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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Abstract

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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Materials

Basic Protocol 1: Processing and Isolation of CD34+ Umbilical Cord Blood Hematopoietic Stem Cells

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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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Figures

Videos

Literature Cited

Literature Cited
   Adams, G.B. 2008. Deconstructing the hematopoietic stem cell niche: Revealing the therapeutic potential. Regen. Med. 3:523‐530. doi: 10.2217/17460751.3.4.523.
   Aizman, I. , Tate, C.C. , McGrogan, M. , and Case, C.C. 2009. Extracellular matrix produced by bone marrow stromal cells and by their derivative, SB623 cells, supports neural cell growth. J. Neurosci. Res. 87:3198‐3206. doi: 10.1002/jnr.22146.
   Atkinson, K. 2000. Clinical Bone Marrow and Stem Cell Transplantation, 2nd ed. Cambridge University Press, New York.
   Banu, N. , Rosenzweig, M. , Kim, H. , Bagley, J. , and Pykett, M. 2001. Cytokine‐augmented culture of haematopoietic progenitor cells in a novel three‐dimensional cell growth matrix. Cytokine 13:349‐358. doi: 10.1006/cyto.2001.0836.
   Behar, R.Z. , Bahl, V. , Wang, Y. , Weng, J.‐H. , Lin, S.C. , and Talbot, P. 2012. Adaptation of stem cells to 96‐well plate assays: Use of human embryonic and mouse neural stem cells in the MTT assay. Curr. Protoc. Stem Cell Biol. 23:1C.13.1‐1C.13.21.
   Bottaro, D.P. , Liebmann‐Vinson, A. , and Heidaran, M.A. 2002. Molecular signaling in bioengineered tissue microenvironments. Ann. N.Y. Acad. Sci. 961:143‐153. doi: 10.1111/j.1749‐6632.2002.tb03068.x.
   Broxmeyer, H.E. , Cooper, S. , Hass, D.M. , Hathaway, J.K. , Stehman, F.B. , and Hangoc, G. 2009. Experimental basis of cord blood transplantation. Bone Marrow Transplant. 44:627‐633. doi: 10.1038/bmt.2009.285.
   Celebi, B. , Mantovani, D. , and Pineault, N. 2011. Effects of extracellular matrix proteins on the growth of haematopoietic progenitor cells. Biomed. Mater. 6:055011. doi: 10.1088/1748‐6041/6/5/055011.
   Chen, X.D. , Dusevich, V. , Feng, J.Q. , Manolagas, S.C. , and Jilka, R.L. 2007. Extracellular matrix made by bone marrow cells facilitates expansion of marrow‐derived mesenchymal progenitor cells and prevents their differentiation into osteoblasts. J. Bone Miner. Res. 22:1943. doi: 10.1359/jbmr.070725.
   Chou, S. , Chu, P. , Hwang, W. , and Lodish, H. 2010. Expansion of human cord blood hematopoietic stem cells for transplantation. Cell Stem Cell 7:427‐428. doi: 10.1016/j.stem.2010.09.001.
   Coligan, J.E. , Kruisbeek, A.M. , Margulies, D.H. , Shevach, E.M. , and Strober, W. (eds.) 2014. Current Protocols in Immunology. John Wiley & Sons, Hoboken, N.J.
   Dellatore, S.M. , Garcia, A.S. , and Miller, W.M. 2008. Mimicking stem cell niches to increase stem cell expansion. Curr. Opin. Biotechnol. 19:534‐540. doi: 10.1016/j.copbio.2008.07.010.
   Evans, N.D. , Gentleman, E. , Chen, X. , Roberts, C.J. , Polak, J.M. , and Stevens, M.M. 2010. Extracellular matrix‐mediated osteogenic differentiation of murine embryonic stem cells. Biomaterials 31:3244‐3252. doi: 10.1016/j.biomaterials.2010.01.039.
   Franke, K. , Pompe, T. , Bornhauser, M. , and Werner, C. 2007. Engineered matrix coatings to modulate the adhesion of CD133+ human hematopoietic progenitor cells. Biomaterials 28:836‐843. doi: 10.1016/j.biomaterials.2006.09.031.
   Guilak, F. , Cohen, D.M. , Estes, B.T. , Gimble, J.M. , Liedtke, W. , and Chen, C.S. 2009. Control of stem cell fate by physical interactions with the extracellular matrix. Cell Stem Cell 5:17‐26. doi: 10.1016/j.stem.2009.06.016.
   Hamilton, R. and Campbell, F.R. 1991. Immunochemical localization of extracellular materials in bone marrow of rats. Anat. Rec. 231:218‐224. doi: 10.1002/ar.1092310210.
