Directed Differentiation of Human Embryonic Stem Cells as Spin Embryoid Bodies and a Description of the Hematopoietic Blast Colony Forming Assay

Elizabeth S. Ng1, Richard P. Davis1, Tanya Hatzistavrou1, Edouard G. Stanley1, Andrew G. Elefanty1

1 Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, Victoria, Australia
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1D.3
DOI:  10.1002/9780470151808.sc01d03s4
Online Posting Date:  January, 2008
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Abstract

This unit describes a protocol for the differentiation of human embryonic stem cells (hESCs). To generate spin embryoid bodies (EBs), known numbers of hESCs are deposited into low‐attachment, round‐bottomed 96‐well plates in a serum‐free medium supplemented with growth factors. The cells are then aggregated by centrifugation, initiating formation of EBs of uniform size. The spin EBs generated using this technique differentiate efficiently and synchronously along the lineages preferentially induced by the combinations of growth factors to which the cells are exposed. The 96‐well format permits an assessment of the effects of different combinations of growth factors in the same experiment, facilitating the optimization of differentiation conditions for any given cell type. Up to 40 plates can be set up within a couple of hours by one experimenter, and aliquots of the differentiating EBs can be harvested at intervals and subjected to analyses using a variety of techniques. Curr. Protoc. Stem Cell Biol. 4:1D.3.1‐1D.3.23. © 2008 by John Wiley & Sons, Inc.

Keywords: human embryonic stem cells; hESCs; differentiation; serum‐free differentiation; spin EBs

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Generation of Spin Embryoid Bodies (Stage 1)
  • Basic Protocol 2: Extended Differentiation of Spin EBs (Stage 2)
  • Support Protocol 1: Propagation and Expansion of Human Embryonic Stem Cells in Bulk Culture Prior to Differentiation
  • Support Protocol 2: Harvesting EBs for Further Analysis
  • Support Protocol 3: Identification of Hematopoietic Progenitor Cells in Spin EBs Using a Methylcellulose Colony Forming Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Generation of Spin Embryoid Bodies (Stage 1)

  Materials
  • hESCs in 75‐cm2 or 150‐cm2 tissue culture flasks at enzymatic passages between 5 and 20 (see protocol 3) in hESC medium (see recipe for medium)
  • 75‐ or 150‐cm2 gelatinized (see recipe for gelatization of flasks) tissue culture flasks seeded with mitotically inactivated MEF at 2.0 × 104/cm2 for maintenance of hESC or 0.8–1.2 × 104/cm2 for passaging hESC prior to differentiation
  • Sterile water
  • Serum‐free differentiation medium (see recipe)
  • Cytokines required for differentiation (see reciperecipes)
  • CMF‐PBS (Invitrogen)
  • TrypLE Select cell dissociation enzyme (Invitrogen; see recipe)
  • 0.4% Trypan blue (Fluka)
  • 96‐well round‐bottomed low‐attachment plates and lids (Nunc cat. no. 262162 and lids cat. no. 264122; or Costar cat. no. 3788)
  • 14‐ml sterile centrifuge tubes
  • Tissue culture microscope with phase contrast objectives and phase rings
  • Hemacytometer (Neubauer)
  • Reagent reservoir, optional
  • Multichannel pipets (Finnpipettes, Thermo Electron), optional
  • 96‐well plate spinner attachment for tissue culture centrifuge

Basic Protocol 2: Extended Differentiation of Spin EBs (Stage 2)

  Materials
  • Sterile water
  • 0.1% (w/v) gelatin (see recipe)
  • Spin EBs plated in round‐bottomed, low‐attachment plates (see protocol 1)
  • Differentiation medium (see recipe) with required stage 2 growth factors (see reciperecipes)
  • 96‐well flat‐bottomed, tissue culture–treated plates
  • Flexible tubing attached to a vacuum source (e.g., VacSax, VacSax Ltd.)
  • Stereomicroscope (optional)
  • Sterile reagent reservoir
  • Multichannel pipettors (Finnpipettes, Thermo Electron)

Support Protocol 1: Propagation and Expansion of Human Embryonic Stem Cells in Bulk Culture Prior to Differentiation

  Materials
  • hESCs in 75‐cm2 tissue culture flasks at enzymatic passage 2 or greater
  • Phosphate‐buffered saline without Ca2+ and Mg2+ (CMF‐PBS), sterile
  • 1× TrypZean (TZ solution; Sigma; see recipe) or 1× TrypLE Select (TS solution; Invitrogen; see recipe), sterile
  • Soybean trypsin inhibitor (required if TrypZean is used; Sigma; see recipe), sterile
  • hESC medium (see recipe), sterile
  • 75‐cm2 or 150‐cm2 gelatinized flasks (see recipe) seeded with mitotically inactivated MEFs at 2 × 104 MEFs/cm2 (mitotically inactivated primary mouse embryonic fibroblasts, MEFs; Conner, ; unit 1.3)
  • Tissue culture microscope with phase contrast objectives and phase rings
  • 14‐ml centrifuge tubes
  • Cell scraper (optional), sterile
  • Refrigerated centrifuge

Support Protocol 2: Harvesting EBs for Further Analysis

  Materials
  • Stage 1 cultures (see protocol 1) or stage 2 cultures (see protocol 2)
  • Appropriate lysis buffer for nucleic acid extraction, flow cytometry buffer, or differentiation medium
  • Phosphate buffered saline without Ca2+ and Mg2+ (CMF‐PBS)
  • TrypLE Select (see recipe)
  • 10‐, 14‐, or 50‐ml centrifuge tubes, sterile
  • P200 Gilson pipetman with sterile (plugged) 200‐µl tips
  • Dissecting microscope, optional
  • Multichannel pipettor
  • Pasteur pipets and pipet‐aid
  • 23‐G 1‐in. needle
  • 3‐ml syringe

