Isolation of tumor spheres and mesenchymal stem-like cells from a single primitive neuroectodermal tumor specimen

Abstract

PURPOSE: It has been reported that cancer stem cells (CSCs) can be isolated from primitive neuroectodermal tumor (PNET) specimens. Moreover, mesenchymal stem-like cells (MSLCs) have been isolated from Korean glioma specimens. Here, we tested whether tumor spheres and MSLCs can be simultaneously isolated from a single PNET specimen, a question that has not been addressed. METHODS: We isolated single-cell suspensions from PNET specimens, then cultured these cells using methods for MSLCs or CSCs. Cultured cells were analyzed for surface markers of CSCs using immunocytochemistry and for surface markers of bone marrow-derived mesenchymal stem cells (BM-MSCs) using fluorescence-activated cell sorting (FACS). Tumor spheres were exposed to neural differentiation conditions, and MSLCs were exposed to mesenchymal differentiation conditions. Possible locations of MSLCs within PNET specimens were determined by immunofluorescence analysis of tumor sections. RESULTS: Cells similar to tumor spheres and MSLCs were independently isolated from one of two PNET specimens. Spheroid cells, termed PNET spheres, were positive for CD133 and nestin, and negative for musashi and podoplanin. PNET spheres were capable of differentiation into immature neural cells and astrocytes, but not oligodendrocytes or mature neural cells. FACS analysis revealed that adherent cells isolated from the same PNET specimen, termed PNET-MSLCs, had surface markers similar to BM-MSCs. These cells were capable of mesenchymal differentiation. Immunofluorescence labeling indicated that some CD105(+) cells might be closely related to endothelial cells and pericytes. CONCLUSION: We showed that both tumor spheres and MSLCs can be isolated from the same PNET specimen. PNET-MSLCs occupied a niche in the vicinity of the vasculature and could be a source of stroma for PNETs.