Isolation of Human Embryonic Stem Cell–Derived Teratomas for the Assessment of Pluripotency

Karin Gertow1, Stefan Przyborski2, Jeanne F. Loring3, Jonathan M. Auerbach4, Olga Epifano4, Timo Otonkoski5, Ivan Damjanov6, Lars Ährlund‐Richter7

1 Monash Immunology and Stem Cell Laboratories, Monash University, Australia, 2 School of Biological and Biomedical Science, University of Durham, Durham, United Kingdom, 3 Burnham Institute for Medical Research, LaJolla, California, 4 GlobalStem Inc., Rockville, Maryland, 5 Hospital for Children and Adolescents and the Biomedicum Stem Cell Center, University of Helsinki, Finland, 6 Department of Pathology, The University of Kansas, School of Medicine, Kansas City, 7 Department of Woman and Child Health, Karolinska Institute, Stocholm, Sweden.
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1B.4
DOI:  10.1002/9780470151808.sc01b04s3
Online Posting Date:  October, 2007
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Abstract

This unit describes protocols on how to assess the developmental potency of human embryonic stem cells (hESCs) by performing xenografting into immunodeficient mice to induce teratoma formation. hESCs can be injected under the testis capsule, or alternatively into the kidney or subcutaneously. Teratomas that develop from grafted hESCs are surgically removed, fixed in formaldehyde, and paraffin embedded. The tissues in the teratoma are analyzed histologically to determine whether the hESCs are pluripotent and form tissues derived from of all three embryonic germ layers (ectoderm, mesoderm, and endoderm). Teratomas can also be fixed in Bouin's or cryosectioned for analysis, and they can be analyzed by immunohistochemistry for tissue markers. Methods for these procedures are included in this unit. Curr. Protoc. Stem Cell Biol. 3:1B.4.1‐1B.4.29. © 2007 by John Wiley & Sons, Inc.

Keywords: human embryonic stem cells; pluripotency; teratoma; immunodeficient mice

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Injection of hESC Under the Testis Capsule in Immunodeficient Mice
  • Alternate Protocol 1: Injection of hESC Under the Kidney Capsule
  • Alternate Protocol 2: Subcutaneous Injection of hESC
  • Support Protocol 1: Preparation of hESC for Injection
  • Support Protocol 2: Anesthesia for the Mouse
  • Basic Protocol 2: Excision and Fixation for Paraffin Embedding of the hESC Teratoma
  • Alternate Protocol 3: Bouin's Fixation of Teratomas
  • Alternate Protocol 4: Tissue Cryopreservation and Preparation for Cryo–Microtome Sectioning
  • Basic Protocol 3: Evaluation of Tissue Formation and Demonstration of the Presence of Embryonic Germ Layers in the hESC Teratoma
  • Basic Protocol 4: Paraformaldehyde Fixation and Preparation of Tissues for Immunohistochemistry
  • Basic Protocol 5: Preparation of Tissues for mRNA Expression Analysis
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Injection of hESC Under the Testis Capsule in Immunodeficient Mice

  Materials
  • hESCs (see protocol 4)
  • Mice (immunodeficient; either from immunosuppressive treatment, or genetic mutation; see )
  • 70% ethanol
  • 0.015 mg/ml Temgesic (Buprenorphinum)
  • Stereomicroscope for harvesting hESCs in the animal surgery room (optional depending on procedure used for the harvest of hESCs; see protocol 4)
  • Sterile paper tissue
  • Electric clippers
  • Sterile drapes
  • 2 curved forceps
  • Small surgical scissors
  • Dissecting microscope
  • 1‐ml syringe (e.g., U‐100 Micro‐Fine 12.7‐mm; Becton Dickinson), or a Hamilton syringe
  • Needle holder
  • Culture dish
  • Resorbable sutures (e.g., Ethicon, Vicryl V422 4‐0)
  • 9‐mm stainless steel wound clips (autoclips from MikRon Precision)
  • Clip applier
  • Additional reagents and equipment for anesthetizing the recipient mouse ( protocol 5)
NOTE: Protocols for surgical opening of the abdomen require the use of sterile instruments, surgical gloves, and aseptic procedures to minimize the risk of post‐surgical infection.

Alternate Protocol 1: Injection of hESC Under the Kidney Capsule

  • Phosphate buffered saline, calcium‐ and magnesium‐free (CMF‐PBS)
  • 21‐G needle
NOTE: Subcutaneous injection of cells does not require anesthesia.

Alternate Protocol 2: Subcutaneous Injection of hESC

  Materials
  • Cultures of hESC
  • Enzyme for harvesting
  • Dissecting microscope or stereomicroscope
  • 1‐ml insulin syringe
  • Mouth pipet
  • Thick needle
  • Equipment for enzymatic splitting
  • Stereomicroscope
  • Centrifuge

Support Protocol 1: Preparation of hESC for Injection

  Materials
  • Recipient animals
  • Anesthetics (in compliance with local guidelines for major surgery): e.g., isoflurane (1‐chloro‐2.2.2‐trifluoroethyl difluoromethyl ether)
  • Ophthalmic ointment or artificial tears
  • Hypnorm (fentanyl/fluanisone)
  • Anesthesia unit: induction chamber (0.8‐liter) and maintenance mask (e.g., Univentor 400; http://www.univentor.com)
  • Surgical tape

Support Protocol 2: Anesthesia for the Mouse

  Materials
  • Mouse with teratoma
  • 70% ethanol
  • Sodium pentobarbitone (for perfusion fixation experiments)
  • 4% (w/v) buffered formaldehyde or paraformaldehyde (PFA) in saline
  • Paraffin wax
  • Sterile paper tissue
  • Scissors
  • Forceps curved and straight
  • 10‐cm dish
  • Razor blade
  • 50‐ml tubes
  • Additional reagents and equipment for euthanizing the mouse (Donovan and Brown, ) and fixation, embedding, and sectioning tissues (http://home.primus.com.au/royellis/histo.html or Hofman, )
CAUTION: Formaldehyde is an irritant; avoid skin contact and inhalation of its vapors.

