Abstract
Induced pluripotent stem (iPS) cells can be directly generated from somatic cells by overexpression of defined transcription factors. iPS cells can perpetually self-renew and differentiate into all cell types of an organism. iPS cells were first generated through infection with retroviruses that contain reprogramming factors. However, development of an exogene-free iPS cell generation method is crucial for future therapeutic applications, because integrated exogenes result in the formation of tumors in chimeras and regain pluripotency after differentiation in vitro. Here, we describe a method to generate iPS cells by transfection of plasmid vectors and to convert partially reprogrammed cells into fully reprogrammed iPS cells by switching from mouse ESC culture conditions to KOSR-based media with bFGF. We also describe basic methods used to characterize fully reprogrammed iPS cells.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factor. Cell 126(4):663–676
Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5):861–872
Okita K, Ichisaka T, Yamanaka S (2007) Generation of germline-competent induced pluripotent stem cells. Nature 448(7151):313–317
Kim MJ, Choi HW, Jang HJ, Chung HM, Arauzo-Bravo MJ, Schöler HR, Do JT (2013) Conversion of genomic imprinting by reprogramming and redifferentiation. J Cell Sci 126(11):2516–2524
Park IH, Arora N, Huo H, Maherali N, Ahfeldt T, Shimamura A, Lensch MW, Cowan C, Hochedlinger K, Daley GQ (2008) Disease-specific induced pluripotent stem cells. Cell 134(5):877–886
Soldner F, Hockemeyer D, Beard C, Gao Q, Bell GW, Cook EG, Hargus G, Blak A, Cooper O, Mitalipova M, Isacson O, Jaenisch R (2009) Parkinson’s disease patient-derived induced pluripotent stem cells free of viral reprogramming factors. Cell 136(5):964–977
Dimos JT, Rodolfa KT, Niakan KK, Weisenthal LM, Mitsumoto H, Chung W, Croft GF, Saphier G, Leibel R, Goland R, Wichterle H, Henderson CE, Eggan K (2008) Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 321(5893):1218–1221
Ebert AD, Yu J, Rose FF Jr, Mattis VB, Lorson CL, Thomson JA, Svendsen CN (2009) Induced pluripotent stem cells from a spinal muscular atrophy patient. Nature 457(7227):277–280
Choi HW, Kim JS, Choi S, Hong YJ, Kim MJ, Seo HG, Do JT (2014) Neural stem cells differentiated from iPS cells spontaneously regain pluripotency. Stem Cells 32(10):2596–2604
Mikkelsen TS, Hanna J, Zhang X, Ku M, Wernig M, Schorderet P, Bernstein BE, Jaenisch R, Lander ES, Meissner A (2008) Dissecting direct reprogramming through integrative genomic analysis. Nature 454(7200):49–55
Chan EM, Ratanasirintrawoot S, Park IH, Manos PD, Loh YH, Huo H, Miller JD, Hartung O, Rho J, Ince TA, Daley GQ, Schlaeger TM (2009) Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells. Nat Biotechnol 27(11):1033–1037
Kim JS, Choi HW, Choi S, Seo HG, Moon SH, Chung HM, Do JT (2014) Conversion of partially reprogrammed cells to fully pluripotent stem cells is associated with further activation of stem cell maintenance- and gamete generation-related genes. Stem Cells Dev. doi:10.1089/scd.2014.0020
Sridharan R, Tchieu J, Mason MJ, Yachechko R, Kuoy E, Horvath S, Zhou Q, Plath K (2009) Role of the murine reprogramming factors in the induction of pluripotency. Cell 136(2):364–377
Acknowledgment
This work was supported by the Biomedical Technology Development Program and Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT and Future Planning (grant nos. 20110019489 and 2013R1A1A2011394).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Kim, J.S., Choi, H.W., Hong, Y.J., Do, J.T. (2014). Generation of Partially Reprogrammed Cells and Fully Reprogrammed iPS Cells by Plasmid Transfection. In: Turksen, K., Nagy, A. (eds) Induced Pluripotent Stem (iPS) Cells. Methods in Molecular Biology, vol 1357. Humana Press, New York, NY. https://doi.org/10.1007/7651_2014_161
Download citation
DOI: https://doi.org/10.1007/7651_2014_161
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3054-8
Online ISBN: 978-1-4939-3055-5
eBook Packages: Springer Protocols