Overview of Very Small Embryonic‐Like Stem Cells (VSELs) and Methodology of Their Identification and Isolation by Flow Cytometric Methods

Ewa K. Zuba‐Surma1, Mariusz Z. Ratajczak1

1 Stem Cell Biology Institute, University of Louisville, Louisville, Kentucky
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.29
DOI:  10.1002/0471142956.cy0929s51
Online Posting Date:  January, 2010
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Abstract

The protocols presented here describe the procedures employed to identify and isolate very small embryonic‐like stem cells (VSELs) using flow cytometric technologies including fluorescence‐activated cell sorting (FACS). We describe the recommended steps in detail for their successful identification and isolation from adult tissues. These protocols were initially established to isolate such cells from murine bone marrow (BM) and human cord blood (CB) and may also be employed to isolate these primitive cells from other adult organs and embryonic tissues. Here, we focus on some critical parameters/key points required for the successful identification and purification of these rare cells by employing classical flow cytometry. In the last part of this unit, we also discuss a novel flow cytometric tool, ImageStream, an imaging flow cytometer, which allows better identification and morphological analysis of sorted cells. Curr. Protoc. Cytom. 51:9.29.1‐9.29.15. © 2010 by John Wiley & Sons, Inc.

Keywords: VSELs; flow cytometry; ImageStream; Oct‐4; pluripotent stem cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Identification and Isolation of VSELs from Adult Murine Bone Marrow (BM) by Classical Flow Cytometry
  • Basic Protocol 2: Identification and Isolation of VSELs from Human Umbilical Cord Blood by Classical Flow Cytometry
  • Basic Protocol 3: Identification of Murine and Human VSELs by Imaging Cytometry
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Identification and Isolation of VSELs from Adult Murine Bone Marrow (BM) by Classical Flow Cytometry

  Materials
  • 8 to 10 adult C57BL/6 mice (4‐ to 8‐week‐old; Jackson Laboratory)
  • Medium used for bone marrow (BM) isolation and staining including:
    • RPMI 1640 (no serum; Invitrogen)
    • RPMI 1640 with 2% fetal bovine serum (FBS; Invitrogen)
  • Lysing buffer (BD PharmLyse; BD Biosciences, cat. no. 555899)
  • Antibodies used for staining include rat monoclonal antibodies against murine epitopes, predominantly directly conjugated with fluorochromes (listed in Table 9.29.1)
  • Flow Cytometry Size Calibration Kit microspheres (Invitrogen)
  • 100‐mm plastic tissue‐grade dishes (BD Biosciences)
  • 50‐ml plastic tissue‐grade culture tubes (BD Biosciences)
  • 5‐ml syringes and 25‐G needles (BD Biosciences)
  • 70‐ and 40‐µm strainer/mesh filters (BD Biosciences)
  • Centrifuge
  • 5‐ml round‐bottom tubes (BD Biosciences)
  • Flow cytometer
  • Additional reagents and equipment for staining cells (units 6.2& 6.21)
    Table 9.9.1   MaterialsAntibodies Employed in Staining for Identification and Sorting of Murine BM‐Derived VSELs by Flow Cytometry

    Antibody Clone Fluorochrome Vendor
    antiCD45 30‐F11 APC‐Cy7 BD Pharmingen
    anti‐Ly‐6A/E (Sca‐1) E13‐161.7 biotin BD Pharmingen
    Streptavidin PE‐Cy5 BD Pharmingen
    anti‐CD45R/B220 RA3‐6B2 PE BD Pharmingen
    anti‐Gr1 RB6‐8C5 PE BD Pharmingen
    anti‐TCR β H57‐597 PE BD Pharmingen
    anti‐TCR γδ GL3 PE BD Pharmingen
    anti‐CD11b M1/70 PE BD Pharmingen
    anti‐Ter119 TER‐119 PE BD Pharmingen
    Isotype controls:
    Rat IgG2b, κ A95‐1 APC‐Cy7 BD Pharmingen
    Rat IgG2a, κ R35‐95 PE‐Cy5 BD Pharmingen
    Rat IgG2a, κ R35‐95 PE BD Pharmingen

Basic Protocol 2: Identification and Isolation of VSELs from Human Umbilical Cord Blood by Classical Flow Cytometry

  Materials
  • Anticoagulated cord blood (CB) sample (harvested fresh from the umbilical cord vein immediately after delivery and collected into a medium supplemented with anticoagulant)
  • Lysing buffer (BD PharmLyse; BD Biosciences, cat. no. 555899)
  • RPMI 1640 medium with 2% fetal bovine serum (FBS; Invitrogen)
  • Antibodies used for staining include mouse monoclonal antibodies against human epitopes that are predominantly directly conjugated with fluorochromes (listed in Table 9.29.2)
  • Flow Cytometry Size Calibration Kit microspheres (Invitrogen)
  • 50‐ml plastic tissue culture–grade tubes (BD Biosciences)
  • Centrifuge
  • 5‐ml round‐bottom tubes (BD Biosciences)
  • 40‐µm strainer/mesh filters (BD Biosciences)
  • Flow cytometer
    Table 9.9.2   MaterialsAntibodies Employed in Staining for Identification and Sorting of Human CB‐Derived VSELs by Flow Cytometry

