Preparation of Defined Human Embryonic Stem Cell Populations for Transcriptional Profiling

Qi Zhou1, Hun Chy1, Andrew L. Laslett2

1 Commonwealth Scientific and Industrial Research Organization (CSIRO), Molecular and Health Technologies, Clayton, Australia, 2 Department of Anatomy and Developmental Biology, Monash University, Clayton, Australia
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1B.7
DOI:  10.1002/9780470151808.sc01b07s14
Online Posting Date:  July, 2010
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Abstract

This unit describes a useful approach to preparing highly reproducible samples of human embryonic stem cell (hESC) total RNA suitable for transcriptional profiling from heterogeneous mixtures of cells containing undifferentiated hESC and differentiated cell types. In this unit, fluorescence‐activated cell sorting (FACS) is used to sub‐fractionate hESC populations on the basis of their levels of co‐expression of two previously published hESC surface markers, CD9(TG30) and GCTM‐2. This sub‐fractionation allows for the separation of undifferentiated hESC (CD9hi, GCTM‐2hi) from the early stages in hESC differentiation (CD9neg or low, GCTM‐2neg or low). Curr. Protoc. Stem Cell Biol. 14:1B.7.1‐1B.7.15. © 2010 by John Wiley & Sons, Inc.

Keywords: human embryonic stem cells; cell surface markers; fluorescence‐activated cell sorting

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Enzymatic Passaging of Cultures of hESCs
  • Alternate Protocol 1: Starting hESC Bulk Culture from Maintenance Culture
  • Basic Protocol 2: Harvesting hESCs from Bulk Cultures
  • Basic Protocol 3: Immunofluorescent Staining of Surface Antigens on Live hESCs for FACS
  • Alternate Protocol 2: Combined Detection of OCT‐4 Intracellular Expression and Other Cell Surface Markers on Fixed hESC by Flow Cytometry
  • Basic Protocol 4: RNA Isolation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Enzymatic Passaging of Cultures of hESCs

  Materials
  • hESCs in culture
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS)
  • Collagenase I solution (4 mg/ml in DMEM/F‐12)
  • Knock‐out serum replacement (KSR) medium (see recipe)
  • Mouse embryonic fibroblast (MEF) feeder cells (Pera et al., ; unit 1.3) plated on gelatinized flasks at 1.2 × 104 cells/cm2
  • Inverted microscope
  • Dissecting microscope
  • 37°C incubator
  • 10‐ml pipet
  • Cell scraper, optional
  • 15‐ml tubes
  • Centrifuge

Alternate Protocol 1: Starting hESC Bulk Culture from Maintenance Culture

  Materials
  • Mechanically passaged hESCs in organ culture dishes
  • PBS with Ca++ and Mg++ (PBS+)
  • Dispase solution (10 mg/ml in hESC medium; see recipe), ice cold
  • Gelatinized 25‐cm2 flasks seeded with 1.2 × 104 cells/cm2 inactivated MEFs (unit 1.3)
  • Knock‐out serum replacement (KSR) medium (see recipe)
  • Dissecting microscope with warm stage
  • 4‐well culture dish (Nunc, cat. no. 176740)
  • Pulled glass capillary cutter or a 26‐G needle attached to a 3‐ml syringe or a sterilized ultra sharp splitting blade
  • Micropipet tips
  • 20‐µl or 200‐µl micropipet tip
  • 1.5‐ml microcentrifuge tubes

Basic Protocol 2: Harvesting hESCs from Bulk Cultures

  Materials
  • hESCs in bulk cultures ( protocol 1)
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS)
  • TrypLE Express (Invitrogen, cat. no. 12604)
  • 20% (v/v) fetal bovine serum (FBS; Invitrogen, cat. no. 16000‐044) in DMEM/F12 medium (chilled to 4°C)
  • 37°C incubator
  • 35‐µm cell strainer (BD Biosciences, cat. no. 352235)
  • Centrifuge
  • Additional reagents and equipment for counting cells using a hemacytometer (unit 1.3)

Basic Protocol 3: Immunofluorescent Staining of Surface Antigens on Live hESCs for FACS

