Analysis and Manipulation of Hematopoietic Progenitor and Stem Cells from Murine Embryonic Tissues

Alexander Medvinsky1, Samir Taoudi1, Sandra Mendes2, Elaine Dzierzak2

1 Institute for Stem Cell Research, University of Edinburgh, Edinburgh, United Kingdom, 2 Erasmus Medical Center, Department of Cell Biology, The Netherlands
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2A.6
DOI:  10.1002/9780470151808.sc02a06s4
Online Posting Date:  January, 2008
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Abstract

Hematopoietic development begins in several locations in the mammalian embryo: yolk sac, aorta‐gonad‐mesonephros region (AGM), and the chorio‐allantoic placenta. Generation of the most potent cells, adult definitive hematopoietic stem cells (HSCs), occurs within the body of the mouse embryo at midgestation in the AGM region. Similarly, at the equivalent developmental time in the human embryo, the AGM region has been shown to contain multipotent progenitors. Hence, the mouse embryo serves as an excellent model to study hematopoietic development. To further studies on the ontogeny of the adult hematopoietic system, the focus of this unit is on the experimental methods used in analysis of the AGM region. Curr. Protoc. Stem Cell Biol. 4:2A.6.1‐2A.6.25. © 2008 by John Wiley & Sons, Inc.

Keywords: developmental hematopoiesis; hematopoietic stem cells; embryo; AGM; lineage differentiation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Dissection of Mouse Embryonic Tissues from Day 9 to 12 Mouse Embryos
  • Support Protocol 1: Generating Mouse Embryos
  • Support Protocol 2: Staging Embryos
  • Basic Protocol 2: Preparation of Cell Suspension from Tissues of Midgestation Mouse Embryos
  • Basic Protocol 3: Explant Culture of Embryonic Tissues
  • Basic Protocol 4: Preparation of Embryonic Cells for Flow Cytometry
  • Basic Protocol 5: Hematopoietic (Myeloid) Clonogenic Assay
  • Basic Protocol 6: Long‐Term Repopulation Assay
  • Support Protocol 3: Preparation of Acidified Drinking Water for Irradiated Mice
  • Basic Protocol 7: Endothelial Assay
  • Support Protocol 4: Maintenance of OP9 Cells
  • Support Protocol 5: Visualization of Endothelial Tubules
  • Mesenchymal Lineage Differentiation Assays
  • Basic Protocol 8: Osteogenic Differentiation of Mesenchymal Cells
  • Alternate Protocol 1: Adipogenic Differentiation of Mesenchymal Cells
  • Alternate Protocol 2: Chondrogenic Differentiation of Mesenchymal Cells
  • Support Protocol 6: Histological Staining with Alizarin Red for Identification of Bone Tissue
  • Support Protocol 7: Histological Staining with Oil Red O Stain for Identification of Adipocytes
  • Support Protocol 8: Histological Staining with Toluidine Blue Stain for Identification of Cartilage
  • Support Protocol 9: Immunostaining Sections of Micro‐Mass Cultures with Anti‐Collagen Type II for Identificaton of Cartilage
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Dissection of Mouse Embryonic Tissues from Day 9 to 12 Mouse Embryos

  Materials
  • Pregnant female mice of chosen background strain
  • 70% ethanol
  • Medium I (medium for embryo collection; see recipe)
  • Medium II (medium for dissections; see recipe)
  • Surgical scissors (two pairs treated with 70% alcohol)
  • Fine, straight watchmaker's forceps (two pairs)
  • 60 × 15–mm and 35 × 15–mm plastic tissue culture petri dishes
  • 150‐W cold light source equipped with double gooseneck fiber‐optic system
  • Dissection microscope (magnification range from 7× to 40× with a flat, black background stage; Leica, Zeiss, or Olympus)
  • Fine, curved watchmaker's forceps
  • Dissection needles: sharpened tungsten wire 0.375‐mm diameter (Agar Scientific Ltd.) attached to metal holders typically used for bacterial culture inoculation (alternatively, 29‐G needles attached to micro‐fine insulin syringes, e.g., U‐100, Beckton‐Dickinson)
  • Device for sharpening dissection needles (an electrolytic device for sharpening tungsten needles described in Hogan and Beddington, , or alternatively, a sharpening stone)
NOTE: For dissections, always use room temperature solutions. When necessary to maintain sterility during tissue isolation, wash dissection tools with 70% alcohol and wipe with a tissue. Excess blood should first be removed from tools by wiping with distilled water.

Support Protocol 1: Generating Mouse Embryos

  Materials
  • Collagenase type I (see recipe)
  • Embryonic tissues ( protocol 1)
  • Medium II (see recipe)
  • Medium III (see recipe), room temperature and ice cold
  • 10‐ml round‐bottom transparent polystyrene tubes (Sterilin)
  • 37°C water bath with shaking
  • Vacuum aspirator
NOTE: Keep cell suspensions strictly on ice.