   Hofmeister, C.C. , Zhang, J. , Knight, K.L. , Le, P. , and Stiff, P.J. 2007. Ex vivo expansion of umbilical cord blood stem cells for transplantation: Growing knowledge from the hematopoietic niche. Bone Marrow Transplant. 39:11‐23. doi: 10.1038/sj.bmt.1705538.
   Itoh, K. , Tezuka, H. , Sakoda, H. , Konno, M. , Nagata, K. , Uchiyama, T. , Uchino, H. , and Mori, K.J. 1989. Reproducible establishment of hemopoietic supportive stromal cell lines from murine bone marrow. Exp. Hematol. 17:145‐153.
   Kanai, M. , Hirayama, F. , Yamaguchi, M. , Ohkawara, J. , Sato, N. , Fukazawa, K. , Yamashita, K. , Kuwabara, M. , Ikeda, H. , and Ikebuchi, K. 2000. Stromal cell‐dependent ex vivo expansion of human cord blood progenitors and augmentation of transplantable stem cell activity. Bone Marrow Transplant. 26:837‐844. doi: 10.1038/sj.bmt.1702634.
   Keller, U. , Gotze, K.S. , Duyster, J. , Schmidt, B. , Rose‐John, S. , and Peschel, C. 2002. Murine stromal cells producing hyper‐interleukin‐6 and Flt3 ligand support expansion of early human hematopoietic progenitor cells without need of exogenous growth factors. Leukemia 16:2122‐2128. doi: 10.1038/sj.leu.2402660.
   Kleinman, H.K. , Philp, D. , and Hoffman, M.P. 2003. Role of the extracellular matrix in morphogenesis. Curr. Opin. Biotechnol. 14:526‐532. doi: 10.1016/j.copbio.2003.08.002.
   Philp, D. , Chen, S.S. , Fitzgerald, W. , Orenstein, J. , Margolis, L. , and Kleinman, H.K. 2005. Complex extracellular matrices promote tissue‐specific stem cell differentiation. Stem Cells 23:288‐296. doi: 10.1634/stemcells.2002‐0109.
   Sutherland, D.R. , Anderson, L. , Keeney, M. , Nayar, R. , and Chin‐Yee, I. 1996. The ISHAGE guidelines for CD34+ cell determination by flow cytometry. International Society of Hematotherapy and Graft Engineering. J. Hematother. 5:213‐226. doi: 10.1089/scd.1.1996.5.213.
   Till, J.E. and McCulloch, E. 1961. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat. Res. 14:213‐222. doi: 10.2307/3570892.
   Tiwari, A. , Lefevre, C. , Kirkland, M.A. , Nicholas, K. , and Pande, G. 2013. Comparative gene expression profiling of stromal cell matrices that support expansion of hematopoietic stem/progenitor cells. J. Stem Cell Res. 3:152.
   Tiwari, A. , Tursky, M.L. , Mushahary, D. , Wasnik, S. , Collier, F.M. , Suma, K. , Kirkland, M.A. , and Pande, G. 2012. Ex vivo expansion of haematopoietic stem/progenitor cells from human umbilical cord blood on acellular scaffolds prepared from MS‐5 stromal cell line. J. Tissue Eng. Regen. Med. doi: 10.1002/term.1479.
   Topp, K.S. , Tablin, F. , and Levin, J. 1990. Culture of isolated bovine megakaryocytes on reconstituted basement membrane matrix leads to proplatelet process formation. Blood 76:912‐924.
   Tursky, M.L. , Collier, F.M. , Ward, A.C. , and Kirkland, M.A. 2012. Systematic investigation of oxygen and growth factors in clinically valid ex vivo expansion of cord blood CD34(+) hematopoietic progenitor cells. Cytotherapy 14:679‐685. doi: 10.3109/14653249.2012.666851.
   Wasnik, S. , Tiwari, A. , Kirkland, M.A. , and Pande, G. 2012. Osteohematopoietic stem cell niches in bone marrow. Int. Rev. Cell Mol. Biol. 298:95‐133. doi: 10.1016/B978‐0‐12‐394309‐5.00003‐1.
   Weissman, I.L. 2000. Stem cells: Units of development, units of regeneration, and units in evolution. Cell 100:157‐168. doi: 10.1016/S0092‐8674(00)81692‐X.
   Yao, C.L. and Hwang, S.M. 2007. Ex vivo expansion of hematopoietic stem cells from human cord blood in serum‐free conditions. Methods Mol. Biol. 407:165‐175. doi: 10.1007/978‐1‐59745‐536‐7_13.
   You, M. , Peng, G. , Li, J. , Ma, P. , Wang, Z. , Shu, W. , Peng, S. , and Chen, G.Q. 2011. Chondrogenic differentiation of human bone marrow mesenchymal stem cells on polyhydroxyalkanoate (PHA) scaffolds coated with PHA granule binding protein PhaP fused with RGD peptide. Biomaterials 32:2305‐2313. doi: 10.1016/j.biomaterials.2010.12.009.
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