Isolation of Human Umbilical Cord Blood Aldehyde Dehydrogenase–Expressing Progenitor Cells that Modulate Vascular Regenerative Functions In Vitro and In Vivo

David M. Putman1, David A. Hess1

1 Vascular Biology Research Group, Robarts Research Institute, Department of Physiology and Pharmacology, Western University, London, Ontario, Canada
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2A.10
DOI:  10.1002/9780470151808.sc02a10s25
Online Posting Date:  May, 2013
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Abstract

This unit describes the isolation and application of human umbilical cord blood progenitor cells to modulate vascular regenerative functions using in vitro co‐culture systems and in vivo transplantation models. Using aldehyde dehydrogenase as a marker of stem cell function, blood‐derived progenitors can be efficiently purified form human umbilical cord blood using flow cytometry. We describe in vitro approaches to measure cell‐mediated effects on the survival, proliferation, and tube‐forming function of endothelial cells using growth‐rate assays and Matrigel tube‐forming assays. Additionally, we provide a detailed protocol for inducing acute unilateral hindlimb ischemia in immune‐deficient mice to assess progenitor cell–modulated effects on vascular regeneration by tracking the recovery of blood flow using noninvasive laser Doppler perfusion imaging. Collectively, we present combined in vitro and in vivo transplantation strategies for the pre‐clinical assessment of human progenitor cell–based therapies to treat ischemic disease. Curr. Protoc. Stem Cell Biol. 25:2A.10.1‐2A.10.19. © 2013 by John Wiley & Sons, Inc.

Keywords: aldehyde dehydrogenase; stem cells; umbilical cord blood; critical limb ischemia; angiogenesis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Umbilical Cord Blood Progenitor Cells with High Aldehyde Dehydrogenase Activity
  • Support Protocol 1: Cell Separation Based on Aldehyde Dehydrogenase Activity
  • Basic Protocol 2: Co‐Culture of UCB ALDHhi Cells to Assess Paracrine Effects on Endothelial Cell Survival, Proliferation, and Function
  • Support Protocol 2: Preparing Conditioned Medium to Assess Paracrine Support of Endothelial Cell Function
  • Basic Protocol 3: Murine Femoral Artery Ligation and Excision and Quantification of Vascular Regeneration In Vivo
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Umbilical Cord Blood Progenitor Cells with High Aldehyde Dehydrogenase Activity

  Materials
  • Clamped placenta and umbilical cord obtained from live birth
  • RosetteSephuman UCB progenitor enrichment cocktail (Stem Cell Technologies, cat. no. 15026; optional)
  • Dulbecco's phosphate‐buffered saline DPBS without Ca2+ or Mg2+ (Invitrogen, cat. no. 14190‐250)
  • Ficoll‐Hypaque gradient solution (density 1.077 g/liter, Stem Cell Technologies)
  • Fetal bovine serum (FBS, HyClone)
  • Ammonium chloride solution (Stem Cell Technologies, cat. no. 07850)
  • Aldefluor kit (Stem Cell Technologies, cat. no. 01700)
  • 50‐ml heparinized syringe (250 U) with 16‐gauge needle
  • 15‐ or 50‐ml conical tubes
  • Beckman GPR centrifuge with horizontal rotor
  • Additional reagents and equipment for counting cells by trypan blue exclusion (unit 1.3)

Support Protocol 1: Cell Separation Based on Aldehyde Dehydrogenase Activity

  Materials
  • Aldefluor kit (Stem Cell Technologies, cat. no. 01700) containing:
    • Dry Aldefluor reagent, 50 µg
    • DEAB inhibitor, 1.5 mM, in 95% ethanol, 1 ml
    • 2 N HCl, 1.5 ml
    • DMSO, 1.5 ml
    • Aldefluor assay buffer, 4 × 25 ml
  • Dulbecco's phosphate‐buffered saline (DPBS; without Ca2+ or Mg2+; Invitrogen, cat. no. 14190) plus 5% FBS
  • Hemacytometer and inverted light microscope (Zeiss), or automated cell counter
  • High‐speed fluorescence activated cell sorter (FACS Aria III; Becton Dickenson)
  • 15‐ml conical tubes containing 2 ml DPBS (without Ca2+ or Mg2+; Invitrogen, cat. no. 14190) plus 5% FBS (for sorted cell collection)
  • Additional reagents and equipment for counting cells by trypan blue exclusion (unit 1.3)
NOTE: Store kit reagents at 4° to 8°C according to the manufacturer's instructions. Do not freeze. Aliquots of activated Aldefluor reagent can be frozen and stored at −20°C for up to 1 year.NOTE: The following protocols have been optimized for lineage depleted UCB samples. The protocol has been slightly modified from the manufacturer's instructions to conserve reagents.