Support Protocol 2: Staging Embryos

  Materials
  • Explant medium: myeloid long‐term culture medium (Stem Cell Technologies cat. no. M5300) supplemented with hydrocortisone succinate (Sigma) at a final concentration of 10−5 M
  • Embryonic tissues ( protocol 1)
  • PBS or sterile water (Sigma)
  • 70% ethanol
  • Collagenase type I (see recipe)
  • Stainless‐steel wire mesh supports (see recipe)
  • 6‐well tissue culture plates
  • Straight and curved fine‐tipped watchmaker's forceps
  • 0.65‐µm membrane filters (Millipore Durapore)
  • Scalpel blade
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 2: Preparation of Cell Suspension from Tissues of Midgestation Mouse Embryos

  Materials
  • Single‐cell suspension from embryonic tissues (see protocol 4)
  • FACS wash buffer: ice‐cold 7% FBS/CMF‐PBS (Sigma cat. no. D8537)
  • Fc‐block (anti‐CD16/32 antibodies/Fc‐γ III/II receptor) (BD Bioscience; Clone 2.4G2)
  • Appropriate experimental antibodies (Table 2.6.1)
  • 7‐Amino‐actinomycin D (7‐AAD; eBioscience; Table 2.6.2)
  • 40‐µm nylon cell strainer (BD Falcon)
  • 5.0‐ml polystyrene tubes (BD Falcon)
  • Refrigerated swing‐out centrifuge
  • Flow cytometer (e.g., FACSCalibur, BD Biosciences)
    Table 2.0.1   MaterialsList of Useful Primary AntibodiesList of Useful Secondary Reagents

    Antigen Clone Isotype Working concentration Supplier
    α4‐Integrin 9C10 Rat IgG2α,κ 2.0 µg/ml Pharmingen
    AA4.1 AA4.1 Rat IgG2β,κ 2.0 µg/ml eBioscience
    c‐Kit 2B8 Rat IgG2β,κ 2.0 µg/ml Pharmingen
    CD16/32 2.4G2 Rat IgG2β,κ 2.0 µg/ml Pharmingen
    CD34 RAM34 Rat IgG2α,κ 2.0 µg/ml Pharmingen
    CD41 MWReg30 Rat IgG1,κ 2.0 µg/ml Pharmingen
    CD45 30‐F11 Rat IgG2β,κ 2.0 µg/ml Pharmingen
    Flk‐1 Avas‐12α Rat IgG2α,κ 2.0 µg/ml Pharmingen
    Ly‐5.1 A20 Mouse IgG2α,κ 2.0 µg/ml eBioscience
    Ly‐5.2 104 Mouse IgG2α,κ 2.0 µg/ml eBioscience
    Mac‐1 M1/70 Rat IgG2β,κ 2.0 µg/ml Pharmingen
    PECAM‐1 MEC 13.3 Rat IgG2α,κ 2.0 µg/ml Pharmingen
    Sca‐1 D7 Rat IgG2α,κ 2.0 µg/ml eBioscience
    Ter119 TER‐119 Rat IgG2β,κ 2.0 µg/ml eBioscience
    Tie‐2 TEK4 Rat IgG1,κ 2.0 µg/ml eBioscience
    VE‐cadherin 11D4.1 Rat IgG2α,κ 6.0 µg/ml Pharmingen
    Reagent Clone aa Working concentration Supplier
    7‐AAD n/a 0.5 µg/ml eBioscience
    Anti‐rat IgG Polyclonal 2.0 mg/ml Southern Biotech
    Mouse IgG2α,κ G155‐178 As appropriate Pharmingen
    Rat IgG1,κ eBRG1 As appropriate eBioscience
    Rat IgG2α,κ R35‐95 As appropriate Pharmingen
    Rat IgG2β,κ A95‐1 As appropriate Pharmingen
    Streptavidin (fluorochrome‐conjugated) n/a 0.2 µg/ml Pharmingen

    Table 2.0.2   MaterialsList of Useful Primary AntibodiesList of Useful Secondary Reagents

