Brain tumor stem cells maintain overall phenotype and tumorigenicity after in vitro culturing in serum-free conditions

Abstract

Traditional in vitro culturing of tumor cells has been shown to induce changes so that cultures no longer represent the tumor of origin. Serum-free culturing conditions are used in a variety of cancers to propagate stem-like cells in vitro. Limited reports, however, exist on the effects of such propagation. We have compared cells from brain tumor biopsies cultivated under serum-free conditions at passages 2 and 10 to describe the effects of in vitro culturing. We were able to establish cell lines from 7 of 10 biopsies from patients with glioblastoma. The cell lines adapted to conditions and had 2.2 times increased population doubling rate at later passages. Karyotyping and comparative genomic hybridization analysis revealed that all examined cell lines had cytogenetic aberrations commonly found in glioblastomas, and there were only minor differences between tumor and early and late passages in the same culture. Whole-transcriptome analysis shows that tumors had interindividual differences. Changes in the overall expression patterns through passaging were modest, with a significant change in only 14 genes; the variation among cultures was, however, reduced through passages. The ability to differentiate differed among tumors but was maintained throughout passaging. The cells initiated tumors upon transplantation to immunodeficient mice with differing phenotypes, but a given cell culture maintained tumor phenotype after serial cultivation. The cultures established maintained individual characteristics specific to culture identity. Thus, each cell culture reflects an image of the tumor--or a personalized model--from which it was derived and remains representative after moderate expansion.

Expansion of Multipotent Stem Cells from the Adult Human Brain

“…For subsequent neurosphere formation (cell passage), the neurospheres were dissociated into single cells when they reached 12–15 cells in diameter, by incubating in papain followed by mechanical resuspension and cultured further in the presence of the aforementioned mitogens. This suspension culture method is the standard method used to propagate tumor-derived spheres in our laboratory [7], [8], [9], [23], [24] and is called ‘VML’ culture protocol.…”

“…We have access to a continuing supply of adult human brain samples from both diseased and ‘normal’ people. Already our lab has investigated harvest, culture and differentiation of human central nervous system (CNS)-derived cells [5], [6], [7], [8], [9], [10], [11]. For these experiments we have employed methods originally derived from well established rodent protocols for the ‘neurosphere assay’ [12], [13].…”

Human breast cancer metastases to the brain display GABAergic properties in the neural niche

“…Low-passage cells derived from metastatic brain tumor resections maintained their original tissue morphology when grown in serum-free "brain-like" conditions and 3D Matrigel cultures ( Fig. S1) (25). Within the intratumoral space in metastatic brain tissue, cells of glial origin interdigitated with the breast tumor cells (26).…”

The homing of human cord blood stem cells to sites of inflammation

“…The complexity of glioma tumors is increased by the discovery of differences in the phenotype of cancer stem cells derived from tumors of equivalent histological class and grade. 36,37 It has been reported that neural stem cells engineered to deliver tumor necrosis factor-related, apoptosis-inducing ligand inhibits the growth of brain tumors. 8 In the present study, we elucidated the pathogenic-specific mechanism of hUCBSC.…”