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Gene expression analyses of GSC11 cell in stem cell culture conditions or in differentiation conditions for 1, 3, or 7 days

ABSTRACT: Although fetal bovine serum (FBS) induces the differentiation of cancer stem cells, the underlying mechanism by which this is accomplished has not been clarified. Whether reactive oxygen species affect the differentiation of cancer stem cells in solid tumors as they do in normal stem cells is not known. This study aimed to determine the role of reactive oxygen species in the FBS-induced differentiation of glioblastoma stem cells. We found that FBS activated the oxidative stress response system in glioblastoma stem cells (GSCs). The resulting differentiated cells showed tremendous increases in mitochondrial superoxide and oxygen consumption, accompanied by a loss in stem cell markers and a gain in differentiation markers. The antioxidant N-Acetyl-Cysteine (NAC) inhibited the mitochondrial superoxide increase and prevented the glioblastoma stem cells from differentiating. It appears that FBS-induced cancer stem cell differentiation is caused by mitochondrial activation, which depends on increases in levels of mitochondrial superoxide. GSC11 cells were cultured in stem cell medium or differentiated medium for 1, 3, or 7 days in triplicate. Total RNA was extracted from 12 samples. Microarray experiment and data analysis were done at Dept. of Systems Biology, MDACC (Houston, USA)

ORGANISM(S): Homo sapiens

SUBMITTER: Kim Sangbae

PROVIDER: S-ECPF-GEOD-28220 | biostudies-other |

REPOSITORIES: biostudies-other

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Project description:BackgroundThe use of equine bone marrow mesenchymal stem cells (BMSC) is a novel method to improve fracture healing in horses. However, additional research is needed to identify optimal culture conditions and to determine the mechanisms involved in regulating BMSC differentiation into osteoblasts. The objectives of the experiments were to determine: 1) if autologous or commercial serum is better for proliferation and differentiation of equine BMSC into osteoblasts, and 2) the expression of key transcription factors during the differentiation of equine BMSC into osteoblasts. Equine BMSC were isolated from the sterna of 3 horses, treated with purchased fetal bovine serum (FBS) or autologous horse serum (HS), and cell proliferation determined. To induce osteoblast differentiation, cells were incubated with L-ascorbic acid-2-phosphate and glycerol-2-phosphate in the presence or absence of human bone morphogenetic protein2 (BMP2), dexamethasone (DEX), or combination of the two. Alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation, was determined by ELISA. Total RNA was isolated from differentiating BMSC between d 0 to 18 to determine expression of runt-related transcription factor2 (Runx2), osterix (Osx), and T-box3 (Tbx3). Data were analyzed by ANOVA.ResultsRelative to control, FBS and HS increased cell number (133 ± 5 and 116 ± 5%, respectively; P < 0.001) and 5-bromo-2'-deoxyuridine (BrdU) incorporation (167 ± 6 and 120 ± 6%, respectively; P < 0.001). Treatment with DEX increased ALP activity compared with control (1,638 ± 38%; P < 0.001). In the absence and presence of Dex, BMP-2 did not alter ALP activity (P > 0.8). Runt-related transcription factor2 expression increased 3-fold (P < 0.001) by d 6 of culture. Osterix expression increased 9-fold (P < 0.05) by d 18 of culture. Expression of Tbx3 increased 1.8-fold at d 3 (P < 0.01); however expression was reduced 4-fold at d 18 (P < 0.01).ConclusionsDexamethasone, but not BMP-2, is required for differentiation of equine BMSC into osteoblasts. In addition, expression of Runx2 and osterix increased and expression of Tbx3 is reduced during differentiation.

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Gene expression analyses of GSC11 cell in stem cell culture conditions or in differentiation conditions for 1, 3, or 7 days

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