Isolation of Hematopoietic Stem Cells from Human Cord Blood

Taina Jaatinen1, Jarmo Laine1

1 Finnish Red Cross Blood Service, Helsinki
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2A.2
DOI:  10.1002/9780470151808.sc02a02s1
Online Posting Date:  June, 2007
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Abstract

Enrichment of hematopoietic stem cells is based on the expression of certain surface antigens, such as CD34 and CD133, or on the lack of expression of lineage‐specific antigens. Immunomagnetic positive selection of CD34+ or CD133+ cells is performed using paramagnetic microbeads conjugated to specific monoclonal antibodies (anti–human CD34 or anti–human CD133). In negative selection of lineage‐negative (Lin) cells, the unwanted cells are labeled with antibodies against known markers for mature hematopoietic cells (CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD66b, and glycophorin A) and retained in the column. Unlabeled cells pass through the column and are collected as the Lin cell fraction. Immunomagnetic cell sorting system MACS is a fast and gentle method to enrich hematopoietic stem cells. Viable and highly pure cells can be separated to be used in various downstream applications, such as flow cytometry and cell culture. Curr. Protoc. Stem Cell Biol. 1:2A.2.1‐2A.2.9. © 2007 by John Wiley & Sons, Inc.

Keywords: cord blood; fresh; cryopreserved; hematopoietic; CD34; CD133; Lin

     
 
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Table of Contents

  • Basic Protocol 1: Isolation of CD34+ or CD133+ Cells from Human Cord Blood
  • Basic Protocol 2: Isolation of Lin− Cells from Human Cord Blood
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation of CD34+ or CD133+ Cells from Human Cord Blood

  Materials
  • Mononuclear cells from human cord blood (unit 2.1)
  • Labeling buffer (see recipe), degassed
  • Direct CD34 Progenitor Cell Isolation Kit (no. 130‐046‐702, Miltenyi Biotec) or CD133 Cell Isolation Kit (no. 130‐050‐801, Miltenyi Biotec) containing:
    • FcR blocking reagent
    • MicroBeads
  • 10‐ml centrifuge tubes
  • Centrifuge (preferably a swinging‐bucket rotor)
  • MACS columns (MS, no. 130‐042‐201 or LS, no. 130‐042‐401; Miltenyi Biotech)
  • MACS separator (MiniMACS, no. 130‐042‐102 or MidiMACS, no 130‐042‐302; Miltenyi Biotec)
  • Additional reagents and equipment for preparing mononuclear cell suspension (unit 2.1)

Basic Protocol 2: Isolation of Lin− Cells from Human Cord Blood

  Materials
  • Mononuclear cells from human cord blood (unit 2.1)
  • Labeling buffer (see recipe), degassed
  • StemSep Human Progenitor Enrichment Kit (StemCell Technologies) containing:
    • Progenitor Enrichment Cocktail with monoclonal antibodies against CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD66b, and glycophorin A
    • StemSep magnetic colloid
  • MACS LD column (no. 130‐042‐901, Miltenyi Biotech)
  • Magnetic cell separator MidiMACS (no. 130‐042‐302, Miltenyi Biotec)
  • 10 ml centrifuge tubes
  • Additional reagents and equipment for preparing mononuclear cell suspension (unit 1.2)
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Figures

Videos

Literature Cited

Literature Cited
   Aroviita, P., Teramo, K., Hiilesmaa, V., and Kekomaki, R. 2005. Cord blood hematopoietic progenitor cell concentration and infant sex. Transfusion 45:613‐621.
   Broxmeyer, H.E., Douglas, G.W., Hangoc, G., Cooper, S., Bard, J., English, D., Arny, M., Thomas, L., and Boyse, E.A. 1989. Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells. Proc. Natl. Acad. Sci. U.S.A. 86:3828‐3832.
   Grewal, S.S., Barker, J.N., Davies, S.M., and Wagner, J.E. 2003. Unrelated donor hematopoietic cell transplantation: Marrow or umbilical cord blood? Blood 101:4233‐4244.
   Hao, Q.L., Shah, A.J., Thiemann, F.T., Smogorzewska, E.M., and Crooks, G.M. 1995. A functional comparison of CD34+ CD38− cells in cord blood and bone marrow. Blood 86:3745‐3753.
   Hogan, C.J., Shpall, E.J., McNulty, O., McNiece, I., Dick, J.E., Shultz, L.D., and Keller, G. 1997. Engraftment and development of human CD34(+)‐enriched cells from umbilical cord blood in NOD/LtSz‐scid/scid mice. Blood 90:85‐96.
   Kekarainen, T., Mannelin, S., Laine, J., and Jaatinen, T. 2006. Optimization of immunomagnetic separation for cord blood‐derived hematopoietic stem cells. B.M.C. Cell. Biol. 7:30.
   Vormoor, J., Lapidot, T., Pflumio, F., Risdon, G., Patterson, B., Broxmeyer, H.E., and Dick, J.E. 1994. Immature human cord blood progenitors engraft and proliferate to high levels in severe combined immunodeficient mice. Blood 83:2489‐2497.
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