Derivation of Transgene‐Free Induced Pluripotent Stem Cells from a Single Drop of Blood

Hong Yu Chen1, Hong‐Kee Tan1, Yuin‐Han Loh2

1 Epigenetics and Cell Fates Laboratory, A*STAR Institute of Molecular and Cell, Biology, 2 Department of Biological Sciences, National University of Singapore
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 4A.9
DOI:  10.1002/cpsc.12
Online Posting Date:  August, 2016
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Abstract

Human‐induced pluripotent stem cells (hiPSCs) have great potential for future use in therapeutic regenerative medicine. Based on the current protocol for deriving hiPSCs, invasive procedures such as skin biopsies and venipuncture are required for obtaining donor samples. Herein, we present a detailed protocol for deriving hiPSCs from human finger‐prick (FP) blood. In this method, the transgene‐free hiPSCs can be easily generated from only 10 µl of FP blood. The finger‐pricked iPSCs (FPiPSCs) show all the pluripotency markers and can be easily differentiated into various cell lineages. The time required for deriving the FPiPSCs is relatively short—10 to 15 days for FP blood expansion and 20 to 30 days for reprogramming. This method can be easily adapted for setting up a large scale iPSC bank as it requires only 10 µl of the donor FP blood, which can be easily collected. © 2016 by John Wiley & Sons, Inc.

Keywords: reprogramming; induced pluripotent stem cell; transgene free iPSCs; iPSC banking

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Expansion of Erythroid Progenitor Cells from a Single Drop of Finger‐Prick Blood
  • Basic Protocol 2: Reprogramming Erythroid Progenitor Cells Derived from Finger‐Prick Blood Culture
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Expansion of Erythroid Progenitor Cells from a Single Drop of Finger‐Prick Blood

  Materials
  • Donor
  • 10× RBC lysis buffer (eBioscience, cat. no. 00‐4300‐54)
  • Distilled water
  • Finger‐Prick blood expansion medium (FPBEM; see recipe)
  • Alcohol swab (Guardian, cat. no. 220114 or any other sources)
  • Finger pricker (Accu‐Check Safe‐T‐Pro Plus Lancet, cat.no. 3448622 or any others sources)
  • Microtainer tubes with K2EDTA (BD, cat. no. 365974)
  • 0‐2μm syringe filter (Minisart, cat. no. 16534‐K)
  • 10‐ and 50‐ml syringes (BD, cat. no. 302149, 300144, respectively)
  • 15‐ml conical tubes (BD Falcon, cat. no. 352096)
  • 2‐, 5‐, and 10‐ml Serological pipets (Corning)
  • 10‐, 20‐, 200‐, and 1000‐μl micropipets (Gilson)
  • Pipet controller (Integra Bioscience)
  • Vortex mixer
  • 24‐well tissue culture plates
  • 37°C, 5% CO 2 incubator
  • Counting Chambers, Dark‐Line (Marienfeld‐Superior, cat. no. 0610010)

Basic Protocol 2: Reprogramming Erythroid Progenitor Cells Derived from Finger‐Prick Blood Culture

  Materials
  • CytoTune ‐iPS Sendai Reprogramming Kit (Cytotune 1.0) (Life Technology, cat. no. A1378001)
  • CytoTune ‐iPS 2.0 Sendai Reprogramming Kit (Cytotune 2.0) (Life Technology, cat. no. A16517)
  • Ice
  • Finger‐Prick blood expansion medium (FPBEM; see recipe)
  • Gelatin (from porcine skin; Sigma, cat. no. G1890‐100 G)
  • CF‐1 MEF (γ‐irradiated; Global Stem, cat. no. GSC‐6201 G or please refer to Lin and Talbot, for a more detailed description on the preparation of mitotically inactivated mouse embryonic fibroblast feeder)
  • MEF medium (see recipe)
  • Human stem cell medium (hSCM; see recipe)
  • D‐PBS
  • StainAlive TRA‐1‐60 Antibody (DyLight 488), Mouse anti‐Human (Optional) (Stemgent, cat. no. 09‐0068)
  • DMEM/F‐12, HEPES, no phenol red (Optional) (Invitrogen/Gibco, cat. no. 11039021)
  • Y‐27632 (Stemcell Technologies, cat. no. 72304)
  • mTeSR1 (Stemcell Technologies, cat. no. 05850)
  • ½ mTeSR/½ CM (see recipe)
  • 2‐, 5‐, 10‐, 25‐, and 50‐ml serological pipets (Corning)
  • 10‐, 20‐, 200‐, and 1000‐μl micropipets (Gilson)
  • Pipet controller (Integra Bioscience)
  • 12‐well tissue culture treated plates (Corning, cat. no. 3513)
  • 24‐well tissue culture treated plates (Corning, cat. no. 3536)
  • Centrifuge
  • 37°C, 5% CO 2 incubator
  • 6‐well tissue culture treated plates (Corning, cat. no. 3516)
  • 15‐ml conical tubes
  • Inverted phase contrast microscope with epifluorescent, 4×, 10×, 20×, 40× objective lens (Nikon)
  • Marker pen
  • 200‐μl pipets
  • Stereomicroscope (Nikon)
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Figures

Videos

Literature Cited

Literature Cited
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