Culture, Expansion, and Differentiation of Murine Megakaryocytes from Fetal Liver, Bone Marrow, and Spleen

Harald Schulze1

1 Universitätsklinikum Würzburg, Chair of Experimental Biomedicine: Experimental Hemostaseology, Würzburg
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 22F.6
DOI:  10.1002/0471142735.im22f06s112
Online Posting Date:  February, 2016
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Abstract

Megakaryocytes (MKs) are the source of circulating platelets and are readily recognized by their large size and distinctive morphology. Their poor representation in hematopoietic tissues often requires considerable ex vivo expansion to generate cells for biochemical and cell biological studies. These experimental protocols describe the assessment of megakaryocytic potential within hematopoietic precursor cells in the bone marrow by colony‐forming assays and expansion and enrichment of MKs from cultured fetal liver or spleen or bone marrow cells. Although these MKs are not synchronized in their maturation, they can be enriched over an albumin step gradient, and one‐third to one‐half of recovered cells will typically elaborate proplatelets, the immediate precursors of blood platelets. Both protocols require recombinant thrombopoietin (TPO) as a growth factor. Support protocols describe methods for preparing fetal liver cells, identifying mature rodent MKs by staining for acetylcholinesterase activity, and staining (May‐Grünwald‐Giemsa) mixed populations on cytocentrifuged blood cell preparations. © 2016 by John Wiley & Sons, Inc.

Keywords: megakaryocytes; platelets; hematopoietic progenitors; thrombopoietin; fetal liver culture

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Megakaryocyte Colony‐Forming Unit (CFU‐MK) Assay for Hematopoietic Progenitors
  • Basic Protocol 2: Preparing a Megakaryocyte Suspension Culture from Murine Fetal Livers
  • Support Protocol 1: Preparing Single‐Cell Suspensions from Fetal Mouse Livers
  • Support Protocol 2: Acetylcholinesterase Staining of Megakaryocyte Cells
  • Support Protocol 3: May‐Grünwald‐Giemsa Histologic Staining of Megakaryocyte Cells
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Megakaryocyte Colony‐Forming Unit (CFU‐MK) Assay for Hematopoietic Progenitors

  Materials
  • Adult mice (for bone marrow or spleen cells only) or single‐cell suspension of mouse fetal liver cells (from appropriate developmental stage; see protocol 3)
  • PBS ( appendix 2A), sterile
  • DMEM containing 10% (v/v) fetal bovine serum (FBS)
  • MethoCult M3231 (StemCell Technologies) or the following methylcellulose medium components (for colony‐forming assay without acetylcholinesterase staining only):
    • Methylcellulose (MethoCult; StemCell Technologies)
    • Iscove's modified Dulbecco medium (IMDM; Life Technologies)
    • FBS (Hyclone)
    • BSA (Sigma or Serological)
    • 2‐Mercaptoethanol (2‐ME)
  • Recombinant thrombopoietin (TPO; R&D Systems)
  • Collagen gel (e.g., MegaCult‐C; Stem Cell Technologies), for colony‐forming assay with acetylcholinesterase staining only
  • Surgical equipment, for harvesting spleen and femur only, including:
    • Sharp scissors
    • Forceps, for spleen cells only
  • 15‐ml conical‐bottom tubes
  • 3‐ml syringes and 23‐G needles
  • 35‐mm petri dishes
  • 3‐ml round‐bottom tubes, sterile
  • 15‐cm petri dish
  • 37°C, 5% CO 2 incubator, humidified
  • Additional reagents (i.e., isoflurane) and equipment for mouse euthanasia (unit 1.8; Donovan and Brown, ) and counting viable cells ( appendix 3B; Strober, )

Basic Protocol 2: Preparing a Megakaryocyte Suspension Culture from Murine Fetal Livers