    Antigen Clone Isotype Working concentration Supplier
    α4‐Integrin 9C10 Rat IgG2α,κ 2.0 µg/ml Pharmingen
    AA4.1 AA4.1 Rat IgG2β,κ 2.0 µg/ml eBioscience
    c‐Kit 2B8 Rat IgG2β,κ 2.0 µg/ml Pharmingen
    CD16/32 2.4G2 Rat IgG2β,κ 2.0 µg/ml Pharmingen
    CD34 RAM34 Rat IgG2α,κ 2.0 µg/ml Pharmingen
    CD41 MWReg30 Rat IgG1,κ 2.0 µg/ml Pharmingen
    CD45 30‐F11 Rat IgG2β,κ 2.0 µg/ml Pharmingen
    Flk‐1 Avas‐12α Rat IgG2α,κ 2.0 µg/ml Pharmingen
    Ly‐5.1 A20 Mouse IgG2α,κ 2.0 µg/ml eBioscience
    Ly‐5.2 104 Mouse IgG2α,κ 2.0 µg/ml eBioscience
    Mac‐1 M1/70 Rat IgG2β,κ 2.0 µg/ml Pharmingen
    PECAM‐1 MEC 13.3 Rat IgG2α,κ 2.0 µg/ml Pharmingen
    Sca‐1 D7 Rat IgG2α,κ 2.0 µg/ml eBioscience
    Ter119 TER‐119 Rat IgG2β,κ 2.0 µg/ml eBioscience
    Tie‐2 TEK4 Rat IgG1,κ 2.0 µg/ml eBioscience
    VE‐cadherin 11D4.1 Rat IgG2α,κ 6.0 µg/ml Pharmingen
    Reagent Clone aa Working concentration Supplier
    7‐AAD n/a 0.5 µg/ml eBioscience
    Anti‐rat IgG Polyclonal 2.0 mg/ml Southern Biotech
    Mouse IgG2α,κ G155‐178 As appropriate Pharmingen
    Rat IgG1,κ eBRG1 As appropriate eBioscience
    Rat IgG2α,κ R35‐95 As appropriate Pharmingen
    Rat IgG2β,κ A95‐1 As appropriate Pharmingen
    Streptavidin (fluorochrome‐conjugated) n/a 0.2 µg/ml Pharmingen

     an/a, not applicable.

Basic Protocol 3: Explant Culture of Embryonic Tissues

  Materials
  • MethoCult GF M3434: complete methylcellulose‐based medium with cytokines (SCF, IL‐3, IL‐6, erythropoietin) for detection of BFU‐E, CFU‐GM, CFU‐G, CFU‐M, and CFU‐GEMM‐formed colonies (100 ml, Stem Cell Technologies cat. no. 03434)
  • Cell suspension
  • 7% FBS/CMF‐PBS (Sigma)
  • DPBS, sterile
  • 7‐ml Bijou tubes (Sterilin)
  • 2 and 10‐ml syringes
  • 18‐G needles
  • Neubauer hemacytometer
  • 30‐ and 140‐mm Petri dishes (non‐adherent surface)
  • 37°C, 5% CO 2 incubator
  • Inverted microscope
  • Gridded 60‐mm Petri dish
  • Additional reagents and equipment for trypan blue staining (unit 1.3)

Basic Protocol 4: Preparation of Embryonic Cells for Flow Cytometry

  Materials
  • Adult recipient Ly5.1 C57Bl6 mice
  • Ly5.2 C57Bl6 embryos
  • CMF‐PBS (Ca2+/Mg2+‐free; Sigma)
  • Adult Ly5.1/2 C57Bl6 mice bone marrow cells
  • Mouse food
  • Acidified water containing neomycin ( protocol 9)
  • 200 mg/liter EDTA/PBS (Sigma)
  • PharmLyse (BD Biosciences) or preferred red blood cell lysis buffer
  • FACS wash buffer: 3% (v/v) FBS/CMF‐PBS
  • Anti‐Ly5.1 and Ly5.2 monoclonal antibodies conjugated with alternative fluorochromes
  • 137Cesium irradiator (e.g., Gammacell GC40, MDS Nordion)
  • Mice cages with heating pads
  • Mouse holder with opening allowing extension of the tail
  • 1‐ml plastic syringes with 27‐G needles
  • 1.5‐ml microcentrifuge tubes
  • Swing‐out centrifuge
  • Flow cytometer (e.g., FACSCalibur, BD Biosciences)

Basic Protocol 5: Hematopoietic (Myeloid) Clonogenic Assay

  Materials
  • Concentrated HCl
  • Neomycin (Sigma)

Basic Protocol 6: Long‐Term Repopulation Assay

  Materials
  • OP9 cells (see protocol 11)
  • Dissected E11.5 embryonic tissues
  • Endothelial growth medium (see recipe)
  • Anti‐PECAM‐1 antibody (see protocol 12)
  • 4‐well tissue culture flat bottom plates (Nunc)

