gms | German Medical Science

60. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie (DGNC)
Joint Meeting mit den Benelux-Ländern und Bulgarien

Deutsche Gesellschaft für Neurochirurgie (DGNC) e. V.

24. - 27.05.2009, Münster

The influence of in vitro conditions for the enrichment of stem-like cell population in primary human brain tumor cultures

Meeting Abstract

  • U.D. Kahlert - Labor für Molekulare Neurochirurgie, Abt. Stereotaktische Neurochirurgie, Universitätsklinikum Freiburg
  • D. Maciaczyk - Labor für Molekulare Neurochirurgie, Abt. Stereotaktische Neurochirurgie, Universitätsklinikum Freiburg
  • G. Nikkhah - Labor für Molekulare Neurochirurgie, Abt. Stereotaktische Neurochirurgie, Universitätsklinikum Freiburg
  • J. Maciaczyk - Abt. Allgemeine Neurochirurgie, Universitätsklinikum Freiburg

Deutsche Gesellschaft für Neurochirurgie. 60. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie (DGNC), Joint Meeting mit den Benelux-Ländern und Bulgarien. Münster, 24.-27.05.2009. Düsseldorf: German Medical Science GMS Publishing House; 2009. DocP06-03

DOI: 10.3205/09dgnc307, URN: urn:nbn:de:0183-09dgnc3070

Veröffentlicht: 20. Mai 2009

© 2009 Kahlert et al.
Dieser Artikel ist ein Open Access-Artikel und steht unter den Creative Commons Lizenzbedingungen (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.de). Er darf vervielfältigt, verbreitet und öffentlich zugänglich gemacht werden, vorausgesetzt dass Autor und Quelle genannt werden.


Gliederung

Text

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.