Abstract
This chapter provides a method for reprogramming human dermal fibroblasts into induced pluripotent stem cells (iPSCs) using three lentiviruses containing cDNAs for OCT4 and SOX2, KLF4 and C-MYC, and NANOG and LIN28, respectively. Lentiviral vectors are based on the human immunodeficiency virus (HIV) and provide an effective means for the delivery, integration, and expression of exogenous genes in mammalian cells. Lentiviruses are attractive gene delivery vehicles as they are able to infect both proliferating and nonproliferating cells. Lentiviruses stably integrate into the genome without incurring cellular toxicity and can maintain sustained transgene expression during prolonged host cell proliferation and differentiation. In this protocol, we describe how to prepare lentiviruses, stably transduce human fibroblasts, and identify bona fide iPSC colonies based on morphological similarity to human embryonic stem cell (ESC) colonies and live-cell immunological staining using cell-surface markers of human PSCs such as Tra-1-60 and Tra-1-81.
Key words
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Takahashi, K. & Yamanaka, S. (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126: 663–676.
Takahashi, K. et al. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131: 861–872.
Yu, J. et al. (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318: 1917–1920.
Belmonte, J.C., Ellis, J., Hochedlinger, K., & Yamanaka, S. (2009) Induced pluripotent stem cells and reprogramming: seeing the science through the hype. Nat. Rev. Genet. 10: 878–883.
Cowan, C.A., Atienza, J., Melton, D.A., & Eggan, K. (2005) Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells. Science 309: 1369–1373.
Stojkovic, M. et al. (2005) Derivation of a human blastocyst after heterologous nuclear transfer to donated oocytes. Reprod. Biomed. Online. 11: 226–231.
Chan, E.M., Ratanasirintrawoot, S., Park, I-H., Manos, P.D., Loh, Y-H., Huo, H., Miller, J.D., Hartung, O., Rho, J., Ince, T.A., Daley, G.Q. & Schlaeger, T.M. (2009) Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells. Nat Biotechnol. Nov; 27(11): 1033–7.
Acknowledgments
This work has been funded by the National Institutes of Health (T15HL074286, R21MH087925, R01HD059967). The NCI Preclinical Repository supplied FGF-2. We also recognize Richard Pepple for proof-reading this chapter.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Nethercott, H.E., Brick, D.J., Schwartz, P.H. (2011). Derivation of Induced Pluripotent Stem Cells by Lentiviral Transduction. In: Schwartz, P., Wesselschmidt, R. (eds) Human Pluripotent Stem Cells. Methods in Molecular Biology, vol 767. Humana Press. https://doi.org/10.1007/978-1-61779-201-4_6
Download citation
DOI: https://doi.org/10.1007/978-1-61779-201-4_6
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-200-7
Online ISBN: 978-1-61779-201-4
eBook Packages: Springer Protocols