Basic Protocol 2: Excision and Fixation for Paraffin Embedding of the hESC Teratoma

  • Bouin's fixative: 70% (v/v) saturated picric acid (Sigma); 25% (v/v) of 37% to 40% formaldehyde; 5% (v/v) glacial acetic acid (Sigma)
  • Additional reagents and equipment for fixation, embedding, and sectioning tissues (http://home.primus.com.au/royellis/histo.html) and cryosectioning (Hofman, )

Alternate Protocol 3: Bouin's Fixation of Teratomas

  Materials
  • Freshly excised teratoma tissue
  • Cryomount ( e.g., TissueTek OCT; Sakura)
  • Liquid nitrogen
  • Razor blade
  • Specimen holder/Cryomould
  • Long forceps
  • Cryotube
  • Cryomicrotome
  • SuperFrost+ slides
CAUTION: Use protective gloves and glasses when handling liquid nitrogen. Use long forceps to place holders/cryotubes in liquid nitrogen.

Alternate Protocol 4: Tissue Cryopreservation and Preparation for Cryo–Microtome Sectioning

  Materials
  • hESC‐derived teratomas
  • 4% (w/v) paraformaldehyde (Sigma; 4% (w/v) formaldehyde may also be used)
  • Phosphate‐buffered saline (PBS, Sigma)
  • 60%, 70%, 90%, and 95% ethanol
  • Absolute ethanol
  • Histoclear (Sigma) or xylene
  • Paraffin wax and appropriate molds
  • 10 mM citrate buffer (Sigma), pH 6
  • Blocking/washing solution: 1% (w/v) bovine serum albumin (BSA, Sigma)/0.2% (v/v) Triton‐X‐100 (Sigma)/5% (v/v) normal goat serum (Sigma) in PBS
  • Primary antibody
  • Secondary labeled (e.g., FITC‐conjugated) antibody
  • Mounting medium: DPX (Sigma) or aqueous mountant (Vectorlabs)
  • Rotary microtome
  • Microscope slides and coverslips (electrostatically charged for improved section adhesion, Sigma)
  • Microwave oven
  • Fluorescence microscope
  • Digital camera and associated imaging software
  • Additional reagents and equipment for fixation, embedding, and sectioning tissues (http://home.primus.com.au/royellis/histo.html or Hofman, )

Basic Protocol 3: Evaluation of Tissue Formation and Demonstration of the Presence of Embryonic Germ Layers in the hESC Teratoma

  Materials
  • hESC‐derived teratoma, surgically removed
  • Liquid nitrogen (alternatively dry ice/ethanol bath)
  • RNA purification kit (Ambion)
  • Reverse transcriptase
  • Labeled nucleotide
  • Primer composed of oligo(dT) fused to a bacteriophage T7 promoter
  • T7 polymerase
  • Uridine triphosphate (UTP) and Biotin‐16‐UTP (e.g., Perkin Elmer Life and Analytical Sciences)
  • RNA amplification kit (e.g., the Illumina RNA Amplification kit; Ambion)
  • Amersham Fluorolink streptavidin‐Cy3 (GE Healthcare Bio‐Sciences)
  • Cryostat
  • 1.5‐ml nuclease‐free microcentrifuge tubes
  • Microarray chip (e.g., the Refseq 6 BeadChip; Illumina, Inc)
  • Confocal scanner and software (e.g., Illumina BeadArray Reader confocal scanner and software (Illumina BeadArray)
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Figures

Videos

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Key References
   Hofman, F. 2002. See above.
  Detailed knowledge on histology and collections of protocols used in histology.
   Van Zutphen, L.F.M., Baumans, V., Beynes, A.C. 2001. Principles of Laboratory Animal Science; Revised edition. Amsterdam, Netherlands.
  This book covers the main theoretical aspects of laboratory animal science.
   Waynforth and Flecknell, 1999. See above.
  This book covers standard surgical procedures
Internet Resources
  http://iacuc.cwru.edu/policy/nihpolicies/surguide.htm
  To learn more about animal experimentation, particularly rodent surgery, the authors recommend NIH Guidelines for Rodent Surgery.
  http://home.primus.com.au/royellis/histo.html
  Detailed knowledge on histology and collections of protocols used in histology.
  http://www.ncbi.nlm.nih.gov/geo
  Published expression patterns of adult tissues.
  http://www.stemcellcommunity.org
  Provides expression profiles of the same genes in the HESCs.
  http://www‐stat.stanford.edu/∼tibs/SAM/
  Further reading on Significance Analysis for Microarrays (SAM) used to obtain lists of genes that are up or down regulated within a given microarray dataset.
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