Using MicroRNAs to Enhance the Generation of Induced Pluripotent Stem Cells

Zhonghan Li1, Tariq M. Rana1

1 Program for RNA Biology, Sanford‐Burnham Medical Research Institute, La Jolla, California
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 4A.4
DOI:  10.1002/9780470151808.sc04a04s20
Online Posting Date:  March, 2012
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Abstract

Somatic cells reprogrammed to acquire an ES‐like state are termed iPS cells. In this unit, a protocol to use microRNAs as enhancers to increase the reprogramming efficiency is described. Mouse embryonic fibroblasts (MEFs) are isolated from E13.5 mouse embryos and seeded for reprogramming by defined factors. microRNA mimics are transfected into MEFs at two time points during this process to enhance the overall reprogramming efficiency. Two standard protocols for characterization of these miR‐iPSCs, embryoid body formation and teratoma formation, are also provided. By using this method, the investigators can obtain a significantly higher number of bona‐fide iPSC colonies and miR‐iPSCs can be derived at a faster rate than with non‐treated cells. Curr. Protoc. Stem Cell Biol. 20:4A.4.1‐4A.4.14. © 2012 by John Wiley & Sons, Inc.

Keywords: reprogramming; microRNA; iPSC; embryonic body; teratoma

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Mouse Embryonic Fibroblasts (MEFs)
  • Basic Protocol 2: Use microRNAs as an Enhancer to Reprogram Mouse Embryonic Fibroblasts
  • Basic Protocol 3: Derivation and Characterization of miR‐iPSC Colonies
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Mouse Embryonic Fibroblasts (MEFs)

  Materials
  • Female mice, B6;129S4‐Pou5f1tm2Jae/J (13 to 14 days gestation)
  • Phosphate buffered saline (PBS; Invitrogen, cat. no. 10010023)
  • 0.25% trypsin (Invitrogen, cat. no. 25200)
  • MEF derivation medium (see recipe)
  • MEF culture medium (see recipe)
  • Cryopreservation medium (see recipe)
  • Liquid nitrogen tank
  • Absorbent paper towels
  • Alcohol pads
  • Dissecting scissors, sterile
  • Sterile, disposable petri dishes (Sarstedt, cat. no. 82.1473.001)
  • Watchmaker's forceps
  • Sterile, disposable 10‐ml pipets (Costar, cat. no. 4488)
  • Castro‐Viejo scissors
  • 37°C, 5% CO 2 incubator
  • 50‐ml conical tubes (Costar, cat. no. 430828)
  • 75‐cm2 flasks (BD Falcon, cat. no. 353136)
  • 1.7‐ml cryopreservation tubes (cryovial, Corning, cat. no. 430488)
  • Isopropanol freezing container

Basic Protocol 2: Use microRNAs as an Enhancer to Reprogram Mouse Embryonic Fibroblasts

  Materials
  • PLAT‐E cells (Cell Biolabs, cat. no. RV‐101)
  • 10% FBS medium without selection drug (see recipe)
  • PLAT‐E culture medium (see recipe)
  • pMX vectors (Oct4, Sox2, Klf4, cMyc; Addgene)
  • Lipofectamine (Invitrogen, cat. no. 18324012)
  • PLUS reagent (Invitrogen, cat. no. 11514015)
  • Basal DMEM medium
  • 0.1% (w/v) gelatin solution
  • MEFs (CF‐1, Oct4‐GFP, etc.; see protocol 1)
  • microRNA mimics (miR‐93, 106b, Dharmacon; Li et al., )
  • Opti‐MEM (Invitrogen, cat. no. 37985070)
  • Lipofectamine 2000 (Invitrogen, cat. no. 11668019)
  • MEF culture medium (see recipe)
  • mES culture medium (see recipe)
  • 15‐ml conical tubes (Costar, cat. no. 430790)
  • 37°C water bath
  • Bench top centrifuge
  • 150‐cm2 tissue culture flasks (BD Biosciences, cat. no. 355001)
  • 37°C, 5% CO 2 incubator
  • 10‐cm tissue culture plates (Corning, cat. no. 430167)
  • Syringe filters, 0.22‐ and 0.45‐µm (Millipore, cat. nos. SLGP033RS and SLHV033RS)
  • 12‐well tissue culture plates (BD Biosciences, cat. no. 353043)

Basic Protocol 3: Derivation and Characterization of miR‐iPSC Colonies

  Materials
  • MEFs (CF‐1, Oct4‐GFP, etc.; see protocol 1)
  • 0.25% trypsin/EDTA
  • MEF culture medium (see recipe)
  • PBS
  • 0.1% (w/v) gelatin solution
  • mES culture medium (see recipe)
  • 15% ES‐screened FBS, 10% DMSO in DMEM (Invitrogen, cat. no. 11995065)
  • mEB formation medium (see recipe)
  • 4% paraformaldehyde
  • 4‐ to 6‐week‐old female athymus nude mice
  • Avertin
  • Alcohol pads
  • Zinc formalin solution (Fisher, cat. no. 23313096)
  • 150‐cm2 flasks
  • 50‐ml conical tubes
  • Irradiator (RS2000, Rad Source)
  • 12‐well tissue culture plates (BD Biosciences, cat. no. 353043)
  • Microscope
  • Pasteur pipets
  • 96‐well tissue culture plates
  • 20‐µl Gilson pipet
  • 37°C incubator
  • 10‐cm petri dishes (SARSTED, cat. no. 821473001)
  • 6‐well tissue culture plates (BD Biosciences, cat. no. 353046)
  • Additional reagents and equipment for MEFs (see protocol 1)
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Figures

Videos

Literature Cited

Literature Cited
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