    Antibody Clone Fluorochrome Vendor
    anti‐CD45 HI30 PE BD Biosciences
    anti‐CD19 HIB19 FITC BD Biosciences
    anti‐CD2 RPA‐2.10 FITC BD Biosciences
    anti‐CD3 UCHT1 FITC BD Biosciences
    anti‐CD14 M5E2 FITC BD Biosciences
    anti‐CD66b G10F5 FITC BD Biosciences
    anti‐CD24 ML5 FITC BD Biosciences
    anti‐CD16 3G8 FITC BD Biosciences
    anti‐CD56 NCAM16.2 FITC BD Biosciences
    anti‐CD235a GA‐R2 FITC BD Biosciences
    anti‐AC133 AC133 APC Miltenyi Biotec
    Isotype controls:
    Mouse IgG1, κ MOPC‐21 PE BD Biosciences
    Mouse IgG1, κ MOPC‐21 FITC BD Biosciences
    Mouse IgG2a, κ G155‐178 FITC BD Biosciences
    Mouse IgG2b, κ 27‐35 FITC BD Biosciences
    Mouse IgM, κ MM‐30 FITC BioLegend
    Mouse IgG1, κ MOPC‐21 APC BD Biosciences

Basic Protocol 3: Identification of Murine and Human VSELs by Imaging Cytometry

  Materials
  • Murine BM or human CB are processed as described in Basic Protocols protocol 11 and protocol 22 to obtain TNCs
  • RPMI 1640 medium with 2% fetal bovine serum (FBS; Invitrogen)
  • Antibodies and dyes used for staining (including antibodies described for classical flow cytometry in Tables 9.29.1 and 9.29.2, as well as 7‐AAD DNA dye for nuclear staining)
  • 2% (w/v) paraformaldehyde (see recipe)
  • 1× phosphate‐buffered saline (PBS), calcium‐ and magnesium‐free (CMF‐PBS; Invitrogen)
  • 0.1% Triton X‐100 solution (see recipe)
  • Centrifuge
  • 0.5‐ml microcentrifuge tubes
  • 40‐µm strainer/mesh filters (BD Biosciences)
  • ImageStream system 100 (Amnis), the standard imaging flow cytometer equipped with blue laser (488 nm)
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Figures

Videos

Literature Cited

Literature Cited
   Basiji, D.A., Ortyn, W.E., Liang, L., Venkatachalam, V., and Morrissey, P. 2007. Cellular image analysis and imaging by flow cytometry. Clin. Lab. Med. 27:653‐670.
   Kucia, M., Ratajczak, J., and Ratajczak, M.Z. 2005. Are bone marrow stem cells plastic or heterogeneous—that is the question. Exp. Hematol. 33:613‐623.
   Kucia, M., Reca, R., Campbell, F.R., Zuba‐Surma, E., Majka, M., Ratajczak, J., and Ratajczak, M.Z. 2006. A population of very small embryonic‐like (VSELs) CXCR4(+)SSEA‐1(+)Oct‐4+ stem cells identified in adult bone marrow. Leukemia 20:857‐869.
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   Ratajczak, M.Z., Machalinski, B., Wojakowski, W., Ratajczak, J., and Kucia, M. 2007. A hypothesis for an embryonic origin of pluripotent Oct‐4+ stem cells in adult bone marrow and other tissues. Leukemia 21:860‐867.
   Ratajczak, M.Z., Kucia, M., Reca, R., Majka, M., Janowska‐Wieczorek, A., and Ratajczak, J. 2004. Stem cell plasticity revisited: CXCR4‐positive cells expressing mRNA for early muscle, liver and neural cells ‘hide out’ in the bone marrow. Leukemia 18:29‐40.
   Wagers, A.J. and Weissman, I.L. 2004. Plasticity of adult stem cells. Cell 116:639‐648.
   Wojakowski, W., Tendera, M., Kucia, M., Zuba‐Surma, E., Paczkowska, E., Ciosek, J., Halasa, M., Krol, M., Kazmierski, M., Buszman, P., Ochala, A., Ratajczak, J., Machalinski, B., and Ratajczak, M.Z. 2009. Mobilization of bone marrow‐derived Oct‐4+ SSEA‐4+ very small embryonic‐like stem cells in patients with acute myocardial infarction. J. Am. Coll. Cardiol. 53:1‐9.
   Zuba‐Surma, E.K., Kucia, M., Abdel‐Latif, A., Lillard, J.J., and Ratajczak, M.Z. 2007a. The ImageStream System: a key step to a new era in imaging. Folia Histochem. Cytobiol. 45:279‐290.
   Zuba‐Surma, E.K., Kucia, M., and Ratajczak, M.Z. 2007b. ImageStream technology—a step further than flow cytometry. Adv. Cell Biol. 34:361‐375.
   Zuba‐Surma, E., Kucia, M., Izabela Klich, I., Greco, N., Laughlin, M., Paul, P., Ratajczak, M., and Ratajczak, J. 2008a. Optimization of isolation and further molecular and functional characterization of SSEA‐4+/Oct‐4+/CD133+/CXCR4+/LINneg/CD45neg Very Small Embryonic‐Like (VSELs) stem cells isolated from umbilical cord blood. Blood 112:807.
   Zuba‐Surma, E.K., Kucia, M., Abdel‐Latif, A., Dawnn, B., Hall, B., Singh, R., Lillard, J.W., and Ratajczak, M.Z. 2008b. Morphological characterization of Very Small Embryonic‐Like stem cells (VSELs) by ImageStream system analysis. J. Cell. Mol. Med. 12:292‐303.
   Zuba‐Surma, E.K., Kucia, M., Wu, W., Klich, I., Lillard, J.W., Jr., Ratajczak, J., and Ratajczak, M.Z. 2008c. Very small embryonic‐like stem cells are present in adult murine organs: ImageStream‐based morphological analysis and distribution studies. Cytometry A 73A:1116‐1127.
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