Optimization of reprogramming culture conditions for the generation of induced pluripotent stem cells from Col1a1 4F2A‐Oct4‐GFP mice with high efficiency
| Autor: | Sheng-Chieh Hsu, Yaa-Jyuhn James Meir, Mei-Chun Liu, Fang-Chi Hsiao, Ying-Hsuan Lee, Po-Ying Lin, Hung-Chi Chen, Wen-Bin Len, Tsai-Chen Lin, Pei-Ling Pan |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Genetically modified mouse Somatic cell Recombinant Fusion Proteins Induced Pluripotent Stem Cells Context (language use) Biology Biochemistry Collagen Type I Mice 03 medical and health sciences Transforming Growth Factor beta Animals Mesenchymal–epithelial transition Transgenes Induced pluripotent stem cell Molecular Biology Cells Cultured Mice Knockout fungi Mesenchymal stem cell Teratoma Cell Biology Fibroblasts Cellular Reprogramming Embryonic stem cell Coculture Techniques Culture Media Cell biology Collagen Type I alpha 1 Chain 030104 developmental biology Gene Expression Regulation Gamma Rays Doxycycline embryonic structures Reprogramming Cell Division Transcription Factors |
| Zdroj: | The FEBS Journal. 285:1667-1683 |
| ISSN: | 1742-4658 1742-464X |
| DOI: | 10.1111/febs.14436 |
| Popis: | 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 (MEFCol1a14F2A-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 MEFCol1a14F2A-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 γMEFICR feeders. Interestingly, the use of SB431542 with the γMEFICR -adopted system could eliminate this difference. RT-PCR-based comparative analysis further demonstrated that TGF-β expression was 10-fold higher in γMEFICR 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 MEFCol1a14F2A-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 MEFCol1a14F2A-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. |
| Databáze: | OpenAIRE |
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