  Materials
  • Harvested bulk hESCs ( protocol 3)
  • Mouse anti‐CD9 (TG30) monoclonal antibody (Millipore, cat. no. MAB4427)
  • Mouse anti‐GCTM2 monoclonal antibody (0.4 mg/ml; kind gift from Martin Pera)—a commercially available alternative, which detects the same antigen is the antibody TG343 (Millipore, cat. no. MAB4346)
  • PE rat anti‐mouse CD 90.2 (0.2 mg/ml; BD Pharmingen, cat. no. 553014)
  • Mouse IgG2a isotype control (0.5 mg/ml; BD Pharmingen, cat. no. 554121)
  • Mouse IgM isotype control (0.5 mg/ml; BD Pharmingen, cat. no. 553472)
  • 20% (v/v) fetal bovine serum (FBS) in DMEM/F12 medium (chilled to 4°C)
  • Alexa Fluor 488 goat anti–mouse IgG2a (2 mg/ml; Invitrogen, cat. no. A21131)
  • Alexa Fluor 647 goat anti–mouse IgM (2 mg/ml; Invitrogen, cat. no. A21238)
  • R‐phycoerythrin goat anti–mouse IgG2a conjugate (1.0 mg/ml; Invitrogen, cat. no. P21139)
  • Propidium iodide solution (1.0 mg/ml; Sigma, cat. no. P4864)
  • Spherotech 8 peak Ultra Rainbow beads
  • Centrifuge
  • 35‐µm cell strainer (BD Biosciences, cat. no. 352235)
  • FACSVantage DiVa (Becton Dickinson) or equivalent
NOTE: Both primary and secondary antibody staining and washing are carried out in 20% FBS medium prechilled to 4°C. For 1.5 × 107 cells (recommended cell number for test stainings; fewer cells can be used in control tubes, e.g., 50,000 cells/tube); 10 ml and 2 ml medium are used for primary and secondary antibody staining respectively.

Alternate Protocol 2: Combined Detection of OCT‐4 Intracellular Expression and Other Cell Surface Markers on Fixed hESC by Flow Cytometry

  Materials
  • Harvested hESCs ( protocol 3)
  • 2% (w/v) paraformaldehyde (PFA) solution in CMF‐PBS, pH 7.4
  • 10% (v/v) goat serum in CMF‐PBS
  • Phosphate‐buffered serum, calcium‐ and magnesium‐free (CMF‐PBS)
  • 0.1% (v/v) Triton X‐100 solution in CMF‐PBS
  • Mouse anti‐Oct4 monoclonal antibody(1 mg/ml; Chemicon, cat. no. MAB4401)
  • R‐phycoerythrin goat anti‐mouse IgG1 conjugate (1 mg/ml; Invitrogen, cat. no. P21129)
  • Mouse IgG1 isotype control (0.5 mg/ml; BD Pharmingen, cat. no. 553447)
  • Centrifuge
  • 35‐µm cell strainer (BD Biosciences, cat. no. 352235)

Basic Protocol 4: RNA Isolation

  Materials
  • hESC bulk cultures ( protocol 3)
  • RNeasy Mini kit (Qiagen, cat. no. 74104)
  • RNase‐free H 2O
  • Nanodrop ND 1000 spectrophotometer (Thermo Fisher Scientific)
  • RNase‐free tubes (PCR tubes)
NOTE: Keep all reagents, working solutions, and master mixes on ice during these procedures.
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Figures

Videos

Literature Cited

Literature Cited
   Boyer, L.A., Lee, T.I., Cole, M.F., Johnstone, S.E., Levine, S.S., Zucker, J.P., Guenther, M.G., Kumar, R.M., Murray, H.L., Jenner, R.G., Gifford, D.K., Melton, D.A., Jaenisch, R., and Young, R.A. 2005. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122:947‐956.
   Chambers, I., Colby, D., Robertson, M., Nichols, J., Lee, S., Tweedie, S., and Smith, A. 2003. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113:643‐655.
   Filipczyk, A.A., Laslett, A.L., Mummery, C., and Pera, M.F. 2007. Differentiation is coupled to changes in the cell cycle regulatory apparatus of human embryonic stem cells. Stem Cell Res. 1:45‐60.
   Hough, S.R., Laslett, A.L., Grimmond, S.B., Kolle, G., and Pera, M.F. 2009. Metastable states of pluripotency in human embryonic stem cells. PLOS ONE 4:e7708.
   Kolle, G., Ho, M., Zhou, Q., Chy, H.S., Krishnan, K., Cloonan, N., Bertoncello, I., Laslett, A.L., and Grimmond, S.M. 2009. Identification of human embryonic stem cell surface markers by combined membrane‐polysome translation state array analysis and immunotranscriptional profiling. Stem Cells 27:2446‐2456.
   Laslett, A.L., Filipczyk, A., and Pera, M.F. 2003. Characterization and culture of human embryonic stem cells. Trends Cardiovasc. Med. 13:295‐301.
   Laslett, A.L., Grimmond, S., Gardiner, B., Stamp, L., Lin, A., Hawes, S.M., Wormald, S., Nikolic‐Paterson, D., Haylock, D., and Pera, M.F. 2007. Transcriptional analysis of early lineage commitment in human embryonic stem cells. BMC Dev. Biol. 7:12.
   Mitsui, K., Tokuzawa, Y., Itoh, H., Segawa, K., Murakami, M., Takahashi, K., Maruyama, M., Maeda, M., and Yamanaka, S. 2003. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113:631‐642.
   Pera, M.F., Filipczyk, A., Hawes, S.M., and Laslett, A.L. 2003. Isolation, characterization, and differentiation of human embryonic stem cells. Methods Enzymol. 365:429‐446.
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