Support Protocol 3: Preparation of Acidified Drinking Water for Irradiated Mice

  Materials
  • OP9 cells (developed by Nakano et al., )
  • Culture medium (see recipe), prewarmed
  • CMF‐PBS
  • Dissociation solution (see recipe)
  • 10% DMSO (BDH)
  • 10‐ml tube
  • 75‐cm2 tissue culture flasks (Iwaki)
  • 10‐ml plastic pipets
  • 1‐ml cryotubes (Nunc)
  • 37°C, 5% CO 2 incubator

Basic Protocol 7: Endothelial Assay

  Materials
  • Cultures of endothelial cells (OP9 stroma; protocol 11) in 4‐well plates (Nunc)
  • CMF‐PBS
  • 2% (w/v) paraformaldehyde(PFA)/PBS, pH 7.4 (Sigma)
  • 0.1% (v/v) Nonidet P40 (NP40)/PBS (Sigma)
  • 10% FBS/PBS
  • Anti‐PECAM‐1 antibody (BD Bioscience)
  • Secondary anti‐rat IgG antibody conjugated with alkaline phosphatase (AP) (Southern Biotechnology Associates)
  • 0.1 M Tris·Cl, pH 8.2
  • 0.125 M Levamisol (Vector)
  • Vector Blue alkaline phosphatase substrate kit III (Vector)
  • 4‐well tissue culture flat‐bottom plates (Nunc)
  • Microscope with camera attached

Support Protocol 4: Maintenance of OP9 Cells

  Materials
  • Primary cell suspension (see protocol 4)
  • Osteogenic differentiation medium (see recipe)
  • DPBS
  • 4% (w/v) paraformaldehyde (PFA) in PBS
  • Alkaline phosphatase staining kit (Sigma Diagnostics cat. no. 85L1) or alizarin red (see protocol 16)
  • 6‐well plates
  • 37°C, 5% CO 2 humidified incubator

Support Protocol 5: Visualization of Endothelial Tubules

  • Adipogenic differentiation medium I (see recipe)
  • Adipogenic differentiation medium II (see recipe)

Basic Protocol 8: Osteogenic Differentiation of Mesenchymal Cells

  • Chondrogenic differentiation medium (see recipe)
  • Toluidine blue stain (see protocol 18)
  • Tissue tek
  • 15‐ml polypropylene tubes
  • Plastic molds
  • Cryostat

Alternate Protocol 1: Adipogenic Differentiation of Mesenchymal Cells

  Materials
  • Alizarin red (Sigma cat. no. A5533)
  • 1 M NaOH
  • Cultures of cell to be tested for differentiation
  • PBS
  • 4% (w/v) paraformaldehyde (PFA)
  • 45‐µm filter

Alternate Protocol 2: Chondrogenic Differentiation of Mesenchymal Cells

  Materials
  • Oil red O (Sigma cat. no. 75087)
  • Cultures of cells to be tested for differentiation in 6‐well plates
  • PBS
  • 4% (w/v) paraformaldehyde (PFA)

Support Protocol 6: Histological Staining with Alizarin Red for Identification of Bone Tissue

  Materials
  • Toluidine blue (Sigma cat. no. 89640)
  • Cryostat sections of micro‐mass cultures
  • 4% paraformaldehyde (PFA)
  • 45‐µm filters

Support Protocol 7: Histological Staining with Oil Red O Stain for Identification of Adipocytes

  Materials
  • Cryosections of micro‐mass cultures
  • 4% (w/v) PFA
  • PBS/0.05% (v/v) Tween 20
  • Collagen type II antibody (CIIC1, Developmental Studies Hybridoma Bank)
  • Anti‐immunoglobulin‐HRP (Dako)
  • Chromogen diaminobenzidine (DAB, Dako)
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Figures

Videos

Literature Cited

Literature Cited
   Bertrand, J.Y., Giroux, S., Golub, R., Klaine, M., Jalil, A., Boucontet, L., Godin, I., and Cumano, A. 2005. Characterization of purified intraembryonic hematopoietic stem cells as a tool to define their site of origin. Proc. Natl. Acad. Sci. U.S.A. 102:134‐139.
   Cumano, A., Furlonger, C., and Paige, C.J. 1993. Differentiation and characterization of B‐cell precursors detected in the yolk sac and embryo body of embryos beginning at the 10‐ to 12‐somite stage. Proc. Natl. Acad. Sci. U.S.A. 90:6429‐6433.
   Cumano, A., Ferraz, J.C., Klaine, M., Di Santo, J.P., and Godin, I. 2001. Intraembryonic, but not yolk sac hematopoietic precursors, isolated before circulation, provide long‐term multilineage reconstitution. Immunity 15:477‐485.
   de Bruijn, M.R.T.R., Speck, N.A., Peeters, M.C.E., and Dzierzak, E. 2000. Definitive hematopoietic stem cells first emerge from the major arterial regions of the mouse embryo. EMBO J. 19:2465‐2474.
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