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, null, 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, 2002, 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.

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

 Materials
  • Collagenase type I (see recipe)
  • Embryonic tissues (Basic 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.

Basic Protocol 3: Explant Culture of Embryonic Tissues

 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 (Basic 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% CO2 incubator unless otherwise specified.

Basic Protocol 4: Preparation of Embryonic Cells for Flow Cytometry

 Materials
  • Single-cell suspension from embryonic tissues (see Basic Protocol 2)
  • FACS wash buffer: ice-cold 7% FBS/CMF-PBS (Sigma cat. no. D8537)
  • Fc-block (anti-CD16/32 antibodies/Fc- III/II receptor) (BD Bioscience; Clone 2.4G2)
  • Appropriate experimental antibodies (Table 2A.6.1)
  • 7-Amino-actinomycin D (7-AAD; eBioscience; Table 2A.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 2A.6.1 List of Useful Primary Antibodies
    AntigenCloneIsotypeWorking concentrationSupplier
    4-Integrin9C10Rat IgG2,2.0 µg/mlPharmingen
    AA4.1AA4.1Rat IgG2,2.0 µg/mleBioscience
    c-Kit2B8Rat IgG2,2.0 µg/mlPharmingen
    CD16/322.4G2Rat IgG2,2.0 µg/mlPharmingen
    CD34RAM34Rat IgG2,2.0 µg/mlPharmingen
    CD41MWReg30Rat IgG1,2.0 µg/mlPharmingen
    CD4530-F11Rat IgG2,2.0 µg/mlPharmingen
    Flk-1Avas-12Rat IgG2,2.0 µg/mlPharmingen
    Ly-5.1A20Mouse IgG2,2.0 µg/mleBioscience
    Ly-5.2104Mouse IgG2,2.0 µg/mleBioscience
    Mac-1M1/70Rat IgG2,2.0 µg/mlPharmingen
    PECAM-1MEC 13.3Rat IgG2,2.0 µg/mlPharmingen
    Sca-1D7Rat IgG2,2.0 µg/mleBioscience
    Ter119TER-119Rat IgG2,2.0 µg/mleBioscience
    Tie-2TEK4Rat IgG1,2.0 µg/mleBioscience
    VE-cadherin11D4.1Rat IgG2,6.0 µg/mlPharmingen
     
    Table 2A.6.2 List of Useful Secondary Reagents
    ReagentCloneaWorking concentrationSupplier
    7-AADn/a0.5 µg/mleBioscience
    Anti-rat IgGPolyclonal2.0 mg/mlSouthern Biotech
    Mouse IgG2,G155-178As appropriatePharmingen
    Rat IgG1,eBRG1As appropriateeBioscience
    Rat IgG2,R35-95As appropriatePharmingen
    Rat IgG2,A95-1As appropriatePharmingen
    Streptavidin (fluorochrome-conjugated)n/a0.2 µg/mlPharmingen
     an/a, not applicable.

Basic Protocol 5: Hematopoietic (Myeloid) Clonogenic Assay

 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% CO2 incubator
  • Inverted microscope
  • Gridded 60-mm Petri dish
  • Additional reagents and equipment for trypan blue staining (unit 1C.3)

Basic Protocol 6: Long-Term Repopulation Assay

 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 (Support Protocol 3)
  • 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)

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

 Materials
  • Concentrated HCl
  • Neomycin (Sigma)

Basic Protocol 7: Endothelial Assay

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

Support Protocol 4: Maintenance of OP9 Cells

 Materials
  • OP9 cells (developed by Nakano et al., 1994)
  • 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% CO2 incubator

Support Protocol 5: Visualization of Endothelial Tubules

 Materials
  • Cultures of endothelial cells (OP9 stroma; Support Protocol 4) 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
Basic Protocol 8:  Osteogenic Differentiation of Mesenchymal Cells
 Materials
  • Primary cell suspension (see Basic Protocol 2)
  • 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 Support Protocol 6)
  • 6-well plates
  • 37°C, 5% CO2 humidified incubator
Alternate Protocol 1:  Adipogenic Differentiation of Mesenchymal Cells
 Additional Materials (also see Basic Protocol 8)
  • Adipogenic differentiation medium I (see recipe)
  • Adipogenic differentiation medium II (see recipe)
Alternate Protocol 2:  Chondrogenic Differentiation of Mesenchymal Cells
 Additional Materials (also see Basic Protocol 8)
  • Chondrogenic differentiation medium (see recipe)
  • Toluidine blue stain (see Support Protocol 8)
  • Tissue tek
  • 15-ml polypropylene tubes
  • Plastic molds
  • Cryostat
Alternate Protocol 2:  Chondrogenic 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)
Alternate Protocol 2:  Chondrogenic Differentiation of Mesenchymal Cells
 Materials
  • Toluidine blue (Sigma cat. no. 89640)
  • Cryostat sections of micro-mass cultures
  • 4% paraformaldehyde (PFA)
  • 45-µm filters
Alternate Protocol 2:  Chondrogenic Differentiation of Mesenchymal Cells
 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

