We have analyzed the expression of PDGFRα, A2B5, O4 and CD44 as surface markers for glial precursor cells 
, and CD133 for stem cells 
on freshly prepared glioma cells using flow cytometry. In addition, we analyzed the expression of EGFR, which is expressed in immature astrocytes and is critically important for astrocyte development 
. EGFR is also frequently overexpressed in high-grade glioma cells 
. Six low-grade and 17 high-grade glioma specimens were analyzed. As shown by representative dot-plot profiles for low-grade glioma specimens (), intermingled cells of hematopoietic origin can be distinguished via their CD45 expression. PDGFRα, A2B5, O4, and CD44 were found to be expressed in nearly all CD45 negative cells. The concomitant expression of these progenitor cell markers was observed in nearly all low-grade glioma specimens analyzed (). These data, combined with the homogenous GFAP staining and the absence of NSE/synaptophysin staining ( and ), suggest that low-grade glioma cells are reminiscent of glial-progenitor cells. In 4 out of 6 low-grade glioma specimens, CD133 expression was observed at lower frequencies, but these CD133+
cells are predominantly of blood vessel endothelial origin (see below). The expression of EGFR was found in specimens from 2 of the 6 low-grade gliomas. We also analyzed the expression of CD24. Absence of CD24 expression in combination with positive staining on CD133, or PNA or ABCG2/Bcrp1 was used to enrich primitive neural cells with “stem cell-like” properties 
. CD24 expression was detected in 4 out of 6 low-grade gliomas.
Low-grade glioma cells concurrently express multiple cell surface markers characteristic of adult human glial progenitors.
Expression of neural stem cell and glial progenitor surface markers in low- and high-grade glioma specimens analyzed.
Co-expression of neuronal and glial markers in high-grade glioma cells
Neuronal marker expression was not detected in low-grade gliomas, but in high-grade gliomas.
As in low-grade glioma cells, high-grade glioma cells analyzed in this study maintained the expression profiles for PDGFRα, A2B5, O4 and CD44 ( and ). In all these high-grade glioma cases, GFAP expression was detected with inter- as well as intra-glioma variation ( and ). The expression of CD24 was observed in 7 of 16 high-grade gliomas analyzed. In striking contrast to the low-grade glioma cells, distinct cell populations with high intensity of CD133 expression (ranging from 22 to 90% of the CD45− cell fraction) was detected in 12 of the 17 high-grade glioma specimens. In addition, EGFR over-expression was detected in 11 of the 17 high-grade glioma specimens analyzed. Of note, no detectable difference in cell surface marker expression profile was observed between the secondary (patients ID 15 and 16) and de novo GBM specimens analyzed ().
Maintenance of glial progenitor-like phenotype, but enhanced CD133 expression in high-grade gliomas.
As gliomas, particularly the high-grade gliomas, are highly vascularized, newly formed blood vessel endothelial cells may also express CD133 
. To ascertain the glioma origin of the CD133+
cell fraction in our low-grade and high-grade glioma specimens, we subsequently performed CD133 expression analysis in combination with CD45 and CD31 staining. By doing so, it is possible to distinguish glioma cells from both hematopoietic cells (via CD45 expression) and tumor blood vessel endothelial cells (via CD31 expression) 
. As shown in , a small population of CD45−
, but CD133+
cells can be detected in low-grade glioma specimens. Relatively higher frequencies of such cells were found in GBM specimens. However, such CD45−
cells were predominantly CD31+
in low-grade glioma specimens (ranging from 0.3 to 7% of the total living cells, n
4). This indicates that CD133+
cells, if present in low-grade gliomas, are predominantly derived from newly formed blood vessel endothelial cells, and not from the glioma cells. In GBM specimens, the majority of CD133+
cells were of glioma origin, although CD31+
cells (ranging from 0.5 to 10% of the total living cells, n
9) were also found.
Vessel or glioma origin of CD133 expressing cells.
