Hematopoietic Differentiation of Human Embryonic Stem Cells by Cocultivation with Stromal Layers

Katherine L. Hill1, Dan S. Kaufman1

1 Stem Cell Institute and Department of Medicine, University of Minnesota, Minneapolis, Minnesota
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1F.6
DOI:  10.1002/9780470151808.sc01f06s6
Online Posting Date:  September, 2008
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Abstract

Human embryonic stem (hES) cells are of remarkable interest both for the utility of these cells for studying basic human developmental biology and as a potential source for novel therapeutics. Here, we provide detailed methodologies of one of the first systems used to mediate differentiation of hES cells—stromal cell coculture. Use of stromal cells adds the ability to manipulate aspects of the developmental niche that support differentiation into a defined lineage. These methods will allow efficient and reproducible development of hematopoietic progenitor cells, as well as potentially mature hematopoietic cells that are suitable for subsequent in vitro and in vivo studies. Curr. Protoc. Stem Cell Biol. 6:1F.6.1‐1F.6.12. © 2008 by John Wiley & Sons, Inc.

Keywords: human embryonic stem cells; hematopoietic; stromal; cocultivation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Coculture of M210 Stromal Cells and Human ES Cells
  • Basic Protocol 2: Production of Single‐Cell Suspension of Differentiated hES Cells and Selection of Hematopoietic Progenitors Via Magnetic Sorting
  • Support Protocol 1: Culture of hES Cells
  • Support Protocol 2: Culture of M210 Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Coculture of M210 Stromal Cells and Human ES Cells

  Materials
  • M‐210‐B4 cells (ATCC #CRL‐1972) maintained in RPMI‐1640 (Invitrogen, cat. no. 10‐404‐CV) supplemented with 10% fetal bovine serum (FBS; Hyclone, cat. no. SH30088.03) and 1% penicillin‐streptomycin (Invitrogen, cat. no. 15140‐122)
  • Mitomycin C (American Pharmaceutical Partners, cat. no. 109020) divided into 900‐µl aliquots and stored at −80°C
  • M210‐conditioned medium (see recipe)
  • 0.1% (w/v) gelatin in distilled, deionized water and autoclaved for sterility
  • Dulbecco's phosphate‐buffered saline (DPBS) without Ca+ and Mg2+ (CMF‐DPBS)
  • 0.05% (w/v) trypsin/0.53 mM EDTA
  • Undifferentiated H1 and/or H9 human embryonic stem cells (WiCell; Kaufman et al., ) maintained on mouse embryonic fibroblast feeder cells
  • Collagenase type IV (see recipe)
  • ES cell wash medium (see recipe) for rinsing
  • Differentiation medium (see recipe) for coculture
  • 6‐well tissue culture plates
  • 15‐ml conical tube
  • 5‐ml and 10‐ml glass pipets (VWR, cat. nos. 53283‐738 and 53283‐740)

Basic Protocol 2: Production of Single‐Cell Suspension of Differentiated hES Cells and Selection of Hematopoietic Progenitors Via Magnetic Sorting

  Materials
  • hESCs cocultured with preferred stromal cell line (e.g., M210; see protocol 1)
  • Collagenase type IV (see recipe)
  • ES cell wash medium (see recipe) for rinsing
  • Dulbecco's phosphate‐buffered saline (DPBS) without Ca+ and Mg2+ (CMF‐DPBS)
  • 0.05% (w/v) trypsin/0.53mM EDTA (Cellgro/Mediatech, cat. no. 25‐052‐CI)
  • Chick serum (Sigma)
  • EasySep buffer consisting of CMF‐DPBSsupplemented with 2% (v/v) FBS and 1 mM EDTA (stored at 2° to 8°C)
  • EasySep human CD34 selection kit (StemCell Technologies, cat. no. 18056) containing:
    • CD34 positive selection cocktail
    • Magnetic nanoparticle solution
  • FACS buffer (see recipe)
  • Hematopoietic culture medium of choice
  • Appropriate antibodies
  • Appropriately labeled isotype controls
  • Propidium iodide or 7‐AAD
  • 10‐ml glass pipets
  • 15‐ml conical tubes
  • 37°C water bath
  • Vortex
  • 100‐µm cell filter/strainer (Partec CellTrics, cat. no. 04‐0042‐2318)
  • FACS tube, sterile
  • EasySep Magnet (StemCell Technologies, cat. no. 18000)
  • Additional reagents and equipment for counting cells (unit 1.3)

Support Protocol 1: Culture of hES Cells

  • hES cells
  • hES cell medium (see recipe) for undifferentiated cell growth

Support Protocol 2: Culture of M210 Cells

  Materials
  • 0.1% (w/v) gelatin in distilled deionized water and autoclaved for sterility
  • M210 stromal cells
  • M210 culture medium (see recipe)
  • Dulbecco's phosphate‐buffered saline (DPBS) without Ca+ and Mg2+ (CMF‐DPBS)
  • 0.05% (w/v) trypsin/0.53 mM EDTA
  • 75‐cm2 flasks
  • 10‐ml glass pipets
  • 15‐ml conical tubes
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Figures

Videos

Literature Cited

Literature Cited
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