  •  FigureFigure 2A.6.1 Dissection of an E11 mouse conceptus. (A) Embryo with chorionic membrane being removed. (B) Separation of the placenta (PL) from the yolk sac which envelops the embryo. (C) After the yolk sac (YS) is disrupted, it no longer envelops the embryos but is still attached to it through the vitelline vessels. (D) The umbilical cord (UC) is seen connected to the embryo body at one end and the disrupted yolk sac is visible at the head of the embryo. (E) The head and upper region of the embryo body to the forelimbs is severed from the trunk of the embryo. (F) The dorsal tissues, neural tube (NT), and somite tissue (ST) are dissected away from the embryo trunk region. (G) After removal of the dorsal tissues, the dorsal aorta (Ao) is visible along the midline on a view of the dorsal trunk. (H) On the ventral trunk, the umbilical vessels (UC) are visible. The liver (FL) is seen as the pink tissue just above the umbilical cord. (I) A dorsal trunk region view showing the body walls (BW) lateral to the AGM have been dissected away. In this dorsal view, the urogenital ridges (UGR) are laterally juxtaposed to the dorsal aorta (Ao). (J) Ventral view of the AGM (only a small part of the UGR is visible) with overlying ventral tissues; stomach (ST) and liver (FL) and the fetal liver (FL) is still attached. (K) Crudely dissected and separated fetal liver (left) and AGM. (L) Cleanly dissected AGM region viewed from the ventral aspect. Ao = aorta (DA). Urogenital ridges located lateral to the Ao are clearly visible, with the genital ridge/developing gonads overlaying the pronephros and mesonephros (embryonic kidney).
    View Image
  •  FigureFigure 2A.6.2 Sort criteria used to purify populations from the E11.5 AGM region. In the left panel, viable cells are identified on the basis of cell size (forward light scatter) and the exclusion of the nuclear stain 7-amino-actinomycin D (7-AAD). Viable cells in the gated region are then separated into endothelial (VE-cadherin+CD45–), hematopoietic (VE-cadherin–CD45+), stem/progenitor (VE-cadherin+CD45+), and non-endothelial/haematopoietic lineages (VE-cadherin–CD45–) according to the differential plasma membrane expression of VE-cadherin and CD45 (right panel). According to this strategy, LTR-HSCs can be purified to a high frequency in the VE-cadherin+CD45+ population (North et al., 2002; Taoudi et al., 2005).
    View Image
  •  FigureFigure 2A.6.3 Distribution and classification criteria of clonogenic colony forming units-culture (CFU-C) within the AGM region. (A) Distribution of CFU potential within one embryo equivalent (~150,000 viable cells) of cells from the E11.5 AGM region. CFU-C identity is retrospectively ascribed following analysis of lineage potential using MethoCult medium (Stem Cell Technologies). (B) Criteria used for the classification of CFU-C identity: BFU-E produce erythroid cells in the presence of either macrophages or megakaryocytes; CFU-Mac, monocytes/macrophages; CFU-Mast, mast cells; CFU-GM, granulocytes and monocytes/macrophages; CFU-GEMM, granulocytes, monocytes/macrophages, erythroid cells and megakaryocytes (Meg). Colony images (top panels), original magnification 40×; cytospin preparations (bottom panels), original magnification 630×.
    View Image
  •  FigureFigure 2A.6.4 An example of how donor, recipient, and carrier cells can be distinguished following hematopoietic reconstitution. Dead cells and debris are excluded according to the uptake of 7-AAD and forward scatter profile; donor cells can subsequently be identified as Ly5.2/2 cells, recipient as Ly5.1/1, and carrier as Ly5.2/1.
    View Image
  •  FigureFigure 2A.6.5 Endothelial tubule forming potential of flow cytometrically purified AGM region cell populations. (A) In vitro endothelial tubule forming potential of E11.5 AGM region cell populations is largely restricted to the VE-cadherin+CD45– (endothelial) fraction. (B) Example of PECAM-1+ endothelial tubules produced from 5000 VE-cadherin+CD45– cells after 4 days in culture. (C) Example of the extensive vascular networks produced from 20,000 VE-cadherin+CD45– cells. Original magnification of photomicrographs 40×. VE-cad, VE-cadherin.
    View Image
  •  FigureFigure 2A.6.6 Mesenchymal cells from midgestation hematopoietic tissues. Differentiation to (A) osteogenic, (B) adipogenic, and (C) chondrogenic lineages. After 10 to 12 days in osteogenic medium, colonies of cells are positive (blue) when stained for alkaline phosphatase activity. After stimulation in adipogenic medium, colonies contained cells with a distinct adipocyte morphology, which includes the lipid droplets. After 21 days in chondrogenic medium, cells formed a cartilage-like tissue with an extracellular matrix rich in proteoglycans, as detected by toluidine blue staining.
    View Image

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Literature Cited

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