Expansion of Human Hematopoietic Stem/Progenitor Cells on Decellularized Matrix Scaffolds

Abhilasha Tiwari1, Melinda L. Tursky2, Lakshmi P. Nekkanti1, Graham Jenkin1, Mark A. Kirkland3, Gopal Pande4

1 The Ritchie Centre, Hudson Institute of Medical Research, Monash University, Clayton, 2 St. Vincent's Centre for Applied Medical Research, St. Vincent's Hospital, Sydney, New South Wales, 3 Geelong Technology Precinct, Deakin University, Geelong, Victoria, 4 CSIR Centre for Cellular and Molecular Biology (CCMB), Hyderabad
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1C.15
DOI:  10.1002/9780470151808.sc01c15s36
Online Posting Date:  February, 2016
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Umbilical cord blood (UCB) is one of the richest sources for hematopoietic stem/progenitor cells (HSPCs), with more than 3000 transplantations performed each year for the treatment of leukemia and other bone marrow, immunological, and hereditary diseases. However, transplantation of single cord blood units is mostly restricted to children, due to the limited number of HSPC per unit. This unit develops a method to increase the number of HSPCs in laboratory conditions by using cell‐free matrices from bone marrow cells that mimic ‘human‐body‐niche‐like’ conditions as biological scaffolds to support the ex vivo expansion of HSPCs. In this unit, we describe protocols for the isolation and characterization of HSPCs from UCB and their serum‐free expansion on decellularized matrices. This method may also help to provide understanding of the biochemical organization of hematopoietic niches and lead to suggestions regarding the design of tissue engineering–based biomimetic scaffolds for HSPC expansion for clinical applications. © 2016 by John Wiley & Sons, Inc.

Keywords: hematopoietic niche; decellularized biological scaffold; extracellular matrix; umbilical cord blood; stem cell expansion

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Table of Contents

  • Introduction
  • Basic Protocol 1: Processing and Isolation of CD34+ Umbilical Cord Blood Hematopoietic Stem Cells
  • Basic Protocol 2: Preparation of Decellularized Matrix Scaffolds
  • Basic Protocol 3: Expansion of HSPCs on Decellularized Matrices
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Processing and Isolation of CD34+ Umbilical Cord Blood Hematopoietic Stem Cells

  • 150 ml CPD single cord blood bags (Macopharma, cat. no. MSC1201DU)
  • 80% (v/v) ethanol
  • Processing buffer (see recipe)
  • Ficoll‐Paque Plus (GE Healthcare, cat. no. 17‐1440‐03)
  • 1% (w/v) trypan blue (Sigma‐Aldrich, cat. no. T8154)
  • PBS, degassed (see recipe)
  • Dulbecco's PBS (DPBS) without calcium or magnesium (Invitrogen, cat. no. 14190‐250)
  • CD34 Direct Progenitor Cell Isolation Kit 2 × 109 total cells (Miltenyi, cat. no. 130‐046‐702)
  • Staining solution (see recipe)
  • Recovery Cell Culture Freezing Medium (Invitrogen, cat. no. 12648‐010)
  • Liquid N 2
  • 50 μg/ml CD45‐FITC (BD Biosciences, cat. no. 347463)
  • 25 μg/ml CD34‐PE (BD Biosciences, cat. no. 348057)
  • 50 μg/ml 7‐AAD (BD Biosciences, cat. no. 559925)
  • Fixing solution (see recipe)
  • 15‐ and 50‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Centrifuge
  • LS columns (Miltenyi, cat. no. 130‐042‐401)
  • Magnet: MidiMACS or VarioMACS (Miltenyi)
  • Pre‐separation filter (Miltenyi, cat. no. 130‐041‐407)
  • 5‐ml round‐bottom flow cytometry tubes (Grale Scientific, cat. no. P7512T; http://www.grale.com.au/)
  • Cryovials (2‐ml Greiner Bio One; Interpath Sciences; http://www.interpath.com.au/)
  • Mr Frosty controlled‐rate freezer (Interpath Sciences, cat. no. 5100; http://www.interpath.com.au/)
  • FACSCalibur flow cytometer, CellQuest software (Becton Dickinson Biosciences)
  • SepMate‐50 tubes (Stem Cell Technologies, cat. no. 15470)
  • Additional reagents and equipment for counting cells with a hemacytometer and counting viable cells by trypan blue exclusion (e.g., unit ; Behar et al., ) and flow cytometry (Coligan et al., , Chapter 5)