Basic Protocol 2: Co‐Culture of UCB ALDHhi Cells to Assess Paracrine Effects on Endothelial Cell Survival, Proliferation, and Function

  Materials
  • Endothelial cells: HMVEC (Lonza, cat. no. CC‐2543) or HUVEC (Lonza, cat. no. C2519A)
  • EBM‐2 basal medium (Lonza, cat. no. CC‐3156)
  • EGM‐2 MV SingleQuot Kit and growth factors (Lonza, cat. no. CC‐4147): contains a cocktail of growth factors (EGF, VEGF, FGF, IGF‐1) and fetal bovine serum (5% FBS) supplements to be added to basal medium (for HMVEC)
  • EGM‐2 SingleQuot Kit and growth factors (Lonza, cat. no. CC‐4176): contains a cocktail of growth factors (EGF, VEGF, FGF, IGF‐1) and fetal bovine serum (2% FBS) supplements to be added to basal medium (for HUVEC)
  • Dulbecco's phosphate‐buffered saline (DPBS; without Ca2+ or Mg2+; Invitrogen, cat. no. 14190); prepare in advance
  • Trypsin solution [TrypLE Express (1×), no phenol red; Invitrogen, cat. no. 12604‐013]
  • Fetal bovine serum (FBS, HyClone)
  • ALDH‐activity purified UCB cells (ALDHhi and ALDHlo from protocol 1)
  • Matrigel basement membrane matrix, growth factor reduced (BD Biosciences, cat. no. 356231)
  • Tissue culture treated dishes/plates (Corning: 75‐cm2 flasks for expansion, cat. no. 430641; 12‐well plate, cat. no. 3512; 24‐well plate, cat. no. 3524, for co‐culture assays)
  • 50‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Hemacytometer and inverted light microscope (Zeiss), or automated cell counter
  • Beckman GPR centrifuge with horizontal rotor
  • Hanging polycarbonate transwell inserts (3‐µm pore; Corning, cat. no. 3402)
  • Stereomicroscope
  • Additional reagents and equipment for counting cells by trypan blue exclusion (unit 1.3)

Support Protocol 2: Preparing Conditioned Medium to Assess Paracrine Support of Endothelial Cell Function

  • 0.2‐µm syringe filters

Basic Protocol 3: Murine Femoral Artery Ligation and Excision and Quantification of Vascular Regeneration In Vivo

  Materials
  • 100 mg/ml ketamine hydrochloride (100 mg/ml)
  • 20 mg/ml xylazine (20 mg/ml)
  • Isoflurane
  • Oxygen (O 2)
  • Lubricating eye ointment (Allergan Refresh Lacrilube)
  • Hair removal cream (Nair)
  • Bacteriostatic soap
  • Isopropyl alcohol
  • Betadine solution
  • Nexaband liquid topical tissue adhesive (Abbott Animal Health)
  • Buprenorphine
  • OCT tissue embedding medium
  • Liquid nitrogen
  • Dry ice
  • Mouse on Mouse (M.O.M.) Basic Kit (Vector Laboratories)
  • Mouse anti‐human HLA‐A,B,C antibody (appropriate dilution, 1:500; Becton Dickinson, cat. no. 557347)
  • Rat anti‐mouse CD31 antibody (appropriate dilution, 1:100; Becton Dickinson, cat. no. 550274)
  • Rabbit anti‐vWF antibody (appropriate dilution, 1:200; Millipore, cat. no. AB7356)
  • Mobile laboratory animal anesthesia system (VetEquip, cat. no. 901807) including mouse nose cones
  • Surgical tape
  • Electric hair clippers (Wahl)
  • Microsurgical inverted microscope (Nikon SMZ2B)
  • Surgical scissors and forceps
  • Sterile cotton‐tipped applicators
  • Cautery tool (Alcon Surgical Optemp II, http://alconlabs.com/)
  • 5‐0 silk sutures
  • Heat pad
  • Laser Doppler imager (Moor LDI2)
  • Laser Doppler imager (Moor LDI2, http://us.moor.co.uk/)
  • Tissue molds
  • Cryostat microtome
  • DAPI reagent
  • Immunofluorescent microscope (Zeiss AxioScope or similar)
  • Additional reagents and equipment for anesthesia of mice (Donovan and Brown, ), injection of mice (Donovan and Brown, a), euthanasia of mice (Donovan and Brown, b), and immunohistochemistry (Hofman, )
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Figures

Videos

Literature Cited

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Key References
  Putman et al., . See above.
  This paper demonstrates the characterization and application of UCB ALDHhi cells in vitro and in an immune‐deficient mouse model of acute unilateral hindlimb ischemia using the techniques described in detail here.
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