Teratoma Formation: A Tool for Monitoring Pluripotency in Stem Cell Research

Raman V. Nelakanti1, Nigel G. Kooreman2, Joseph C. Wu1

1 Institute of Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, 2 Department of Vascular Surgery, Leiden University Medical Center, Leiden
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 4A.8
DOI:  10.1002/9780470151808.sc04a08s32
Online Posting Date:  February, 2015
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This unit describes protocols for evaluating the pluripotency of embryonic and induced pluripotent stem cells using a teratoma formation assay. Cells are prepared for injection and transplanted into immunodeficient mice at the gastrocnemius muscle, a site well suited for teratoma growth and surgical access. Teratomas that form from the cell transplants are explanted, fixed in paraformaldehyde, and embedded in paraffin. These preserved samples are sectioned, stained, and analyzed. Pluripotency of a cell line is confirmed by whether the teratoma contains tissues derived from each of the embryonic germ layers: endoderm, mesoderm, and ectoderm. Alternatively, explanted and fixed teratomas can be cryopreserved for immunohistochemistry, which allows for more detailed identification of specific tissue types present in the samples. © 2015 by John Wiley & Sons, Inc.

Keywords: pluripotent stem cell; induced pluripotent stem cell; embryonic stem cell; teratoma; pluripotency; tumorigenicity

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Table of Contents

  • Introduction
  • Basic Protocol 1: Injection of Pluripotent Stem Cells in the Gastrocnemius Muscle
  • Support Protocol 1: Preparation of Pluripotent Stem Cells for Injection
  • Basic Protocol 2: Preparation of Teratomas for Histological Analysis
  • Alternate Protocol 1: Cryopreservation of Teratomas and Immunofluorescence Staining
  • Reagents and solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Injection of Pluripotent Stem Cells in the Gastrocnemius Muscle

  • PSCs suspended in Matrigel (see protocol 2Support Protocol), on ice
  • Disinfectant (not ethanol‐based)
  • Immunodeficient mice: NOD‐SCID IL2Rgammanull (NSG)
  • Isoflurane (2‐chloro‐2‐(difluoromethoxy)‐1,1,1‐trifluoroethane, Isothesia, Butler Schein cat. no. 029405)
  • Hair removal cream
  • Iodine solution
  • Isopropyl alcohol wipes or 70% (v/v) ethanol
  • Anesthesia unit or knockdown chamber
  • 37°C heating pad
  • Surgical station
  • Sterile surgical drape
  • Nose cone for anesthesia
  • Electric clippers
  • Gauze or paper towel
  • Surgical tape (optional)

Support Protocol 1: Preparation of Pluripotent Stem Cells for Injection

  • Culture or frozen stock of PSCs
  • Phosphate‐buffered saline (PBS), without calcium and magnesium (Life Technologies cat. no. 14190)
  • Human cell dissociation medium: 0.25 mM EDTA in PBS (store up to 6 months at 4°C)
  • Human PSC culture medium (Essential 8 Medium, Life Technologies cat. no. A1517001)
  • Trypsin‐EDTA (TrypLE Express Enzyme, Life Technologies cat. no. 12605010)
  • Mouse PSC culture medium with 10% fetal bovine serum (FBS)
  • Matrigel matrix, growth factor−reduced, phenol red−free (BD Biosciences cat. no. 356231)
  • 10‐cm cell culture plates
  • 15‐ml tubes
  • Insulin syringes (29‐G × 0.5‐in. needle, 3/10 cc, Terumo Medical cat. no. SS30M2913)
  • Additional reagents and equipment for counting cells

Basic Protocol 2: Preparation of Teratomas for Histological Analysis

  • Mouse with teratoma (see protocol 1)
  • 4% (w/v) paraformaldehyde (PFA) in PBS
  • 70%, 90%, 95%, and 100% (v/v) ethanol solutions
  • Xylene
  • Paraffin
  • Surgical pins, scissors, and forceps
  • 50‐ml Falcon tubes
  • Immunohistochemistry cassettes
  • Microtome
  • Microscope slides
  • Additional reagents and equipment for euthanasia

Alternate Protocol 1: Cryopreservation of Teratomas and Immunofluorescence Staining

  • 30% (w/v) sucrose in PBS
  • Optimum cutting temperature (OCT) medium (Electron Microscopy Sciences cat. no. 62550‐01)
  • Isopropanol
  • Dry ice
  • PBS
  • Permeabilizing solution (see recipe)
  • Blocking solution (see recipe)
  • Primary and secondary antibodies
  • Wash solution (see recipe)
  • Mounting medium
  • 50‐ml Falcon tubes
  • Cryomolds
  • Glass container for freezing
  • Cryostat
  • Microscope slides (Superfrost Plus, Fisher Scientific cat. no. 12‐550‐15)
  • Blow dryer
  • Liquid blocker, Pap pen (Sigma‐Aldrich cat. no. z377821; optional)
  • Washing jars with holder for multiple slides
  • Humidified black staining box
  • Aluminum foil
  • Cover slips
  • Additional reagents and equipment for excising and fixing teratomas (see protocol 3)
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Literature Cited

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