Lentiviral reprogramming of A-T patient fibroblasts to induced pluripotent stem cells

Nayler, Sam, Kozlov, Sergei V., Lavin, Martin F. and Wolvetang, Ernst (2017). Lentiviral reprogramming of A-T patient fibroblasts to induced pluripotent stem cells. In Sergei V. Kozlov (Ed.), ATM kinase: methods and protocols (pp. 401-418) New York, NY, United States: Humana Press; Springer Science+Business Media. doi: 10.1007/978-1-4939-6955-5_29


Author Nayler, Sam
Kozlov, Sergei V.
Lavin, Martin F.
Wolvetang, Ernst
Title of chapter Lentiviral reprogramming of A-T patient fibroblasts to induced pluripotent stem cells
Title of book ATM kinase: methods and protocols
Place of Publication New York, NY, United States
Publisher Humana Press; Springer Science+Business Media
Publication Year 2017
Sub-type Research book chapter (original research)
DOI 10.1007/978-1-4939-6955-5_29
Open Access Status Not yet assessed
Series Methods in Molecular Biology
ISBN 9781493969555
9781493969531
ISSN 1064-3745
1940-6029
Editor Sergei V. Kozlov
Volume number 1599
Chapter number 29
Start page 401
End page 418
Total pages 8
Total chapters 30
Collection year 2018
Language eng
Formatted Abstract/Summary
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.
Keyword Ataxia-Telangiectasia
ATM
DNA damage
Induced pluripotent stem cells
IPSC
Pluripotency
Reprogramming
Q-Index Code B1
Q-Index Status Provisional Code
Institutional Status UQ

 
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Created: Wed, 08 Mar 2017, 13:31:40 EST by Chris Ende on behalf of Aust Institute for Bioengineering & Nanotechnology