,
Hsiao FangChi,
Liu MeiChun,
Pan PeiLing,
Lin TsaiChen,
Lee YingHsuan,
Meir YaaJyuhn James
Publication year - 2018
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.14436
Subject(s) - reprogramming , induced pluripotent stem cell , embryonic stem cell , biology , stem cell , microbiology and biotechnology , context (archaeology) , somatic cell , cell , genetics , gene , paleontology
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 ( iPSC s; approx. 0.005%) under traditional culture conditions. With an optimized culture condition, the iPSC production rate of mouse embryonic fibroblasts ( MEF s) of Col1a1 4F2A‐Oct4‐ GFP mice ( MEF C ol1a1 4F2A‐Oct4‐ GFP ) 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 SB 431542 neither affected the growth rate of MEF C ol1a1 4F2A‐Oct4‐ GFP nor promoted iPSC production. However, the use of the gamma‐irradiated STO‐NEO‐LIF (γ SNL ) cells to serve as feeders for iPSC production resulted in a 5‐fold higher rate of iPSC production than the use of γ MEF ICR feeders. Interestingly, the use of SB 431542 with the γ MEF ICR ‐adopted system could eliminate this difference. RT ‐ PCR ‐based comparative analysis further demonstrated that TGF ‐β expression was 10‐fold higher in γ MEF ICR than in γ SNL 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 C ol1a1 4F2A‐Oct4‐ GFP . Moreover, we found that the initial seeding density is one of the pivotal factors for determining a high efficiency of iPSC generation. The iPSC s efficiently generated from our MEF C ol1a1 4F2A‐Oct4‐ GFP 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 iPSC s with variable efficiencies, which can serve as a tool for interrogating the route taken by cells during somatic reprogramming.