Optimized flow cytometric analysis of central nervous system tissue reveals novel functional relationships among cells expressing CD133, CD15, and CD24.

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.

Lack of SYT-SSX fusion transcripts in malignant peripheral nerve sheath tumors on RT-PCR analysis of 34 archival cases.

The translocation t(X;18) is currently regarded as a specific molecular marker of synovial sarcoma (SS). Recently, however, it has been reported that malignant peripheral nerve sheath tumors expressed this marker in 75% of the cases. To test independently this iconoclastic claim, a molecular analysis for the detection of the SYT-SSX fusion genes was carried out using archival material of 34 consecutive cases diagnosed as malignant peripheral nerve sheath tumors and treated in our Institute from 1998 to 2000. In four of these cases, the molecular analysis on fixed tissues was supplemented with an analysis on fresh frozen tissue. RNA extracted from formalin-fixed paraffin-embedded tissue blocks was evaluated for the presence of SYT-SSX1 and SYT-SSX2 fusion transcripts by RT-PCR. This analysis was extended to a wide variety of normal tissues simultaneously extracted and equally processed. Only two of the cases studied harbored SYT-SSX1 and SYT-SSX2 fusion transcripts, respectively. The diagnostic reevaluation of these two cases in light of the molecular data disclosed that one had the features of a monophasic SS and the other was compatible with that entity. Both of these tumors were strongly immunoreactive for bcl-2, confirming the diagnostic utility of this marker in this instance. Our results reaffirm the specificity of SYT-SSX for SS and suggest that an opposite claim made in a recent study may have been due to a faulty interpretation of the molecular results caused by a contamination of the samples.