Isolation and Visualization of Mouse Placental Hematopoietic Stem Cells

Christos Gekas1, Katrin E. Rhodes1, Hanna K.A. Mikkola1

1 University of California Los Angeles, Los Angeles, California
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2A.8
DOI:  10.1002/9780470151808.sc02a08s6
Online Posting Date:  August, 2008
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Abstract

This unit describes the isolation of hematopoietic stem cells (HSCs) from the mouse placenta. The placenta was recently identified as an important hematopoietic site that generates HSCs de novo and provides a transitory niche for a large pool of HSCs during midgestation. This protocol includes a dissection technique for murine placenta, the mechanical and enzymatic steps of placental tissue dissociation, and phenotypical identification and isolation of HSCs. It also contains a method for immunohistochemical analysis of placenta tissue sections to visualize developing HSCs in the placenta. Curr. Protoc. Stem Cell Biol. 6:2A.1.1‐2A.1.14. © 2008 by John Wiley & Sons, Inc.

Keywords: hematopoietic stem cell (HSC); placenta; fetal; dissection; flow cytometry; FACS sorting; fixed‐frozen sections; immunohistochemistry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Dissection of Murine Placenta
  • Basic Protocol 2: Dissociation of Placental Tissue
  • Basic Protocol 3: Isolation of Placental HSCs by FACS Sorting
  • Basic Protocol 4: Preparation of Placental Tissue for Immunohistochemistry
  • Support Protocol 1: Immunohistochemistry on Fixed Frozen Placental Tissue Sections for HSCs
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Dissection of Murine Placenta

  Materials
  • Pregnant mouse with midgestation embryos
  • Isoflurane
  • 1× Dulbecco's phosphate‐buffered saline with calcium and magnesium (DPBS; Cellgro/Mediatech)
  • 1× DPBS (with calcium and magnesium; Cellgro/Mediatech)/5% (v/v) fetal bovine serum (FBS)
  • Scissors
  • Number 5 or 55 dissection forceps, stainless steel or titanium
  • 35‐mm petri dishes
  • Ice
  • Inverted dissection microscope
  • 15‐ml centrifuge tubes

Basic Protocol 2: Dissociation of Placental Tissue

  Materials
  • 1% (w/v) collagenase stock solution: prepared by dissolving collagenase type IA (from Clostridium histolyticum) lyophilized powder (Sigma) in water; dispense into aliquots and store at −20°C
  • 1× Dulbecco's phosphate‐buffered saline without calcium and magnesium (CMF‐DPBS; Cellgro/Mediatech)
  • Fetal bovine serum (FBS)
  • 10,000 U penicillin and 10,000 µg streptomycin/ml 0.85% saline (P/S; Invitrogen)
  • Dissected placentas ( protocol 1)
  • 15‐ml conical tubes
  • 5‐ml plastic syringes (BD)
  • 16‐G, 18‐G, 20‐G, 22‐G, and 25‐G needles (BD)
  • 50‐µm cell filters (CellTrics, Partec)

Basic Protocol 3: Isolation of Placental HSCs by FACS Sorting

  Materials
  • Dissociated placental cells ( protocol 2)
  • 1× Dulbecco's phosphate‐buffered saline without calcium and magnesium (CMF‐DPBS; Cellgro/Mediatech)
  • 1× red blood cell (RBC) lysis buffer (eBiosciences)
  • Phycoerythrin‐labeled rat‐anti‐mouse c‐kit antibody (c‐kit‐PE; BD Pharmingen)
  • Allophycocyanin‐labeled rat‐anti‐mouse CD34 antibody (CD34‐APC; eBiosciences)
  • 7‐amino‐actinomycin D (7‐AAD; BD Biosciences)
  • Tissue‐culture treated, round‐bottom 96‐well plate (BD; Falcon) or 15‐ml conical tube or 1.5‐ml microcentrifuge tube
  • Centrifuge with rotor adapted for 96‐well plates, optional
  • FACS tubes (5‐ml 12 × 75–mm polypropylene round‐bottom test tube, e.g., BD Falcon)
  • Flow cytometer or a cell sorter (e.g., LSR II, BD; FACSAria, BD)

Basic Protocol 4: Preparation of Placental Tissue for Immunohistochemistry

  Materials
  • Dissected placentas ( protocol 1)
  • 1× Dulbecco's phosphate‐buffered saline without calcium and magnesium (CMF‐DPBS; Cellgro/Mediatech), 4°C
  • 4% (w/v) paraformaldehyde (PFA): dispense into aliquots and store at −20°C
  • 30% (w/v) sucrose solution
  • Optimal cutting temperature solution (O.C.T., Tissue‐Tek)
  • Dry ice
  • Drierite
  • Forceps
  • Tissue culture–treated, round‐bottom 96‐well or 24‐well plates (BD Falcon)
  • Razor blade
  • 10 × 10 × 5–mm disposable vinyl specimen molds (e.g., Cryomolds, Tissue‐Tek)
  • Permanent marker
  • Small plastic bags
  • −80°C freezer

