Generation and Characterization of Human Induced Pluripotent Stem Cells

Mari Ohnuki1, Kazutoshi Takahashi2, Shinya Yamanaka3

1 Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan, 2 Institute for Integrated Cell‐Material Sciences, Kyoto University, Kyoto, Japan, 3 Gladstone Institute of Cardiovascular Disease, San Francisco, California
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 4A.2
DOI:  10.1002/9780470151808.sc04a02s9
Online Posting Date:  June, 2009
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Abstract

This unit describes how to generate human induced pluripotent stem (iPS) cells and evaluate the qualities of the generated iPS cells. The methods for establishment and maintenance of human iPS cells are similar to those for mouse iPS cells but not identical. In addition, these protocols include excellent procedures for passaging and cryopreservation of human iPS cells established by ES cell researchers, which result in an easy way to culture human iPS cells. Moreover, we include methods for characterizing iPS cells for further research. RT‐PCR and immunocytochemistry for detection of pluripotent cell markers, embryoid body differentiation, and teratoma differentiation are used to determine pluripotency in vitro and in vivo, respectively. Curr. Protoc. Stem Cell Biol. 9:4A.2.1‐4A.2.25. © 2009 by John Wiley & Sons, Inc.

Keywords: reprogramming; pluripotency; iPS cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Generation of iPS Cells
  • Support Protocol 1: Preparation of SNL Feeder Cells
  • Support Protocol 2: Preparation of PLAT‐E Packaging Cells
  • Basic Protocol 2: Passage of iPS Cells
  • Basic Protocol 3: Storage of Established iPS Cells
  • Support Protocol 3: RT‐PCR for Detection of Pluripotent Cell Markers
  • Support Protocol 4: Immunocytochemistry for Pluripotent Cell Markers
  • Support Protocol 5: Assessing Pluripotency by In Vitro Differentiation of iPS Cells by Embryoid Body Formation
  • Support Protocol 6: Assessing Pluripotency by In Vivo Differentiation by Teratoma Formation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Generation of iPS Cells

  Materials
  • 293FT cells for producing lentivirus (Invitrogen; see manufacturer‐provided protocol for culture)
  • Dulbecco's phosphate‐buffered saline without calcium and magnesium (CMF‐DPBS: Nacalai Tesque, cat. no. 14249‐95)
  • 0.25% trypsin/1 mM EDTA solution (Invitrogen, cat. no. 25200‐056)
  • 293FT medium (see recipe)
  • OPTI‐MEM I medium (Invitrogen, cat. no. 31985‐062)
  • ViraPower packaging mix (from ViraPower expression system kit; Invitrogen, cat. no. K4990‐00)
  • pLenti6/UbC containing mouse Slc7a1 gene (Addgene; http://www.addgene.org/Shinya_Yamanaka)
  • Lipofectamine 2000 (Invitrogen, cat. no. 11668‐019)
  • 10% FBS medium (see recipe)
  • Human fibroblast cells (available from the following sources:
    • Cell Applications Inc. (http://www.cellapplications.com/)
    • Lonza (http://www.lonza.com/group/en.html)
    • American Type Culture Collection (ATCC, http://www.atcc.org/)
    • European Collection of Cell Cultures (ECACC; http://www.ecacc.org.uk/)
    • Riken Bioresource Center (http://www.brc.riken.jp/)
    • Japanese Collection of Research Bioresources (http://cellbank.nibio.go.jp/)
  • 0.05% trypsin/0.53 mM EDTA solution (Invitrogen, cat. no. 25300‐054)
  • Hexadimethrine bromide (polybrene; Nacalai Tesque, cat. no. 17736‐44)
  • PLAT‐E packaging cells ( protocol 3)
  • pMXs retroviral vectors encoding OCT3/4, SOX2, KLF4, and/or c‐myc (Addgene; http://www.addgene.org/Shinya_Yamanaka):
    • pMXs‐hOCT3/4
    • pMXs‐hSOX2
    • pMXs‐hKLF4
    • pMXs‐hc‐MYC
  • pMXs retroviral vector encoding the green fluorescence protein (GFP) to monitor transfection efficiency and serve as a negative control for iPS cell induction (Cell Biolabs, Inc.)
  • Fugene 6 transfection reagent (Roche, cat. no. 1 814 443)
  • Mitomycin C–treated SNL feeder cell plates, 100‐mm and 24‐well ( protocol 2)
  • hES cell medium (see recipe)
  • Recombinant basic fibroblast growth factor, human (bFGF; Wako, cat. no. 064‐04541)
  • 100‐mm tissue culture dish (Falcon, cat. no. 353003)
  • 0.45‐µm pore size cellulose acetate filter (Whatman, cat. no. FP30/0.45 CA‐S)
  • 96‐well tissue culture plate (Falcon, cat. no. 351172)
  • 24‐well tissue culture plate (Falcon, cat. no. 353047)
  • Additional reagents and equipment for counting cells (unit 1.3) and preparation of PLAT‐E packaging cells ( protocol 3)

