Phenotypic Analysis of Human Embryonic Stem Cells

Mark Ungrin1, Michael O'Connor2, Connie Eaves3, Peter W. Zandstra1

1 IBBME, University of Toronto, Toronto, Canada, 2 StemCell Technologies, Inc, Vancouver, British Columbia, 3 Terry Fox Laboratory, BC Cancer Agency, Vancouver, British Columbia
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1B.3
DOI:  10.1002/9780470151808.sc01b03s2
Online Posting Date:  August, 2007
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Human embryonic stem cells (hESCs) are an important tool for the study of developmental biology and may one day serve as a source of cells for regenerative medicine. As no definitive assay for hESC pluripotency is available, surrogate assays that measure markers or properties that have been correlated with hESC developmental potential are used to measure the effects of test conditions on their propagation and differentiation. This unit presents a range of protocols, including visual inspection, flow cytometry, immunofluorescence, quantitative real‐time reverse‐transcriptase PCR, and a colony‐forming assay, as tools to measure the undifferentiated hESC state. The authors discuss the advantages and limitations of the various protocols, and present expected results and discuss potential problems. The development of quantitative assays of hESC developmental potential are critical for our understanding of hESC biology. Curr. Protoc. Stem Cell Biol. 2:1B.3.1‐1B.3.25. © 2007 by John Wiley & Sons, Inc.

Keywords: hESC; marker; Oct4; SSEA‐3; SSEA‐4; Tra‐1‐60; microscopy; flow cytometry; quantitative real‐time PCR

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Visual Observation of hESC Cultures
  • Basic Protocol 2: Flow Cytometric Measurement of hESC Surface Antigens
  • Alternate Protocol 1: Determining Oct4 Intracellular Expression by Flow Cytometry
  • Support Protocol 1: Preparation of a Single‐Cell Suspension of Viable hESCs
  • Basic Protocol 3: Immunofluorescent Staining of Fixed hESCs on Coverslips
  • Alternate Protocol 2: Immunofluorescence Staining for High‐Content Screening
  • Basic Protocol 4: Quantitative Real‐Time Polymerase Chain Reaction (Q‐RT‐PCR)
  • Basic Protocol 5: hESC Colony Forming Cell Assay
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Visual Observation of hESC Cultures

  • Single‐cell suspensions of hESCs ( protocol 4)
  • Hank's buffered saline solution with 2% (v/v) FBS (HF)
  • Appropriate primary and secondary antibodies
  • 7‐aminoactinomycin‐D (7‐AAD), optional
  • DNase, optional
  • 15‐ml microcentrifuge tubes
  • 40‐µm cell strainer
  • Flow cytometer sample tubes (e.g., BD Falcon 352058)
  • Flow cytometer
  • Additional reagents and equipment for preparing a single‐cell suspension of hESCs ( protocol 4)

Basic Protocol 2: Flow Cytometric Measurement of hESC Surface Antigens

  • Single‐cell suspension of hESCs ( protocol 4)
  • Hank's buffered‐saline solution with 2% (v/v) FBS (HF)
  • IntraPrep Permeabilization Kit (Beckman Coulter) containing:
    • Reagent 1
    • Reagent 2
  • Primary antibody: Mouse anti–mouse Oct3/4 antibody (IgG1 isotype; BD Biosciences, cat. no. 611202;
  • Secondary antibody: Goat anti–mouse IgG (Fc specific)‐FITC conjugate (Sigma‐Aldrich, cat. no. F‐2772;
  • DNase, optional
  • 1.5‐ml microcentrifuge tubes (DiaMed Lab Supplies, no. SPE150‐N)
  • Vortex
  • 40‐µm cell strainer
  • FC sample tubes (e.g., BD Falcon 352058)
  • Flow cytometer
  • Additional reagents and equipment for preparing a single‐cell suspension of hESCs ( protocol 4)

Alternate Protocol 1: Determining Oct4 Intracellular Expression by Flow Cytometry