Support Protocol 1: Immunohistochemistry on Fixed Frozen Placental Tissue Sections for HSCs

  Materials
  • 5‐ to 8‐µm placental sections mounted on glass microscope slides ( protocol 4)
  • 10× GO buffer (see recipe)
  • 1000 U/ml glucose oxidase (GO; Sigma)
  • 1× Dulbecco's phosphate‐buffered saline without calcium and magnesium (CMF‐DPBS; Cellgro/Mediatech)
  • 2.5 µg/ml proteinase K solution: dilute 20 mg/ml proteinase K solution (Amresco) in CMF‐DPBS (Cellgro/Mediatech); store up to 3 months at 4°C
  • Tyramide amplification kit (Invitrogen) including:
    • Tyramide blocking buffer
    • Streptavidin (SA)‐horseradish peroxidase (HRP)
  • CD41 primary antibody (BD Pharmingen)
  • Biotinylated anti‐rat IgG (Vector Laboratories) secondary antibody
  • CMF‐DPBS/0.1% (v/v) Tween 20
  • Vectastain ABC‐AP kit (alkaline phosphatase–based conjugate) or ABC Elite kit (peroxidase‐based conjugate): each kit includes reagent A and reagent B (Vector Laboratories)
  • Appropriate substrate solutions: peroxidase substrate kit DAB (Vector Laboratories) or alkaline phosphatase substrate kit III (Vector Blue, Vector Laboratories) or alkaline phosphatase substrate kit I (Vector Red, Vector Laboratories)
  • Levamisole (Vector Laboratories)
  • CD31/Pecam primary antibody (BD Pharmingen)
  • Cytokeratin primary antibody (Dako Cytomation)
  • Normal blocking buffer: 5% (v/v) normal horse serum (Vector Laboratories)/0.05% (v/v) Tween 20 (Ultra, Sigma)/PBS
  • Avidin/biotin blocking kit (Vector Laboratories)
  • Biotinylated anti‐rabbit IgG (Vector Laboratories)
  • Mounting medium, aqueous‐based mounting medium (e.g., Vectamount AQ, Vector Laboratories)
  • Plastic microscope slide mailers (Fisher)
  • 2.5‐mm Super Pap Pen HT slide markers (Research Products International, Fisher)
  • Humidified chamber (plastic slide box with ¼ in. of water), recommended
  • Vacuum apparatus: glass filter flask with vacuum side arms and plastic tubing (Nalgene 180 PVC, nontoxic and autoclavable)
  • Transfer pipets
  • Microscope
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Figures

Videos

Literature Cited

Literature Cited
   Alvarez‐Silva, M., Belo‐Diabangouaya, P., Salaun J., and Dieterlen‐Lievre, F. 2003. Mouse placenta is a major hematopoietic organ. Development 130:5437‐5444.
   Gekas, C., Dieterlen‐Lievre, F., Orkin, S.H., and Mikkola, H.K. 2005. The placenta is a niche for hematopoietic stem cells. Dev. Cell 8:365‐375.
   Mikkola, H.K. and Orkin, S.H. 2006. The journey of developing hematopoietic stem cells. Development 133:3733‐3744.
   Mikkola, H.K., Fujiwara, Y., Schlaeger, T.M., Traver, D., and Orkin, S.H. 2003. Expression of CD41 marks the initiation of definitive hematopoiesis in the mouse embryo. Blood 101:508‐516.
   Mikkola, H.K., Gekas, C., Orkin, S.H., and Dieterlen‐Lievre, F. 2005. Placenta as a site for hematopoietic stem cell development. Exp. Hematol. 33:1048‐1054.
   Ottersbach, K. and Dzierzak, E. 2005. The murine placenta contains hematopoietic stem cells within the vascular labyrinth region. Dev. Cell 8:377‐387.
   Rhodes, K.E., Gekas, C., Wang, Y., Lux, C.T., Francis, C.S., Chan, D.N., Conway, S., Orkin, S.H., Yoder, M.C., and Mikkola, H.K.A. 2008. The emergence of hematopoietic stem cells is initiated in the placental vasculature in the absence of circulation. Cell Stem Cell 2:252‐263.
   Sanchez, M.J., Holmes, A., Miles, C., and Dzierzak, E. 1996. Characterization of the first definitive hematopoietic stem cells in the AGM and liver of the mouse embryo. Immunity 5:513‐525.
   Weissman, I.L. 2000. Stem cells: Units of development, units of regeneration, and units in evolution. Cell 100:157‐168.
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