Support Protocol 1: Preparation of SNL Feeder Cells

  Materials
  • Frozen vial of SNL feeder cells (McMahon and Bradley, ): available from Dr. Allan Bradley of the Sanger Institute (http://www.sanger.ac.uk/Teams/faculty/bradley/)
  • SNL medium (see recipe)
  • Dulbecco's phosphate‐buffered saline without calcium and magnesium (CMF‐DPBS: Nacalai Tesque, cat. no. 14249‐95)
  • 0.25% trypsin/1 mM EDTA solution (Invitrogen, cat. no. 25200‐056)
  • 0.4 mg/ml mitomycin C (see recipe)
  • Centrifuge
  • Gelatin‐coated 100‐mm tissue culture dish (see recipe)
  • Other gelatin‐coated culture vessels (see recipe) as needed
  • Additional reagents and equipment for counting cells (unit 1.3)

Support Protocol 2: Preparation of PLAT‐E Packaging Cells

  Materials
  • Frozen vial of PLAT‐E packaging cells (Morita et al., ): available from Dr. Toshio Kitamura at the University of Tokyo ( ) or Cell Biolabs, Inc. (http://www.cellbiolabs.com/)
  • 10% FBS medium (see recipe)
  • Puromycin stock solution (see recipe)
  • Blasticidin S stock solution (see recipe)
  • Dulbecco's phosphate‐buffered saline without calcium and magnesium (CMF‐DPBS: Nacalai Tesque, cat. no. 14249‐95)
  • 0.05% (w/v) trypsin/0.53 mM EDTA solution (Invitrogen, cat. no. 25300‐054)
  • 100‐mm tissue culture dishes
  • Centrifuge
  • 15‐ml conical centrifuge tubes

Basic Protocol 2: Passage of iPS Cells

  Materials
  • Human iPS cells at 80% to 90% confluency in a 24‐well plate ( protocol 1)
  • Dulbecco's phosphate‐buffered saline without calcium and magnesium (CMF‐DPBS: Nacalai Tesque, cat. no. 14249‐95)
  • CTK solution (see recipe)
  • hES medium (see recipe)
  • 6‐well plate seeded with mitomycin C–treated SNL cells ( protocol 2)
  • Sterile disposable cell scraper
  • 15‐ml conical centrifuge tube

Basic Protocol 3: Storage of Established iPS Cells

  Materials
  • 10 mM Y‐27632 (Wako, cat. no. 253‐00513)
  • Confluent iPS cells (see Basic Protocols protocol 11 and protocol 42) in 100‐mm dishes
  • Dulbecco's phosphate‐buffered saline without calcium and magnesium (CMF‐DPBS: Nacalai Tesque, cat. no. 14249‐95)
  • CTK solution (see recipe)
  • hES medium (see recipe)
  • DAP123 solution (see recipe)
  • Liquid nitrogen
  • 60‐mm dish seeded with mitomycin C–treated SNL feeder cells ( protocol 2)
  • Sterile disposable cell scraper
  • 2‐ml cryovials
  • Liquid nitrogen tank