Basic Protocol 2: Preparation of Decellularized Matrix Scaffolds

  • MS‐5 cells: established by Dr. Katsuhiko Itoh, Department of Clinical Medical Biology, Kyoto University, Japan (Itoh et al., ); kindly provided to us by Dr. Stewart Fabb, Monash Institute of Pharmaceutical Sciences, Australia [serious researchers may contact Dr. Gopal Pande, Group Head, Centre for Cellular and Molecular Biology, Hyderabad, India ( ) or Prof. Mark Kirkland of Deakin University, Victoria, Australia ( ) to request the cells as a courtesy]
  • MS‐5 complete medium (see reciperecipes)
  • PBS, degassed (see recipe)
  • Dimethylsulfoxide (DMSO)
  • 0.25% trypsin/EDTA (Invitrogen, cat. no. 25200‐056)
  • Liquid N 2
  • Osteogenic medium (see recipe)
  • Cell lysis solution: prepare 0.02 M NH 4OH in Milli‐Q water in fume hood and store up to 3 months at 4°C for up to 3 months
  • Penicillin‐streptomycin (pen‐strep; e.g., Invitrogen)
  • Centrifuge
  • 25‐cm2 tissue culture flasks
  • Mr Frosty controlled‐rate freezer (Interpath Sciences, cat. no. 5100; http://www.interpath.com.au/)
  • 24‐well plates
  • Inverted microscope
  • 15‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Additional reagents and equipment for counting cells with a hemacytometer and counting viable cells by trypan blue exclusion (e.g., unit ; Behar et al., )

Basic Protocol 3: Expansion of HSPCs on Decellularized Matrices

  • 1 mg/ml RetroNectin (RN) stock (Scientifix, cat. no. T100F; http://www.scientifix.com.au/)
  • PBS, degassed (see recipe)
  • CD34+ cells, frozen ( protocol 1)
  • Stemline II medium (Sigma‐Aldrich, cat. no. S0192)
  • Methocult H4434 (Stem Cell Technologies, cat. no. H4434)
  • HSPC expansion medium (see recipe)
  • Staining solution (see recipe)
  • CD34‐FITC (BD Bio Sciences, cat. no. 348053)
  • CD34‐PE (BD Bio Sciences, cat. no. 348057)
  • CD38‐FITC (BD Bio Sciences, cat. no. 555459)
  • CD45‐FITC (BD Bio Sciences, cat. no. 347463)
  • CD45‐PE (BD Bio Sciences, cat. no. 555483)
  • CD133‐PE (Miltenyi Biotech, cat. no. 130‐090‐853)
  • Human IgG secondary antibody—H&L PE conjugated (Abcam, cat. no. ab7006)
  • Human IgG secondary antibody—H&L, FITC conjugated (Abcam, cat. no. ab6854)
  • 7‐AAD (BD Bio Sciences, cat. no. 559925)
  • Cell Dissociation Solution (Sigma Aldrich, cat no. C5789‐100 ML)
  • 12‐ and 24‐well plates
  • Centrifuge
  • 3‐ml syringes
  • 16‐G blunt‐end needles (Stem Cell Technologies, cat. no. 28110)
  • FACSCalibur flow cytometer, CellQuest software (Becton Dickinson Biosciences)
  • Additional reagents and equipment for counting cells with a hemacytometer and counting viable cells by trypan blue exclusion (e.g., unit ; Behar et al., ), staining for flow cytometry ( protocol 1), and flow cytometry (Coligan et al., )
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