Derivation of Induced Pluripotent Stem Cells from Human Peripheral Circulating T Cells

Tomohisa Seki1, Shinsuke Yuasa2, Keiichi Fukuda1

1 Department of Cardiology, Keio University School of Medicine, Tokyo, Japan, 2 Center for Integrated Medical Research, Keio University School of Medicine, Tokyo, Japan
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 4A.3
DOI:  10.1002/9780470151808.sc04a03s18
Online Posting Date:  September, 2011
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Abstract

This unit describes a protocol for the generation of induced pluripotent stem (iPS) cells from human peripheral circulating T cells. Initially, human dermal fibroblasts and retroviral vectors were used to generate human iPS cells. Invasive approaches, such as skin biopsy, and genomic insertion of transgenes into the host genome are not appropriate for routine clinical application. Peripheral circulating T cells are readily available from blood samples of patients and healthy volunteers. For the efficient generation of human iPS cells, efficient introduction of the transgene into host cells is necessary. Using a combination of activated T cell culture and Sendai virus allows for the easy and efficient introduction of transgenes into activated T cells and the generation of human iPS cells without genomic integration of extrinsic genes. The T cell‐derived iPS (TiPS) cells exhibit monoclonal T cell receptor (TCR) rearrangement in their genome, a hallmark of mature terminally differentiated T cells. Curr. Protoc. Stem Cell Biol. 18:4A.3.1‐4A.3.9. © 2011 by John Wiley & Sons, Inc.

Keywords: induced pluripotent stem cells; human; peripheral blood; T cells; Sendai virus

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Generation of iPS Cells from T Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Generation of iPS Cells from T Cells

  Materials
  • Heparinized whole blood from donors
  • Ficoll‐Paque PREMIUM (GE Healthcare, cat. no. 17‐5442‐02; see manufacturer‐provided protocol)
  • Purified NA/LE mouse anti‐human CD3 (BD Pharmingen, cat. no. 555336)
  • D‐PBS(–) (Wako, cat. no. 045‐29795)
  • GT‐T502 medium (KOHJIN BIO, cat. no. 16025020)
  • SeVs are available from DNAVEC Corporation (http://www.dnavec.co.jp/)
    • OCT3/4‐SeV/TSΔF (DNAVEC)
    • SOX2‐SeV/TSΔF (DNAVEC)
    • KLF4‐SeV/TSΔF (DNAVEC)
    • c‐MYC(HNL)‐SeV/TS15ΔF (DNAVEC)
  • Irradiated MEF feeder cells (see Conner, )
  • Human iPS cell medium (see recipe)
  • 0.1% gelatin‐coated 6‐well plate (see recipe), preplated with MEFs (unit 1.2)
  • Collagenase type IV (see recipe)
  • DMEM/F12 (Sigma, cat. no. D6421)
  • DAP213 solution (see recipe)
  • Liquid nitrogen
  • 15‐ml conical tubes
  • Centrifuge
  • 100‐mm tissue culture dish (Falcon, cat. no. 353003)
  • Dissection microscope
  • 20‐ and 1000‐µl pipets
  • 0.2‐ml cryovials
  • Liquid nitrogen tank
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Figures

Videos

Literature Cited

Literature Cited
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