Support Protocol 3: Identification of Hematopoietic Progenitor Cells in Spin EBs Using a Methylcellulose Colony Forming Assay

  Materials
  • Spin EBs at day 4 of differentiation (at least 30 to 60 EBs required)
  • CMF‐PBS (Invitrogen)
  • TrypLE Select (Invitrogen; see recipe)
  • Differentiation medium (see recipe)
  • 0.4% Trypan blue (Fluka)
  • MethoCult, serum‐free (MC, Stem cell Technologies; see recipe)
  • Hematopoietic cytokines (PeproTech, see reciperecipes)
  • Sterile water
  • Sterile 10‐ml polypropylene yellow‐cap tubes
  • 37°C water bath
  • 23‐G, 1‐in. and 18‐G, 1 1/2‐in. needles (Terumo)
  • 3‐ml syringe (Terumo)
  • Sterile FACS tubes (5‐ml polystyrene, 12 × 75–mm) with lids and with cell‐strainer caps (BD Falcon)
  • Nescofilm (Bando Chemical Ind. Ltd) or equivalent
  • Hemacytometer
  • 24‐well low‐attachment plates (Nunc cat. no. 144530)
  • Gilson pipettors (John Morris Scientific)
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Figures

Videos

Literature Cited

Literature Cited
   Conner, D.A. 2000. Mouse embryo fibroblast (MEF) feeder cell preparation. Curr. Protoc. Mol. Biol. 51:23.22.21‐23.22.27.
   Costa, M., Sourris, K., Hatzistavrou, T., Elefanty, A.G., and Stanley, E.G. 2007. Expansion of human embryonic stem cells in vitro. Curr. Protoc. Stem Cell Biol. 1:1C.1.1‐1C.1.7.
   Davis, R.P., Ng, E.S., Costa, M., Mossman, A.K., Sourris, K., Elefanty, A.G., and Stanley, E.G. 2007. Targeting a GFP reporter gene to the Mixl1 locus of human embryonic stem cells identitifies primitive streak‐like cells and enables isolation of primitive hematopoietic precursors. Blood November 21. Epub. ahead of print.
   Evans, M.J. and Kaufman, M.H. 1981. Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154‐156.
   Hirst, C.E., Ng, E.S., Azzola, L., Voss, A.K., Thomas, T., Stanley, E.G., and Elefanty, A.G. 2006. Transcriptional profiling of mouse and human ES cells identifies SLAIN1, a novel stem cell gene. Dev. Biol. 293:90‐103.
   Keller, G., Kennedy, M., Papayannopoulou, T., and Wiles, M.V. 1993. Hematopoietic commitment during embryonic stem cell differentiation in culture. Mol. Cell Biol. 13:473‐486.
   Kennedy, M., D'Souza, S.L., Lynch‐Kattman, M., Schwantz, S., and Keller, G. 2007. Development of the hemangioblast defines the onset of hematopoiesis in human ES cell differentiation cultures. Blood 109:2679‐2687.
   Lacaud, G., Gore, L., Kennedy, M., Kouskoff, V., Kingsley, P., Hogan, C., Carlsson, L., Speck, N., Palis, J., and Keller, G. 2002. Runx1 is essential for hematopoietic commitment at the hemangioblast stage of development in vitro. Blood 100:458‐466.
   Martin, G.R. 1981. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl. Acad. Sci. U. S. A. 78:7634‐7638.
   Ng, E.S., Azzola, L., Sourris, K., Robb, L., Stanley, E.G., and Elefanty, A.G. 2005a. The primitive streak gene Mixl1 is required for efficient haematopoiesis and BMP4‐induced ventral mesoderm patterning in differentiating ES cells. Development 132:873‐884.
   Ng, E.S., Davis, R.P., Azzola, L., Stanley, E.G., and Elefanty, A.G. 2005b. Forced aggregation of defined numbers of human embryonic stem cells into embryoid bodies fosters robust, reproducible hematopoietic differentiation. Blood 106:1601‐1603.
   Pick, M., Azzola, L., Mossman, A., Stanley, E.G., and Elefanty, A.G. 2007. Differentiation of human embryonic stem cells in serum free medium reveals distinct roles for bone morphogenetic protein 4, vascular endothelial growth factor, stem cell factor, and fibroblast growth factor 2 in hematopoiesis. Stem Cells. 25:2206‐2214.
   Reubinoff, B.E., Pera, M.F., Fong, C.Y., Trounson, A., and Bongso, A. 2000. Embryonic stem cell lines from human blastocysts: Somatic differentiation in vitro. Nat. Biotechnol. 18:399‐404.
   Robertson, S.M., Kennedy, M., Shannon, J.M., and Keller, G. 2000. A transitional stage in the commitment of mesoderm to hematopoiesis requiring the transcription factor SCL/tal‐1. Development 127:2447‐2459.
   Smith, J., Wardle, F., Loose, M., Stanley, E.G., and Patient, R. 2007. Germ layer induction in ESC—Following the vertebrate road map. Curr. Protoc. Stem Cell Biol. 1:1D.1.1‐1D.1.22.
   Thomson, J.A., Itskovitz‐Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., and Jones, J.M. 1998. Embryonic stem cell lines derived from human blastocysts. Science 282:1145‐1147.
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