Formation and Hematopoietic Differentiation of Human Embryoid Bodies by Suspension and Hanging Drop Cultures

Chantal Cerdan1, Seok Ho Hong1, Mickie Bhatia1

1 McMaster University, Hamilton, Ontario, Canada
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1D.2
DOI:  10.1002/9780470151808.sc01d02s3
Online Posting Date:  October, 2007
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The in vitro aggregation of human embryonic stem cells (hESCs) into clusters termed embryoid bodies (EBs) allows for the spontaneous differentiation of cells representing endoderm, mesoderm, and ectoderm lineages. This stochastic process results however, in the generation of low numbers of differentiated cells, and can be enhanced to some extent by the addition of exogenous growth factors or overexpression of regulatory genes. In the authors' laboratory, the use of hematopoietic cytokines in combination with the mesoderm inducer bone morphogenetic protein‐4 (BMP‐4) was able to generate up to 90% of CD45+ hematopoietic cells with colony‐forming unit (CFU) activity. This unit describes two protocols that have been successfully applied in the authors' laboratory for the generation of EBs in (1) suspension and (2) hanging drop (HD) cultures from enzymatically digested clumps of undifferentiated hESC colonies. Curr. Protoc. Stem Cell Biol. 3:1D.2.1‐1D.2.16. © 2007 by John Wiley & Sons, Inc.

Keywords: human embryonic stem cells; embryoid body; suspension cultures; hanging drop cultures; differentiation; hematopoiesis

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Table of Contents

  • Introduction
  • Basic Protocol 1: Formation and Hematopoietic Differentiation of Human Embryoid Bodies using Suspension Cultures
  • Alternate Protocol 1: Formation and Hematopoietic Differentiation of Human Embryoid Bodies using Hanging Drop Cultures
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Formation and Hematopoietic Differentiation of Human Embryoid Bodies using Suspension Cultures

  • hESC, undifferentiated clumps
  • Matrigel (10‐ml bottle; BD Biosciences, no. 353234; see recipe)
  • Knockout DMEM (KO‐DMEM; Invitrogen, no. 10829‐018)
  • Undifferentiated hESCs
  • MEFCM (see recipe)
  • Basic fibroblast growth factor (bFGF), recombinant human (see recipe)
  • Collagenase IV (see recipe)
  • EB medium (see recipe)
  • Growth factors/cytokines:
    • Stem cell factor (SCF), recombinant human (see recipe)
    • Flt‐3 ligand (Flt‐3L), recombinant human (see recipe)
    • Interleukin‐3 (IL‐3), recombinant human (see recipe)
    • Interleukin‐6 (IL‐6), recombinant human (see recipe)
    • Granulocyte colony–stimulating factor (G‐CSF), recombinant human (300 µl bottle, Amgen, no. 3105100); aliquot 50 µl/tube and use it at 300 ng/µl (store no longer than 3 months at −30°C; store thawed aliquots no longer than 1 month at 4°C)
    • Bone morphogenetic protein‐4 (BMP‐4), recombinant human (see recipe)
  • Collagenase B (see recipe)
  • Cell dissociation buffer, enzyme‐free PBS‐based (Invitrogen, no.13151‐014)
  • Iscove's modified Dulbecco's medium (IMDM)
  • FACS buffer
  • 100% fetal bovine serum (FBS)
  • 6‐well plates, tissue culture treated, flat bottom (VWR, cat. no. CA62406‐161)
  • 10‐ml pipet
  • 6‐well ultra‐low attachment plates, flat bottom (Fisher, cat. no. CS003471)
  • 15‐ml polypropylene tube
  • 37°C water bath
  • 200‐ or 1000‐µl pipettor
  • 40‐µm cell strainer, nylon (BD Biosciences, no. 352340)
  • Additional reagents and equipment for counting cells by trypan blue exclusion using a hemacytometer (unit 1.3)

Alternate Protocol 1: Formation and Hematopoietic Differentiation of Human Embryoid Bodies using Hanging Drop Cultures

  • hESC colonies in 6‐well plates (see protocol 1)
  • EB medium (see recipe)
  • Collagenase IV (see recipe)
  • Knockout DMEM (KO‐DMEM; Invitrogen, cat. no.10829‐018)
  • 37°C water bath
  • 5‐ or 10‐ml serological pipet
  • 60 × 15–mm polystyrene petri dish (Falcon, cat. no. 25382)
  • 100 × 15–mm polystyrene petri dish (VWR, cat. no. 25384‐088)
  • 5‐ and 9‐in. sterilized glass pipets (VWR, cat. no. 53283‐914 and 14672‐200)
  • Rubber bulb (VWR, cat. no. 82024‐562)
  • 15‐ml conical tube
  • 6‐ and 24‐well plates, ultra‐low‐attachment, flat bottom (Fisher, cat. no. CS003471 and CS003473)
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