Abstract
Generation of induced pluripotent stem (iPS) cells from differentiated cells has traditionally been performed by overexpressing four transcription factors: Oct4, Sox2, Klf4, and c-Myc. However, inclusion of c-Myc in the reprogramming cocktail can lead to expansion of transformed cells that are not fully reprogrammed, and studies have demonstrated that c-Myc reactivation increases tumorigenicity in chimeras and progeny mice. Moreover, chemical inhibition of Wnt signaling has been shown to enhance reprogramming efficiency. Here, we describe a modified protocol for generating iPS cells from murine fibroblasts using chemical inhibition and overexpression of three transcription factors. Using this protocol, we observed robust conversion to iPS cells while maintaining minimal contamination of partially reprogrammed transformed colonies.
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References
Okita K, Ichisaka T, Yamanaka S (2007) Generation of germline-competent induced pluripotent stem cells. Nature 448(7151):313–317
Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, Hochedlinger K, Bernstein BE, Jaenisch R (2007) In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448(7151):318–324
Kidder BL, Hu G, Yu ZX, Liu C, Zhao K (2013) Extended self-renewal and accelerated reprogramming in the absence of Kdm5b. Mol Cell Biol 33:4793–4810. doi:10.1128/MCB.00692-13, MCB.00692-13 [pii]
Silva J, Barrandon O, Nichols J, Kawaguchi J, Theunissen TW, Smith A (2008) Promotion of reprogramming to ground state pluripotency by signal inhibition. PLoS Biol 6(10):e253. doi:10.1371/journal.pbio.0060253, 08-PLBI-RA-2877 [pii]
Marson A, Foreman R, Chevalier B, Bilodeau S, Kahn M, Young RA, Jaenisch R (2008) Wnt signaling promotes reprogramming of somatic cells to pluripotency. Cell Stem Cell 3(2):132–135. doi:10.1016/j.stem.2008.06.019, S1934-5909 (08)00334-2 [pii]
Kidder BL, Yang J, Palmer S (2008) Stat3 and c-Myc genome-wide promoter occupancy in embryonic stem cells. PLoS ONE 3(12):e3932. doi:10.1371/journal.pone.0003932
Chen X, Xu H, Yuan P, Fang F, Huss M, Vega VB, Wong E, Orlov YL, Zhang W, Jiang J, Loh YH, Yeo HC, Yeo ZX, Narang V, Govindarajan KR, Leong B, Shahab A, Ruan Y, Bourque G, Sung WK, Clarke ND, Wei CL, Ng HH (2008) Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 133(6):1106–1117
Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T, Okita K, Mochiduki Y, Takizawa N, Yamanaka S (2008) Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol 26(1):101–106. doi:10.1038/nbt1374, nbt1374 [pii]
Kidder BL, Oseth L, Miller S, Hirsch B, Verfaillie C, Coucouvanis E (2008) Embryonic stem cells contribute to mouse chimeras in the absence of detectable cell fusion. Cloning Stem Cells 10(2):231–248
Ying QL, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J, Cohen P, Smith A (2008) The ground state of embryonic stem cell self-renewal. Nature 453(7194): 519–523. doi:10.1038/nature06968, nature 06968 [pii]
Acknowledgment
The authors would like to thank Allegra Geller for helpful discussions.
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Kidder, B.L. (2014). Generation of Induced Pluripotent Stem Cells Using Chemical Inhibition and Three Transcription Factors. In: Kidder, B. (eds) Stem Cell Transcriptional Networks. Methods in Molecular Biology, vol 1150. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0512-6_15
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DOI: https://doi.org/10.1007/978-1-4939-0512-6_15
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Online ISBN: 978-1-4939-0512-6
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