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TECHNOLOGY DEVELOPMENT

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

^aCenter for Neuroscience Research, Children's Research Institute, Children's National Medical Center, Washington, DC, USA;
^bStem Cell Core Facility, Children's Research Institute, Children's National Medical Center, Washington, DC, USA;
^cR&D Systems Inc., Minneapolis, Minnesota, USA;
^dFlow Cytometry Core Facility, George Washington University Medical Center, Washington, DC, USA

Key Words. Brain • Brain tumors • Flow cytometry • Cell viability • Stem cells • Progenitor cells

Correspondence: David M. Panchision, Ph.D., 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; e-mail: dpanchision{at}cnmcresearch.org

Received on April 28, 2006; accepted for publication on February 20, 2007.

First published online in STEM CELLS EXPRESS  March 1, 2007.


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 CD24^hi 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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