Differentiation of Mouse Embryonic Stem Cells into Blood

Yunglin D. Ma1, Jesse J. Lugus2, Changwon Park3, Kyunghee Choi2

1 Developmental Biology Program, Washington University School of Medicine, St. Louis, Missouri, 2 Molecular Cell Biology Program, Washington University School of Medicine, St. Louis, Missouri, 3 Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1F.4
DOI:  10.1002/9780470151808.sc01f04s6
Online Posting Date:  July, 2008
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Abstract

Embryonic stem (ES) cells can be maintained as pluripotent stem cells or induced to differentiate into many different somatic cell types. As ES‐derived somatic cells can potentially be used for cell transplantation or cell‐based therapy, ES cells have gained much scientific and general public attention. Successful derivation of blood from ES cells for tissue engineering will require a comprehensive understanding of inductive signals and downstream effectors involved in blood lineage development. Ideally, directed differentiation of ES cells into blood and isolation of pure hematopoietic progenitors will enhance our ability to utilize ES‐derived blood cells for future clinical applications. The protocols provided in this unit describe methods of maintaining and differentiating mouse ES cells as well as identifying and isolating hematopoietic progenitors by utilizing flow cytometry and progenitor assays. Curr. Protoc. Stem Cell Biol. 6:1F.4.1‐1F.4.19. © 2008 by John Wiley & Sons, Inc.

Keywords: embryonic stem cells; ES cells; in vitro differentiation; hematopoietic progenitors

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: In Vitro Differentiation of Mouse ES Cells to Blood Lineages in the Presence of Serum
  • Alternate Protocol 1: In Vitro Differentiation of Mouse ES Cells to Blood Lineages in Serum‐Free Medium
  • Support Protocol 1: Preparation of Irradiated Mouse Embryonic Fibroblasts (MEFs) for Use as Feeder Cells
  • Support Protocol 2: Mouse ES Cell Maintenance
  • Basic Protocol 2: Flow Cytometric Analysis of EB Cells
  • Alternate Protocol 2: Cell Sorting and In Vitro Culture of Sorted Cell Populations
  • Basic Protocol 3: Hematopoietic Progenitor Assays
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: In Vitro Differentiation of Mouse ES Cells to Blood Lineages in the Presence of Serum

  Materials
  • Mouse ES cells, 3rd passage or more (up to 5 to 6 passages) after thawing (see protocol 4)
  • ES‐IMDM medium (see recipe)
  • 0.25% trypsin/EDTA (see recipe)
  • Fetal bovine serum (FBS) for differentiation (see recipe)
  • IMDM medium (see recipe)
  • IMDM medium (see recipe) containing 10% (v/v) FBS for differentiation
  • 2% (w/v) eosin in phosphate‐buffered saline (PBS; see recipe)
  • Serum differentiation medium, liquid (see recipe for ES differentiation media)
  • 25‐cm2 tissue culture flasks (Techno Plastic Product AG, cat. no. 90026; http://www.tpp.ch/), gelatinized (see protocol 4, step 1)
  • 14‐ml polypropylene round‐bottom snap‐cap tubes (Becton Dickinson, cat. no. 352059)
  • Hemacytometer
  • 100‐mm bacterial petri dishes (Kord‐Valmark cat. no. 900; http://www.kord‐valmark.com/)
  • Centrifuge (e.g., Sorvall model RT7‐RTH250)
  • Additional reagents and equipment for maintaining ES cells ( protocol 4)

Alternate Protocol 1: In Vitro Differentiation of Mouse ES Cells to Blood Lineages in Serum‐Free Medium

  • Serum replacement (SR, Invitrogen, cat. no. 10828‐028)
  • Serum‐free differentiation medium (see recipe for ES differentiation media)
  • PFHM‐II (Protein‐Free Hybridoma Medium; Invitrogen, cat. no. 12040‐077)

Support Protocol 1: Preparation of Irradiated Mouse Embryonic Fibroblasts (MEFs) for Use as Feeder Cells