In agreement with previous studies demonstrating glioma expression of oligodendrocyte progenitor cell transcription factor Olig1/2 and surface antigen NG2 
, the concomitant expression of A2B5 (a marker for multiple types of progenitor cells in glial lineage 
), PDGFRα (a marker for early oligodendrocyte progenitor cells at O-2A stage 
) and O4 (a marker for late oligodendrocyte progenitor cells 
) suggests that glioma cells, irrespective of their morphological characteristics, are committed to the oligodendrocyte lineage. To further characterize the differentiation stages along the oligodendrocyte lineage downstream of the A2B5 expressing glial progenitor cell level, we analyzed the expression of O1, a marker for pre-myelinating oligodendrocytes 
. In contrast to the high level A2B5 and O4 expression, the frequency of O1+
cells were significantly diminished in all low-grade and the majority of GBM specimens analyzed ( and ). The expression of GFAP and these oligodendrocyte progenitor surface markers suggests that along the oligodendrocyte lineage differentiation hierarchy, A2B5+
glioma cells were compromised in differentiation downstream of A2B5/O4, but upstream of the O1 stage.
Glioma cells are compromised in downstream differentiation at the O4 stage.
To study glioma cell differentiation potential along neuronal lineage, we performed an immunohistochemical staining for pan-neuronal marker NSE and synaptophysin expression in archival specimens ( and ). In low-grade gliomas, no obvious expression of NSE and synaptophysin was detected, but the cells homogeneously expressed GFAP ( and ). We were unable to ascertain whether the few cells weakly stained for NSE were the intermingled normal neuronal cells or the glioma cells (, upper panel). In high-grade glioma cells, a regional NSE expression was clear in all cases analyzed ( and ), although both region-dependent and homogeneous expression patterns were detected. Synaptophysin positivity was detected in 7 high-grade gliomas. These data, in combination with the expression of GFAP and multiple oligodendrocyte lineage progenitor surface markers ( and , , and ), suggest that compared to low-grade glioma cells, high-grade glioma cells maintain glial progenitor-like features, but additionally exhibit enhanced CD133 expression as well as neuronal differentiation potential.
Transformed glial progenitor-like cells can provide a niche environment recruiting normal stem cells or progenitor cells into the tumor mass 
. To investigate whether freshly isolated glioma cells studied here indeed contained transformed cells, karyotype analysis was performed in short-term cultured glioma cells (). We only observed normal karyotype for cells derived from patients 3 and 8. Abnormal karyotypes were observed from cells derived from patients 4, 12, 13, 14, 17, 18 and 20. Of note, cultures derived from low-grade glioma patient 4 and high-grade glioma patient 12 contained about 40% of the cells with a normal karyotype, contrasting the nearly homogenous phenotype between all cells (, ); but cultures derived from high-grade glioma patients 13, 14, 17, 18 and 20 predominantly contained cells with abnormal karyotypes. In addition, fresh cells from patients 14 and 20 formed xenograft glioma following subcutaneous injection into SCID mice 
. Thus, the analyzed glioma cells did contain neoplastic cells and the karyotype normal and abnormal glioma cells showed hitherto indistinguishable phenotype.
Karyotype analysis of short-term cultured glioma cells
Finally, we investigated whether the glial progenitor or stem cell markers expressed on glioma cells could serve as prognostic markers for patient survival. Glioblastoma patients receiving immune therapy and patients with pilocytic astrocytoma or ependymoma, were excluded from the prognosis analysis. We divided all analyzed grade II to IV glioma patients into a CD133+
low group (CD133+
cells less than 30%) and a CD133+
high group (CD133+
cells higher than 30%). The median survival time in the CD133+
high group was 5.0±9.2 months (n
9) compared to more than 22.0±17.3 months (n
10) in the CD133+
low group (P
test; ). Thus, CD133 expression inversely correlates with glioma patient survival time.
CD133 expression correlates inversely with grade II to IV glioma patient survival time.