Article
The influence of in vitro conditions for the enrichment of stem-like cell population in primary human brain tumor cultures
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Published: | May 20, 2009 |
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Objective: Recently demonstrated slowly proliferating stem-like cell population (BTSC) in malignant brain tumors, responsible for tumor initiation and propagation, allowed the identification of the molecular mechanisms underlying their resistance to both chemo- and radiotherapy. Reduction of BTSC population in glioblastoma cell lines by inhibition of Notch, WNT/β-catenin and SHH pathways diminished in vitro proliferation and ability to form tumors in vivo. Our project is focused on defining cell culture conditions for the enrichment of stem-like cell populations in patient-derived glial tumor cultures for further ex vivo manipulations.
Methods: Freshly resected malignant human glial tumors and fetal-derived neural tissue were propagated as free-floating culture in serum-free media with mitogens. Two media-compositions (DMEM/F12 and Neurocult® based) and oxygen concentrations (21% and 3%) were tested. Culture proliferation kinetics was investigated with MTS-Assay, and BTSC population was analyzed using FACS against stem cell marker CD133 and aldehyde dehydrogenase activity (Aldefluor®). Additionally, semi-quantitative PCR allowed the expression analysis of oncogenic and stem cell specific genes.
Results: The growth curve of all investigated tumor- and fetal-derived cell lines showed significantly higher proliferation in DMEM based medium and under atmospheric oxygen concentration. In contrast, exposure to 3% oxygen in Neurocult® based media resulted in significant decrease of proliferation rate. FACS analysis revealed that slower proliferating cultures contained significantly more CD133+ stem-like cells (up to 15% in tumor-derived and 61% in fetal-derived cultures) compared to their fast proliferating counterparts. Aldefluor-Assay showed increase of BTSC in Neurocult® media under lowered oxygen concentration. Semi-quantitative gene expression analysis confirmed higher expression of stem cell specific genes in the latter culture condition. Interestingly, the effect of relative hypoxia (3% O2) on BTSC enrichment was stronger then any changes of the culture media.
Conclusions: Neurocult® based media combined with lowered oxygen concentration increases the stem-like cell population in malignant glial tumor cultures and in vitro propagated fetal-derived neural tissue. Hypoxia seems to play a key role in BTSC enrichment. This protocol allows the generation of significant numbers of BTSC for further genetic manipulations and ex vivo testing of novel therapeutic protocols.