Optimization of reprogramming culture conditions for the generation of induced pluripotent stem cells from Col1a1 4F2A-Oct4-GFP mice with high efficiency

Po Ying Lin, Sheng Chieh Hsu, Hung Chi Chen, Wen Bin Len, Fang Chi Hsiao, Mei Chun Liu, Pei Ling Pan, Tsai Chen Lin, Ying Hsuan Lee, Yaa Jyuhn James Meir*

*Corresponding author for this work

Research output: Contribution to journalJournal Article peer-review

4 Scopus citations


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 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 SB431542 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 (γSNL) cells to serve as feeders for iPSC production resulted in a 5-fold higher rate of iPSC production than the use of γMEF ICR feeders. Interestingly, the use of SB431542 with the γMEF ICR -adopted system could eliminate this difference. RT-PCR-based comparative analysis further demonstrated that TGF-β expression was 10-fold higher in γMEF ICR 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 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 iPSCs 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 iPSCs with variable efficiencies, which can serve as a tool for interrogating the route taken by cells during somatic reprogramming.

Original languageEnglish
Pages (from-to)1667-1683
Number of pages17
JournalFEBS Journal
Issue number9
StatePublished - 05 2018

Bibliographical note

Publisher Copyright:
© 2018 Federation of European Biochemical Societies


  • Col1a1 4F2A-Oct4-GFP mouse
  • iCD chemically defined medium
  • iPSC
  • mesenchymal–epithelial transition
  • TGF-β


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