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 9.29.1 Antibodies Employed in Staining for Identification and Sorting of Murine BM-Derived VSELs by Flow Cytometry
    AntibodyCloneFluorochromeVendor
    antiCD4530-F11APC-Cy7BD Pharmingen
    anti-Ly-6A/E (Sca-1)E13-161.7biotinBD Pharmingen
    StreptavidinPE-Cy5BD Pharmingen
    anti-CD45R/B220RA3-6B2PEBD Pharmingen
    anti-Gr1RB6-8C5PEBD Pharmingen
    anti-TCR H57-597PEBD Pharmingen
    anti-TCR GL3PEBD Pharmingen
    anti-CD11bM1/70PEBD Pharmingen
    anti-Ter119TER-119PEBD Pharmingen
    Isotype controls:
    Rat IgG2b, A95-1APC-Cy7BD Pharmingen
    Rat IgG2a, R35-95PE-Cy5BD Pharmingen
    Rat IgG2a, R35-95PEBD 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 9.29.2 Antibodies Employed in Staining for Identification and Sorting of Human CB-Derived VSELs by Flow Cytometry
    AntibodyCloneFluorochromeVendor
    anti-CD45HI30PEBD Biosciences
    anti-CD19HIB19FITCBD Biosciences
    anti-CD2RPA-2.10FITCBD Biosciences
    anti-CD3UCHT1FITCBD Biosciences
    anti-CD14M5E2FITCBD Biosciences
    anti-CD66bG10F5FITCBD Biosciences
    anti-CD24ML5FITCBD Biosciences
    anti-CD163G8FITCBD Biosciences
    anti-CD56NCAM16.2FITCBD Biosciences
    anti-CD235aGA-R2FITCBD Biosciences
    anti-AC133AC133APCMiltenyi Biotec
    Isotype controls:
    Mouse IgG1, MOPC-21PEBD Biosciences
    Mouse IgG1, MOPC-21FITCBD Biosciences
    Mouse IgG2a, G155-178FITCBD Biosciences
    Mouse IgG2b, 27-35FITCBD Biosciences
    Mouse IgM, MM-30FITCBioLegend
    Mouse IgG1, MOPC-21APCBD 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 1 and 2 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

  •  FigureFigure 9.29.1 Gating strategy for sorting murine BM-derived VSELs by FACS. BM-derived VSELs were isolated from a fraction of murine BM TNCs by FACS. (A) Agranular, small events ranging from 2 to 10 µm are included in gate R1 after comparison with six differently sized bead particles with standard diameters of 1, 2, 4, 6, 10, and 15 µm. (B) BM-nucleated cells are visualized by dot plot showing FSC vs. SSC signals, which are related to the size and granularity/complexity of the cell, respectively. (D) Cells from region R1 are further analyzed for Sca-1 and Lin expression; only Sca-1+/Lin– events are included in region R2. The population from region R2 is subsequently distinguished based on CD45 marker expression into CD45– and CD45+ subpopulations visualized on histogram (panel C, regions R3 and R4, respectively). Sca-1+/Lin–/CD45– cells (VSELs) are sorted as objects enclosed in a logical gate including regions R1, R2, and R3, while Sca-1+/Lin–/CD45+ cells (HSCs) are from a gate including regions R1, R2, and R4. Percentages show the average content of each cellular subpopulation in total BM nucleated cells.
    View Image
  •  FigureFigure 9.29.2 Gating strategy for sorting of human CB-VSELs derived by FACS. CB-VSELs were isolated from the fraction of human CB TNCs by FACS. (A) All events ranging from 2 µm are included in gate R1 after comparison with six differently sized bead particles with standard diameters of 1, 2, 4, 6, 10, and 15 µm. (B) CB-derived TNCs are visualized by dot plot presenting FSC vs. SSC signals. (D) Cells from region R1 are further analyzed for hematopoietic lineages markers expression and Lin– events are included in region R2. (C) The Lin– population from region R2 is subsequently analyzed based on CD133 and CD45 antigen expression and two populations of CD133+ cells are distinguished based on CD45 appearance, i.e., CD133+/Lin–/CD45– cells (VSELs; region R3) and CD133+/Lin–/CD45+ cells (HSCs; region R4). Percentages show the average content of each cellular subpopulation in total CB nucleated cells.
    View Image
  •  FigureFigure 9.29.3 Murine VSELs and artifacts detected in BM samples with ImageStream system. (A) Shows normal nucleated Sca-1+/Lin–/CD45– VSELs identified in murine BM while selected images of falsely “positive” artifacts and cellular debris are shown in B. Each photograph presents a brightfield image, as well as separate fluorescent images related to nuclear image (7-AAD; red) and expression of Sca-1 (FITC, green), Lin (PE; orange), and CD45 (PE-Cy5; yellow). The scale bar indicates 10 µm.
    View Image

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Literature Cited

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