Support Protocol 3: RT‐PCR for Detection of Pluripotent Cell Markers

  Materials
  • Human iPS cells cultured in 6‐well plate (Basic Protocols protocol 11 and protocol 42), 80% to 90% confluent
  • Dulbecco's phosphate‐buffered saline without calcium and magnesium (CMF‐DPBS: Nacalai Tesque, cat. no. 14249‐95)
  • Trizol reagent (Invitrogen, cat. no. 15596‐026)
  • Chloroform (Nacalai Tesque)
  • Isopropanol (Nacalai Tesque)
  • 70% ethanol in nuclease‐free water
  • Nuclease‐free (e.g., Milli‐Q) water
  • Turbo DNA‐free Kit (Ambion, cat. no. AM1907) containing:
    • 10× DNase I buffer
    • Recombinant DNase I
    • DNase Inactivation Reagent
  • ReverTra Ace ‐α‐ kit (Toyobo, cat no. FSK‐101; http://www.toyobo.co.jp/) containing:
    • 5× reverse transcription buffer (containing 25 mM Mg2+)
    • 10 mM dNTPs
    • Recombinant ribonuclease inhibitor (10 U/µl)
    • Reverse transcriptase
    • Oligo dT 20 primer (10 pmol/µl)
  • ExTaq kit (Takara, cat. no. RR001A; http://www.takara‐bio.us) containing:
    • ExTaq DNA polymerase (5 U/µl)
    • 10× ExTaq buffer
    • 2.5 mM dNTPs
  • PCR primers for human ES cell markers (Figure )
  • 15‐ml conical tubes (Falcon)
  • Centrifuge
  • Nanodrop spectrometer (Thermo Scientific)
  • Filtered pipet tips 10‐µl, 200‐µl, and 1000‐µl (RNase‐free, Watson)
  • 1.5‐ml microcentrifuge tubes, RNase‐free
  • 0.2‐ml PCR reaction tubes (Greiner)
  • Thermal cycler (Applied Biosystems)
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, )
NOTE: Use nuclease‐free water to make all the reagents. Milli‐Q water or equivalent grade of ultrapure water can be used for the experiments with RNA. Wear disposable gloves and mask.

Support Protocol 4: Immunocytochemistry for Pluripotent Cell Markers

  Materials
  • Human iPS cells ( protocol 1)
  • 6‐well plates seeded with mitomycin C–treated feeder cells ( protocol 2)
  • hES medium (see recipe)
  • Dulbecco's phosphate‐buffered saline without calcium and magnesium (CMF‐DPBS: Nacalai Tesque, cat. no. 14249‐95)
  • CMF‐DPBS containing 10% (v/v) formalin
  • CMF‐PBS containing 1% (w/v) bovine serum albumin, 5% (v/v) normal goat serum (or donkey serum), and 0.2% (v/v) Triton X‐100 (omit Triton if staining surface antigens)
  • Primary antibodies against desired ES markers (perform all dilutions in CMF‐DPBS containing 1% v/v bovine serum albumin):
    • Anti‐Nanog goat polyclonal (R&D Systems, cat. no. AF1997; use at 1:20 dilution)
    • Anti‐SSEA‐1 mouse IgM (Developmental Studies Hybridoma Bank, cat. no. MC480; use at 1:5 dilution)
    • Anti‐SSEA‐3 rat IgM (Developmental Studies Hybridoma Bank cat. no. MC631; use at 1:5 dilution)
    • Anti‐TRA‐1‐60 mouse IgM (Chemicon, cat. no. MAB4630; use at 1:50 dilution)
    • Anti‐TRA‐1‐81 mouse IgM (Chemicon, cat no. MAB4381; use at 1:50 dilution)
  • Secondary antibody against IgG or IgM of species in which primary antibody was raised, labeled with Alexa Fluor 488 or Alexa Fluor 546; use at 1:500 dilution in CMF‐DPBS containing 1% (w/v) bovine serum albumin
  • 10 mg/ml Hoechst 33342 (H3570, Invitrogen)

Support Protocol 5: Assessing Pluripotency by In Vitro Differentiation of iPS Cells by Embryoid Body Formation