  Materials
  • Mitotically arrested MEFs (PMEF; Specialty Media)
  • 0.25% trypsin/EDTA (see recipe)
  • FBS for ES cell culture (see recipe)
  • IMDM medium (see recipe)
  • Freezing medium: 90% FBS (for ES culture)/10% DMSO
  • Liquid nitrogen
  • 175‐cm2 tissue culture flasks, gelatinized (see protocol 4, step 1)
  • 50‐ml centrifuge tubes
  • Centrifuge
  • Hemacytometer
  • Cryovials
  • Liquid nitrogen freezer
  • Additional reagents and equipment for γ irradiation of MEFs (unit 1.3)

Support Protocol 2: Mouse ES Cell Maintenance

  Materials
  • 0.1% gelatin (see recipe)
  • γ‐irradiated MEFs (see protocol 3)
  • MEF medium (see recipe)
  • ES cells, frozen, passage 12 to 18
  • ES‐DMEM medium (see recipe)
  • ES‐IMDM medium (see recipe)
  • 0.25% trypsin/EDTA (see recipe)
  • ES cell freezing medium (see recipe)
  • 25‐cm2 tissue culture flasks (Techno Plastic Products AG cat. no. 90026; http://www.tpp.ch/)
  • 14‐ml polypropylene round‐bottom tube (Becton Dickinson; cat. no. 352059)
  • Centrifuge (e.g., Sorvall model RT7‐RTH250)
  • Hemacytometer

Basic Protocol 2: Flow Cytometric Analysis of EB Cells

  Materials
  • Mouse EBs ( protocol 1 and protocol 2)
  • 7.5 mM EDTA (BioRad, cat. no. 161‐0729) in PBS, pH 7.4 (see recipe)
  • IMDM medium (see recipe)
  • Washing buffer: 4% (v/v) FBS (for culture) in PBS (see recipe)
  • Primary antibody at appropriate dilution (Table 1.4.1) in washing buffer
  • Secondary antibody (if needed) at appropriate dilution (Table 1.4.1) in washing buffer
  • 50‐ml centrifuge tubes (Fisher, cat. no. 14‐432‐22)
  • Centrifuge with microtiter plate carrier
  • 20‐G needle (Fisher, cat. no. 14826‐5C)
  • Hemacytometer
  • 96‐well plate with V‐bottom wells (Fisher, cat. no. 07‐200‐96)
  • 5‐ml polypropylene tubes (VWR, cat. no. 60818‐500)
  • CellQuest (Becton‐Dickinson) or FlowJo (Tree Star, Inc., http://www.treestar.com) software
  • FACScan or FACSCalibur flow cytometer (BD Biosciences)
  • Additional reagents and equipment for flow cytometry (Robinson et al., )

Alternate Protocol 2: Cell Sorting and In Vitro Culture of Sorted Cell Populations

  • 40‐µm nylon‐mesh cell strainer (BD Falcon, cat. no. 352340)
  • MoFlo cell sorter (BD Biosciences)
  • 14‐ml tubes (Fisher, no. 14‐959‐49B)

Basic Protocol 3: Hematopoietic Progenitor Assays

  Materials
  • Mouse EBs ( protocol 1 or protocol 2)
  • IMDM medium (see recipe)
  • 2× cellulase solution (see recipe)
  • 0.25% trypsin/EDTA (see recipe)
  • Collagenase solution (optional; for older EBs; see recipe)
  • FBS (for differentiation; see recipe)
  • IMDM medium (see recipe) containing 10% (v/v) FBS (for differentiation)
  • 2% (w/v) eosin in phosphate‐buffered saline (PBS; see recipe)
  • Methylcellulose mixes for progenitor cells of interest (see recipe)
  • 50‐ml polypropylene conical tube (Becton Dickinson; cat. no. 352070)
  • 20‐G and 16‐ or 18‐G needles with 3‐ml syringes
  • 14‐ml polypropylene round‐bottom tube (Becton Dickinson; cat. no. 352059)
  • 35‐mm and 150‐mm bacterial dishes (Becton Dickinson; cat. no. 351008 and 351058, respectively)
  • Inverted microscope
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Figures

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Literature Cited

Literature Cited
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