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Optimized Flow Cytometric Analysis of Central Nervous System Tissue Reveals Novel Functional Relationships Among Cells Expressing CD133, CD15, and CD24

  1. David M. Panchision Ph.D.1,*,
  2. Hui-Ling Chen1,
  3. Francesca Pistollato1,
  4. Daniela Papini2,
  5. Hsiao-Tzu Ni3,
  6. Teresa S. Hawley4

Article first published online: 1 MAR 2007

DOI: 10.1634/stemcells.2006-0260

Issue

STEM CELLS

STEM CELLS

Volume 25, Issue 6, pages 1560–1570, June 2007

Additional Information

How to Cite

Panchision, D. M., Chen, H.-L., Pistollato, F., Papini, D., Ni, H.-T. and Hawley, T. S. (2007), Optimized Flow Cytometric Analysis of Central Nervous System Tissue Reveals Novel Functional Relationships Among Cells Expressing CD133, CD15, and CD24. STEM CELLS, 25: 1560–1570. doi: 10.1634/stemcells.2006-0260

Author Information

  1. 1

    Center for Neuroscience Research, Children's Research Institute, Children's National Medical Center, Washington, DC, USA

  2. 2

    Stem Cell Core Facility, Children's Research Institute, Children's National Medical Center, Washington, DC, USA

  3. 3

    R&D Systems Inc., Minneapolis, Minnesota, USA

  4. 4

    Flow Cytometry Core Facility, George Washington University Medical Center, Washington, DC, USA

*Children's National Medical Center, Center for Neuroscience Research, 5th Floor, Suite 5340, 111 Michigan Avenue, N.W., Washington, DC 20010, USA. Telephone: 202-884-2269; Fax: 202-884-4988

Publication History

  1. Issue published online: 2 JAN 2009
  2. Article first published online: 1 MAR 2007
  3. Manuscript Accepted: 20 FEB 2007
  4. Manuscript Received: 28 APR 2006

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Keywords:

  • Brain;
  • Brain tumors;
  • Flow cytometry;
  • Cell viability;
  • Stem cells;
  • Progenitor cells

Abstract

Although flow cytometry is useful for studying neural lineage relationships, the method of dissociation can potentially bias cell analysis. We compared dissociation methods on viability and antigen recognition of mouse central nervous system (CNS) tissue and human CNS tumor tissue. Although nonenzymatic dissociation yielded poor viability, papain, purified trypsin replacement (TrypLE), and two purified collagenase/neutral protease cocktails (Liberase-1 or Accutase) each efficiently dissociated fetal tissue and postnatal tissue. Mouse cells dissociated with Liberase-1 were titrated with antibodies identifying distinct CNS precursor subtypes, including CD133, CD15, CD24, A2B5, and PSA-NCAM. Of the enzymes tested, papain most aggressively reduced antigenicity for mouse and human CD24. On human CNS tumor cells, CD133 expression remained highest after Liberase-1 and was lowest after papain or Accutase treatment; Liberase-1 digestion allowed magnetic sorting for CD133 without the need for an antigen re-expression recovery period. We conclude that Liberase-1 and TrypLE provide the best balance of dissociation efficiency, viability, and antigen retention. One implication of this comparison was confirmed by dissociating E13.5 mouse cortical cells and performing prospective isolation and clonal analysis on the basis of CD133/CD24 or CD15/CD24 expression. Highest fetal expression of CD133 or CD15 occurred in a CD24hi population that was enriched in neuronal progenitors. Multipotent cells expressed CD133 and CD15 at lower levels than did these neuronal progenitors. We conclude that CD133 and CD15 can be used similarly as selectable markers, but CD24 coexpression helps to distinguish fetal mouse multipotent stem cells from neuronal progenitors and postmitotic neurons. This particular discrimination is not possible after papain treatment.

Disclosure of potential conflicts of interest is found at the end of this article.

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