  Materials
  • Single‐cell suspension of mouse fetal liver cells (from appropriate developmental stage; see protocol 3)
  • DMEM containing 10% (v/v) fetal bovine serum (FBS), with and without 50 U/ml penicillin and 50 μg/ml streptomycin
  • Recombinant thrombopoietin (TPO; R&D Systems)
  • 3% and 1.5% (w/v) BSA (Sigma or Serological) in PBS ( appendix 2A), sterile and prewarmed to 37°C
  • 10‐cm tissue culture dishes or 75‐cm2 tissue culture flasks, sterile
  • 37°C, 5% CO 2 incubator, humidified
  • 15‐ml conical‐bottom tubes, sterile
  • Inverted microscope
  • Additional reagents and equipment for acetylcholinesterase staining (see protocol 4), Giemsa staining (see protocol 5), and cytocentrifugation of cells (unit 21.4; Hofman and Taylor, )

Support Protocol 1: Preparing Single‐Cell Suspensions from Fetal Mouse Livers

  Materials
  • Pregnant mice, postcoital days 13.5 to 15.5
  • HBSS ( appendix 2A) without calcium chloride, magnesium chloride, and magnesium sulfate, sterile
  • DMEM containing 10% fetal bovine serum (FBS), 50 U/ml penicillin, and 50 μg/ml streptomycin
  • Recombinant thrombopoietin (TPO; R&D Systems)
  • Surgical instruments (sterile), including:
    • Sharp scissors
    • Two pairs fine‐tip (#5) forceps10‐cm petri dishes, sterile
  • 18‐, 21‐, and 23‐G needles, sterile
  • 10‐ml syringes, sterile
  • Inverted microscope
  • Conical 15‐ or 50‐ml tube, sterile
  • Additional reagents and equipment for mouse euthanasia (unit 1.8; Donovan and Brown, ) and ACK lysis of red cells [for 15.5 days postcoital (dpc) fetal livers only; unit 3.1 (Kruisbeek, )]

Support Protocol 2: Acetylcholinesterase Staining of Megakaryocyte Cells

  Materials
  • Acetylthiocholine iodide (Sigma), store at –20°C
  • 0.1 M dibasic sodium phosphate (pH adjusted to 6.0 with phosphoric acid)
  • 0.1 M sodium citrate (stable up to several weeks at room temperature)
  • 30 mM copper sulfate (stable up to several weeks at room temperature)
  • 5 mM potassium ferricyanide (stable up to several weeks at room temperature), protect from light
  • Cytocentrifuged slides [unit 21.4 (Hofman and Taylor, ); unfixed and unstained; containing megakaryocytes (MKs) of interest (see Basic Protocols protocol 11 and protocol 22)]
  • 95% (v/v) ethanol
  • Harris hematoxylin (e.g., Medical Chemical Corporation) or Gill's formulation hematoxylin (e.g., Vector Laboratories)
  • Saturated Li 2CO 3 solution
  • Coplin jars to hold up to five slides (e.g., Wheaton, Fisher Scientific)
  • Microscope with 40× objective

Support Protocol 3: May‐Grünwald‐Giemsa Histologic Staining of Megakaryocyte Cells

  Materials
  • Cytocentrifuged slides [unit 21.4; Hofman and Taylor, ; unfixed and unstained; containing megakaryocytes (MKs) of interest (see Basic Protocols protocol 11 and protocol 22)]
  • Methanol
  • May‐Grünwald stain (Medical Chemical Corporation), filter through a 0.2‐ or 0.45‐μm filter
  • Giemsa stain (Medical Chemical Corporation), filter through a 0.2‐ or 0.45‐μm filter and dilute 1:15 (v/v) in distilled water
  • Coplin jars to hold up to five slides (e.g., Wheaton, Fisher Scientific)
  • Microscope
  • Mounting medium such as Permount (Fisher Scientific), optional
  • Coverslips, optional
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Figures

Videos

Literature Cited

Literature Cited
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  Strassel, C., Eckly, A., Léon, C., Moog, S., Cazenave, J.P., Gachet, C., and Lanza, F. 2012. Hirudin and heparin enable efficient megakaryocyte differentiation of mouse bone marrow progenitors. Exp. Cell. Res. 318:25‐32. doi: 10.1016/j.yexcr.2011.10.003.
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