[Isolation and characterization of brain tumor stem cells in human medulloblastoma].
ABSTRACT Tumor stem cells have been isolated from several kinds of solid tumors, including primary brain tumors such as glioma and medulloblastoma. This investigation was to establish a simplified culture system to isolate and passage brain tumor stem cells (BTSCs) from human medulloblastoma, observe their proliferation and differentiation, and determine the expression of normal neural stem cell antigens, CD133 and Nestin, in BTSCs.
Eleven specimens of medulloblastoma were acutely dissociated and triturated into single cells without trypsin digestion. The tumor cells were seeded into serum-free DMEM/F12 medium (200,000 viable cells/ml) containing B27 (1:50), basic fibroblast growth factor (bFGF, 20 microg/L), epidermal growth factor (EGF, 20 microg /L), insulin (4 u/L), L-glutamine, and antibiotics. After generation, the primary brain tumor spheres (BTSs) were mechanically dissociated and passaged in the above serum-free medium. The monoclonal formation experiment was performed to determine the proportion of BTSCs in medulloblastoma and to observe the formation of BTSs. The differentiation of BTSCs was induced in mitogen-free DMEM/F12 medium supplemented with 10% fetal bovine serum. The expression of CD133 and Nestin in BTSs was observed with immunofluorescence staining; the distribution of CD133-positive cells in tumor sections was assessed by immunohistochemistry.
In each of the 11 specimens, only a minority of medulloblastoma cells showed the capacity of self-renew and proliferation. These BTSCs generated free-floating neurosphere-like BTSs in the simplified serum-free medium. The proportion of BTSCs with monoclonal formation capacity in primary tumor cells was (31.18+/-6.18)%. The BTSCs attached to poly-L-lysine-coated coverslips and differentiated when the serum-supplemented medium was added. The expression of CD133 and Nestin was detected in BTSCs. CD133-positive cells scattered or formed nest-like aggregations in tumor masses, and accounted for (33.06+/-8.57)% of all tumor cells.
BTSCs, with the capacity of self-renew and proliferation and express CD133 and Nestin, are exist in human medulloblastoma. They could be isolated and cultured in the simplified serum-free medium, and their differentiation could be induced in serum-supplemented medium.
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- "When cultured using stringent conditions in specially-formulated serum-free tissue culture medium with epidermal growth factor and basic fibroblast growth factor, tumor cells grew non-adherently in clumps of cells rather than as monolayers and cells in these tumor-derived “neurospheres” (Figure 1) expressed genes characteristic of neural stem cells including CD133, the transcription factor Sox2, and nuclear and cytoplasmic proteins musashi-1and bmi-1. More recent studies have used CD133 alone or in combination with nestin, an intermediate filament protein expressed in embryonic neuroglial cells, to isolate TSC in MB, to establish an anaplastic MB cell line with stem cell features, and to develop clinically relevant xenograft mouse models of MB and high-grade glioma [26-28]. CD133+ TSC have been identified in other pediatric brain tumors including ependymoma and atypical teratoid/rhabdoid tumor (AT/RT) [29-31]. "
ABSTRACT: Recently, a subpopulation of cells, termed tumor-initiating cells or tumor stem cells (TSC), has been identified in many different types of solid tumors. These TSC, which are typically more resistant to chemotherapy and radiation compared to other tumor cells, have properties similar to normal stem cells including multipotency and the ability to self-renew, proliferate, and maintain the neoplastic clone. Much of the research on TSC has focused on adult cancers. With considerable differences in tumor biology between adult and pediatric cancers, there may be significant differences in the presence, function and behavior of TSC in pediatric malignancies. We discuss what is currently known about pediatric solid TSC with specific focus on TSC markers, tumor microenvironment, signaling pathways, therapeutic resistance and potential future therapies to target pediatric TSC.Cancers 12/2011; 3(1):298-318. DOI:10.3390/cancers3010298
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ABSTRACT: Mounting evidence suggests that gliomas are comprised of differentiated tumor cells and brain tumor stem cells (BTSCs). BTSCs account for a fraction of total tumor cells, yet are apparently the sole cells capable of tumor initiation and tumor renewal. BTSCs have been identified as the CD133-positive fraction of human glioma, whereas their CD133-negative daughter cells have limited proliferative ability and are not tumorogenic. It is well established that the bulk tumor mass escapes immune surveillance by multiple mechanisms, yet little is known about the immunogenicity of the CD133-positive fraction of the tumor mass. We investigated the immunogenicity of CD133-positive cells in two human astrocytoma and two glioblastoma multiforme samples. Flow cytometry analyses revealed that the majority of CD133-positive cells do not express detectable MHC I or natural killer (NK) cell activating ligands, which may render them resistant to adaptive and innate immune surveillance. Incubating CD133-positive cells in interferon gamma (INF-gamma) significantly increased the percentage of CD133-positive cells that expressed MHC I and NK cell ligands. Furthermore, pretreatment of CD133-positive cells with INF-gamma rendered them sensitive to NK cell-mediated lysis in vitro. There were no consistent differences in immunogenicity between the CD133-positive and CD133-negative cells in these experiments. We conclude that CD133-posistive and CD133-negative glioma cells may be similarly resistant to immune surveillance, but that INF-gamma may partially restore their immunogenicity and potentiate their lysis by NK cells.Journal of Neuro-Oncology 07/2007; 83(2):121-31. DOI:10.1007/s11060-006-9265-3 · 3.07 Impact Factor
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ABSTRACT: The IF protein nestin and the RNA-binding protein musashi are expressed by neural progenitor cells during CNS development. Their expression in glial tumors was evaluated by immunohistochemistry, and the histopathological scores correlated with levels of cysteine cathepsins that are known prognostic markers in several tumors. The levels of nestin, musashi, and cathepsins B and L were assessed by immunohistochemical analysis of biopsies from 87 patients with primary CNS tumors. To confirm the immunohistochemical data, nestin expression was analyzed by real-time PCR in 12 brain tumor biopsies. The exact location of nestin-positive cells was determined by mapping the distribution of nestin in a highly invasive human glioma xenograft model. Immunostaining revealed nestin to be expressed in 95.8% and musashi in 80% of the patient biopsies. The total IHC score for nestin was significantly higher in high- than in low-grade tumors (P < .0001). No difference was observed for musashi (P = .11). Real-time PCR of nestin expression confirmed the immunohistochemical data. Nestin expression was shown to be a strong prognostic marker for decreased overall survival (P = .0001), whereas musashi expression has no prognostic significance. Moreover, nestin was shown by Cox regression analysis to be a stronger prognostic marker than cathepsins B and L. IHC staining of nestin in a xenograft model showed that its expression is localized mainly in the invasive tumor cells at the tumor periphery. Nestin is shown to be a strong prognostic marker for glioma malignancy. The presented data links the invasive glioma cells to CNS precursor cells, indicating that the most malignant cells in the gliomas may well be closely related to the glioma stem cells.Surgical Neurology 09/2007; 68(2):133-43; discussion 143-4. DOI:10.1016/j.surneu.2006.10.050 · 1.67 Impact Factor