  • Cultures of hESCs in 6‐well plates (grown to ∼80% or as desired for a specific experiment)
  • TrypLE Express (Invitrogen, no. 12604‐013)
  • PBS (Invitrogen)
  • 37°C incubator
  • 15‐ml conical centrifuge tube
  • Hemacytometer
  • Additional reagents and equipment for counting cells (Phelan, )

Support Protocol 1: Preparation of a Single‐Cell Suspension of Viable hESCs

  • Poly‐lysine (L or D), optional
  • Phosphate‐buffered saline (PBS; Invitrogen)
  • Desired substrate (e.g., Matrigel, mouse embryo fibroblasts)
  • hESCs
  • 3.7% (w/v) formaldehyde in PBS
  • Methanol (chilled to −20°C)
  • Appropriate primary and fluorescently labeled secondary antibodies
  • 3% (w/v) BSA in PBS
  • Mounting medium/antifade
  • Cosmetic nail polish
  • Coverslips
  • Glass petri dish
  • Filter paper
  • Beakers
  • Microscope slides

Basic Protocol 3: Immunofluorescent Staining of Fixed hESCs on Coverslips

  • hESCs
  • 3.7% (w/v) formaldehyde in PBS
  • Phosphate‐buffered saline (PBS; Invitrogen)
  • Methanol (chilled to −20°C)
  • 10% (v/v) FBS in PBS
  • Appropriate primary and secondary antibodies Hoechst 33342 (Sigma‐Aldrich, cat. no. B2261), frozen stock at 1 mg/ml
  • Multi‐well tissue culture plates designed for use in microscopy (Perkin Elmer, Packard View Plate, cat. no. 6005182; Fisher Scientific, Corning 96‐well, cat. no. 07‐200‐729)
  • Multichannel pipet
  • Disposable reagent reservoirs
  • Sterile trough
  • 200‐µl pipet
  • Aluminum foil
  • Cellomics ArrayScan VTI (Cellomics,, or equivalent automated microscopy platform); see also CellProfiler, a freely available open‐source automated image analysis package, (Carpenter et al., )

Alternate Protocol 2: Immunofluorescence Staining for High‐Content Screening

  • hESC cultures
  • Trizol reagent
  • DEPC‐treated water
  • 250 ng/µl random hexamers
  • 5× FS buffer
  • RNase inhibitor
  • 0.1 M DTT
  • dNTP mix
  • Superscript II reverse transcriptase
  • cDNA samples
  • Primer pairs (forward and reverse primers can be combined into one working stock with each primer at 20 pmoles/µl; see Table 1.3.2)
  • SYBR Green Master Mix
  • Nuclease‐free tubes
  • GeneAmp PCR System 9700 (or equivalent)
  • ABI 96‐well optical reaction plate and adhesive cover
  • Applied Biosystems 7500 Real Time PCR System (or equivalent)
  • Additional reagents and equipment for measuring RNA concentration (Gallagher and Desjardins, )
    Table 1.0.2   Materials   Primers Used for the Most Commonly Studied Gene Products d   Primers Used for the Most Commonly Studied Gene Products

    Gene symbol Accession number Forward primer Reverse primer

     dDue to the presence of pseudogenes for some of these genes, some of the above primer sequences also match pseudogene sequences.

Basic Protocol 4: Quantitative Real‐Time Polymerase Chain Reaction (Q‐RT‐PCR)

  • hESCs cultured in 6‐well plates
  • TrypLE Express (Invitrogen, cat. no. 12604‐013)
  • Phosphate‐buffered saline (PBS; Invitrogen)
  • 35‐mm dish containing mouse embryonic fibroblasts or Matrigel with MEF‐conditioned medium
  • Alkaline phosphatase detection kit (Sigma‐Aldrich, cat. no. 86R‐1KT).
  • 15‐ml conical tubes
  • 40‐µm cell strainer
  • Hemacytometer
  • Standard optical microscope
  • Additional reagents and solutions for counting cells using a hemacytometer (Phelan, )
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Literature Cited

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