High Level In Vitro Expansion of Murine Hematopoietic Stem Cells

Sanja Sekulovic1, Suzan Imren2, Keith Humphries1

1 University of British Columbia, Vancouver, British Columbia, Canada, 2 Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2A.7
DOI:  10.1002/9780470151808.sc02a07s4
Online Posting Date:  January, 2008
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Abstract

Development of strategies to extensively expand hematopoietic stem cells (HSCs) in vitro will be a major factor in enhancing the success of a range of transplant‐based therapies for malignant and genetic disorders. In addition to potential clinical applications, the ability to increase the number of HSCs in culture will facilitate investigations into the mechanisms underlying self‐renewal. In this unit, we describe a robust strategy for consistently achieving over 1000‐fold net expansion of HSCs in short‐term in vitro culture by using novel engineered fusions of the N‐terminal domain of nucleoporin 98 (NUP98) and the homeodomain of the hox transcription factor, HOXA10 (so called NUP98‐HOXA10hd fusion). We also provide a detailed protocol for monitoring the magnitude of HSC expansion in culture by limiting dilution assay of competitive lympho‐myeloid repopulating units (CRU Assay). These procedures provide new possibilities for achieving significant numbers of HSCs in culture, as well as for studying HSCs biochemically and genetically. Curr. Protoc. Stem Cell Biol. 4:2A.7.1‐2A.7.14. © 2008 by John Wiley & Sons, Inc.

Keywords: NUP98‐HOX fusion; HSC expansion; CRU assay; multilineage reconstitution

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Ex Vivo Expansion of Murine HSCs in Short‐Term Cultures
  • Basic Protocol 2: Quantitation of Murine HSCs by Limiting Dilution Analysis in Competitively Repopulated Hosts
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Ex Vivo Expansion of Murine HSCs in Short‐Term Cultures

  Materials
  • 2‐ to 4‐month‐old C57Bl/6Ly‐Pep3b [Pep3b (Ly5.1)] mice, bred and maintained at the British Columbia Cancer Research Centre (http://www.bccrc.ca) animal facility according to the guidelines of the Canadian Council on Animal Care
  • 5‐fluorouracil (5‐FU, Mayne Pharma, http://www.maynepharma.com/)
  • Dulbecco's phosphate‐buffered saline without Ca2+ and Mg2+ (CMF‐DPBS, StemCell Technologies, cat. no. 37350)
  • CMF‐PBS containing 2% (v/v) fetal bovine serum (FBS, StemCell Technologies, cat. no. 06250); store up to 1 month at 4°C
  • 0.8% (w/v) NH 4Cl/1 mM EDTA in H 2O (StemCell Technologies, cat. no. 07850), ice cold
  • DMEM with 15% FBS (see recipe) containing 10 ng/ml human IL‐6, 6 ng/ml murine IL‐3, and 100 ng/ml murine SCF (cytokines available from StemCell Technologies)
  • Sca1+lin or c‐kit+Sca1+lin BM cells (to initiate culture with a starting population highly enriched in HSCs, 5‐FU BM can be further purified to obtain these cells; see Ohta et al., )
  • GP+E‐86 retroviral producer cells (Dr. Keith Humphries, Terry Fox Laboratory, BC Cancer Agency, Vancouver, Canada; see annotation to step 7) irradiated with 40 Gy of X rays (or equivalent)
  • DMEM with 15% FBS (see recipe) containing 10 ng/ml human IL‐6, 6 ng/ml murine IL‐3, 100 ng/ml murine SCF (StemCell Technologies), and 5 µg/ml protamine sulfate (Sigma)
  • DMEM wash medium (see recipe)
  • DMEM with 15% FBS (see recipe)
  • Dissecting instruments including scissors and forceps
  • 22‐G, 1‐in. and 26‐G, 0.5‐in. needles and 3‐ml syringes for harvesting bone marrow
  • Tabletop centrifuge
  • Bacteriological petri dishes, standard style, 100 × 20–mm (BD Falcon, cat. no. 351005)
  • 96‐well U‐bottom microtiter plates (BD Falcon, cat. no. 353077)
  • Cell culture dishes, standard tissue culture treated, 100 × 20–mm (BD Falcon, cat. no. 353003)
  • Cell lifters (Corning)
  • 50‐ml conical tubes (BD Falcon, cat. no. 352070)
  • 24‐well flat‐bottom plates (BD Falcon, cat no. 353047)
  • Additional reagents and equipment for intravenous injection of mice (Donovan and Brown, ), euthanasia of mice (Donovan and Brown, ), counting cells (Phelan, ), and CRU assay ( protocol 2)

Basic Protocol 2: Quantitation of Murine HSCs by Limiting Dilution Analysis in Competitively Repopulated Hosts

  Materials
  • 2‐ to 6‐month‐old C57Bl/6‐W41/W41 [W41 (Ly5.2)] mice bred and maintained at the British Columbia Cancer Research Centre animal facility according to the Canadian Council on Animal Care (also available from The Jackson Laboratory)
  • Dulbecco's phosphate‐buffered saline without Ca2+ and Mg2+ (CMF‐DPBS, StemCell Technologies, cat. no. 37350) containing 2% fetal bovine serum (FBS, StemCell Technologies, cat. no. 06250)
  • Acidified water: prepare 0.1 N HCl in sterile distilled water, then dilute this solution 1:100 in the animals’ drinking water
  • 0.8% (w/v) NH 4Cl/1 mM EDTA in H 2O (StemCell Technologies, cat. no. 07850), ice cold
  • CMF‐PBS containing 2% (v/v) fetal bovine serum (FBS, StemCell Technologies, cat. no. 06250); store up to 1 month at 4°C
  • Antibodies (fluorochrome‐conjugated; BD Pharmingen): B220‐PE, Ly6G‐PE, Mac1‐PE, CD4‐PE, CD8‐PE, Ly5.1‐biotin
  • Streptavidin‐APC (BD Pharmingen)
  • CMF‐DPBS containing 2% (v/v) FBS and 1 µg/ml propidium iodide
  • Mouse irradiator (X‐ray or cesium unit or equivalent)
  • Insulin syringes with 28‐G ½‐in. needles (BD)
  • Heparinized capillary tubes (e.g., Fisher Scientific)
  • 14‐ml polypropylene round‐bottom tubes (BD, cat. no. 352059)
  • Tabletop centrifuge
  • 96‐well U‐bottom microtiter plates plate (BD Falcon, cat. no. 353077)
  • 1.4‐ml ScreenMates round‐bottom storage tubes in a snap rack (Matrix Technologies, cat. no. 4246)
  • L‐Calc Software (StemCell Technologies)
  • Additional reagents and equipment for tail‐vein injection of the mouse (Donovan and Brown, ), tail‐vein blood collection from the mouse (Donovan and Brown, ), and flow cytometry (Robinson et al., )
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Figures

Videos

Literature Cited

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