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Lentiviral Reprogramming of A-T Patient Fibroblasts to Induced Pluripotent Stem Cells

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ATM Kinase

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1599))

Abstract

Reprogramming of cells enables generation of pluripotent stem cells and resulting progeny through directed differentiation, making this technology an invaluable tool for the study of human development and disease. Reprogramming occurs with a wide range of efficiency, a culmination of intrinsic and extrinsic factors including the tissue of origin, the passage number and culture history of the target cells. Another major factor affecting reprogramming is the methodology used and the quality of the reprogramming process itself, including for conventional viral-based approaches viral titer and subsequent viral transduction efficiency, including downstream transgene insertion and stoichiometry. Genetic background is an important parameter affecting the efficiency of the reprogramming process with reports that cells from individuals harboring specific mutations are more difficult to reprogram than control counterparts.

Ataxia-Telangiectasia (A-T) fibroblasts underwent reprogramming at reduced efficiency in contrast to their controls. To optimize reprogramming of fibroblasts from patients with A-T, we examined the response of A-T cells to various cell culture conditions after lentiviral transduction with reprogramming factors Oc4/Sox2 (pSIN4-EF2-O2S) and Klf4/c-Myc (pSIN4-CMV-K2M). Parameters included media type (KSR or serum-containing DMEM), treatment with a p53 inhibitor (small-molecule cyclic pifithrin-α), and either a low or high concentration of bFGF. Post-transduction, equivalent numbers of cells from heterozygote and homozygote patients were plated and assessed at regular intervals for survival and proliferation. Our findings indicate that A-T cells responded favorably to the addition of FCS and gradual weaning away from their native media into KSR-containing stem cell media that produced suitable conditions for their reprogramming. We examined a range of properties to identify and isolate good quality iPSCs including the expression status of important stem cell transcription factors/surface proteins, methylation levels at stem cell associated regulatory loci, persistence of transgenes, karyotype status, and teratoma-forming ability.

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Authors and Affiliations

  1. Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, St. Lucia, Brisbane, QLD, 4072, Australia

    Sam Nayler & Ernst Wolvetang

  2. University of Queensland Centre for Clinical Research (UQCCR), University of Queensland, Building 71/918, Royal Brisbane & Women’s Hospital Campus, Herston, Brisbane, QLD, 4029, Australia

    Sergei V. Kozlov

  3. University of Queensland Centre for Clinical Research (UQCCR), The University of Queensland, Herston, Brisbane, QLD, 4029, Australia

    Martin F. Lavin

Authors
  1. Sam Nayler
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  2. Sergei V. Kozlov
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  3. Martin F. Lavin
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  4. Ernst Wolvetang
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Corresponding author

Correspondence to Ernst Wolvetang .

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Editors and Affiliations

  1. University of Queensland Centre for Clinical Research (UQCCR), University of Queensland, Herston, Queensland, Australia

    Sergei V. Kozlov

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Nayler, S., Kozlov, S.V., Lavin, M.F., Wolvetang, E. (2017). Lentiviral Reprogramming of A-T Patient Fibroblasts to Induced Pluripotent Stem Cells. In: Kozlov, S. (eds) ATM Kinase. Methods in Molecular Biology, vol 1599. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6955-5_29

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  • DOI: https://doi.org/10.1007/978-1-4939-6955-5_29

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6953-1

  • Online ISBN: 978-1-4939-6955-5

  • eBook Packages: Springer Protocols

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