  Materials
  • 10 mg/ml HEMA‐MMA (see recipe)
  • Growing human iPS cells (Basic Protocols protocol 11 and protocol 42) at 80% to 90% confluency in 60‐mm dish
  • Dulbecco's phosphate‐buffered saline without calcium and magnesium (CMF‐DPBS: Nacalai Tesque, cat. no. 14249‐95)
  • CTK solution (see recipe)
  • hES medium containing no bFGF (see recipe)
  • CMF‐PBS containing 10% (v/v) formalin (Sigma)
  • CMF‐PBS containing 1% (w/v) bovine serum albumin, 5% (v/v) normal goat serum (or donkey serum), and 0.2% (v/v) Triton X‐100
  • Primary antibodies against desired ES markers for immunohistochemistry (perform all dilutions in CMF‐PBS containing 1% v/v bovine serum albumin):
    • Anti‐α‐fetoprotein mouse IgG (R&D Systems, cat. no. MAB1368; use at 1:100 dilution)
    • Anti‐α‐smooth muscle actin mouse IgG (Dako, cat. no. N1584; use at 1:500 dilution)
    • Anti‐βIII‐tubulin mouse IgG (Chemicon, cat. no. CB412; use at 1:100 dilution)
  • Secondary antibody: anti‐mouse IgG labeled with Alexa Fluor (use at 1:500 dilution in CMF‐DPBS containing 1% w/v bovine serum albumin)
  • 10 mg/ml Hoechst 33342 solution (Invitrogen)
  • 100‐mm tissue culture dish
  • Sterile disposable cell scraper
  • 15‐ml conical centrifuge tubes
  • Gelatin‐coated 6‐well culture plate (see recipe)
  • Additional reagents and equipment for immunohistochemistry ( protocol 7)

Support Protocol 6: Assessing Pluripotency by In Vivo Differentiation by Teratoma Formation

  Materials
  • 10 mM Y‐27632 (Wako, cat. no. 253‐00513)
  • Growing iPS cells (Basic Protocols protocol 11 and protocol 42) at 80% to 90% confluency in 60‐mm dish
  • Dulbecco's phosphate‐buffered saline without calcium and magnesium (CMF‐DPBS: Nacalai Tesque, cat. no. 14249‐95)
  • CTK solution (see recipe)
  • hES medium (see recipe)
  • DMEM/F12 medium (e.g., Invitrogen) supplemented with 10 µM Y‐27632
  • 1.2% tribromoethanol (Avertin): dissolve 2.5 g tribromoethanol in 5 ml butanol, then add 200 ml distilled water; store at 4°C in the dark
  • SCID mice, (7‐ to 8‐weeks, male)
  • 70% ethanol
  • CMF‐PBS containing 10% formalin
  • Sterile disposable cell scrapers
  • 15‐ml conical centrifuge tubes
  • Centrifuge
  • Hamilton syringe
  • 25‐G to 26‐G needle (Terumo)
  • Suture needle with thread
  • Additional reagents and equipment for intraperitoneal injection (Donovan and Brown, ) and euthanasia of the mouse (Donovan and Brown, ), paraffin embedding and sectioning of tissue, and hematoxylin/eosin staining of tissue sections (unit 2.5)
NOTE: All protocols involving live animals must be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must conform to government regulations for the care and use of laboratory animals.
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Figures

  •   FigureFigure 4.A0.1 Schematic diagram of iPS cell generation. A strategy and approximate time table for human iPS cell generation.
  •   FigureFigure 4.A0.2 Images related to iPS cell induction. (A, B) Images of non‐iPS and ES‐like (iPS) cells, respectively. (C) Image of established iPS cells. Images of immunocytochemistry for undifferentiated pluripotent cell markers: (D) SSEA3; (E) TRA‐1‐60; (F) TRA‐1‐81; (G) Nanog; and (H) SSEA1 (negative). Blue indicates nuclei stained with Hoechst 33342. Images of immunocytochemistry for differentiated cell products: (I) α‐fetoprotein; (J) α‐smooth muscle actin; and (K) βIII‐tubulin. Blue indicates nuclei stained with Hoechst 33342. (L) Image of SCID mouse that had iPS cells injected into the testes 3 months earlier. (M) Image of a dissected teratoma. (N) Image of hematoxylin and eosin–stained teratoma section. Bars = 100 µm. Panels D to H illustrate immunohistochemistry for pluripotent cell markers, while panel I illustrates a marker for endoderm, panel J a marker for mesoderm, and panel K a marker for ectoderm.
  •   FigureFigure 4.A0.3 PCR primers and reaction conditions for pluripotent cell marker analysis.

Videos

Literature Cited

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