The use of four‐colour immunofluorescence techniques to identify mesenchymal stem cells

Schieker, Matthias; Pautke, Christoph; Reitz, Katharina; Hemraj, Indradeo; Neth, Peter; Mutschler, Wolf; Milz, Stefan

doi:10.1111/j.1469-7580.2004.00252.x

Cited by 39 publications

(43 citation statements)

References 22 publications

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“…A similar pattern was seen for MES marker CD44 ( Fig. 2A; Schieker et al 2004;Schieker et al 2007). YAP expression did not necessarily correlate with the PN or MES status of the GSCs ( Fig.…”

Section: Taz Is Required For Mes Transition and Aggressive Gliomagenesupporting

confidence: 51%

The transcriptional coactivator TAZ regulates mesenchymal differentiation in malignant glioma

Bhat¹,

Salazar²,

et al. 2011

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Recent molecular classification of glioblastoma (GBM) has shown that patients with a mesenchymal (MES) gene expression signature exhibit poor overall survival and treatment resistance. Using regulatory network analysis of available expression microarray data sets of GBM, including The Cancer Genome Atlas (TCGA), we identified the transcriptional coactivator with PDZ-binding motif (TAZ ), to be highly associated with the MES network. TAZ expression was lower in proneural (PN) GBMs and lower-grade gliomas, which correlated with CpG island hypermethylation of the TAZ promoter compared with MES GBMs. Silencing of TAZ in MES glioma stem cells (GSCs) decreased expression of MES markers, invasion, self-renewal, and tumor formation. Conversely, overexpression of TAZ in PN GSCs as well as murine neural stem cells (NSCs) induced MES marker expression and aberrant osteoblastic and chondrocytic differentiation in a TEAD-dependent fashion. Using chromatin immunoprecipitation (ChIP), we show that TAZ is directly recruited to a majority of MES gene promoters in a complex with TEAD2. The coexpression of TAZ, but not a mutated form of TAZ that lacks TEAD binding, with plateletderived growth factor-B (PDGF-B) resulted in high-grade tumors with MES features in a murine model of glioma. Our studies uncover a direct role for TAZ and TEAD in driving the MES differentiation of malignant glioma.

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Section: Taz Is Required For Mes Transition and Aggressive Gliomagenesupporting

confidence: 51%

The transcriptional coactivator TAZ regulates mesenchymal differentiation in malignant glioma

Bhat¹,

Salazar²,

et al. 2011

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“…2; 163LR group). The reason for this disparity could be explained by the presence of the heterogeneous group of MSCs (telomerase positive and negative) and it clearly warrants further research [30,31]. The difference could also be related to the passage number of MSCs used.…”

Section: Discussionmentioning

confidence: 99%

The Effect of Telomerase Template Antagonist GRN163L on Bone-Marrow-Derived Rat Mesenchymal Stem Cells is Reversible and Associated with Altered Expression of Cyclin d1, cdk4 and cdk6

Tokcaer-Keskin

Dikmen

Ayaloglu-Butun

et al. 2010

Stem Cell Rev and Rep

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Telomerase activity is essential for the continued growth and survival of malignant cells, therefore inhibition of this activity presents an attractive target for anti-cancer therapy. The telomerase inhibitor GRN163L, was shown to inhibit the growth of cancer cells both in vitro and in vivo. Mesenchymal stem cells (MSCs) also show telomerase activity in maintaining their self-renewal; therefore the effects of telomerase inhibitors on MSCs may be an issue of concern. MSCs are multipotent cells and are important for the homeostasis of the organism. In this study, we sought to demonstrate in vitro effects of GRN163L on rat MSCs. When MSCs were treated with 1 µM GRN163L, their phenotype changed from spindle-shaped cells to rounded ones and detached from the plate surface, similar to cancer cells. Quantitative-RT-PCR and immunoblotting results revealed that GRN163L holds MSCs at the G1 state of the cell cycle, with a drastic decrease in mRNA and protein levels of cyclin D1 and its cdk counterparts, cdk4 and cdk6. This effect was not observed when MSCs were treated with a mismatch control oligonucleotide. One week after GRN163L was removed, mRNA and protein expressions of the genes, as well as the phenotype of MSCs returned to those of untreated cells. Therefore, we concluded that GRN163L does not interfere with the self-renewal and differentiation of MSCs under short term in vitro culture conditions. Our study provides additional support for treating cancers by administrating GRN163L without depleting the body's stem cell pools.

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“…spectrally overlapping fluorescent dyes (e.g. Alexa 546 and Texas Red), which cannot be separated by using standard filter sets, can be linearly unmixed in the staining of one single cell (Schieker et al, 2004).…”

Section: Multi-colour Imaging Of Cells or Tissue Samplesmentioning

confidence: 99%

“…A seven-colour imaging technique using mainly directly labelled primary antibodies has been reported for the simultaneous detection of up to seven antigens on thymus tissue (Tsurui et al, 2000). With respect to multi-colour staining at the single-cell level, a four-colour labelling method has been described for the identification of mesenchymal stem cells (Schieker et al, 2004). …”

Section: Introductionmentioning

confidence: 99%

Six-colour fluorescent imaging of lymphoid tissue based on colour addition theory

Winkelbach

Lindenmaier

et al. 2006

Acta Histochemica

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SummaryMulti-colour imaging of immunofluorescently stained tissue with the confocal microscope was accomplished by using colour addition theory. This new technique includes several improvements for immunolabelling: (1) the use of the colocalization of two or more markers on one cell for the identification of specific cell populations; (2) the use of the colocalization of two fluorescent dyes from secondary reagents for the identification of the cells; (3) a multistep staining protocol with two primary antibodies originating from the same host species or with two or three biotin-conjugated primary antibodies. After image acquisition, colour segmentation/unmixing are applied to the single multi-colour image to generate multipseudochannels for individual or colocalized fluorescent dyes. With this new technique, we have been able to visualize simultaneously six cell populations in the mouse lymph node and intestine. The efficiency of this method has also been demonstrated in the three-dimensional reconstruction of thick sections from mouse ileum. Our method is simple, efficient and may be indispensable in experimental cell and tissue studies requiring multiple immunolabelling.

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The use of four‐colour immunofluorescence techniques to identify mesenchymal stem cells

Cited by 39 publications

References 22 publications

The transcriptional coactivator TAZ regulates mesenchymal differentiation in malignant glioma

The transcriptional coactivator TAZ regulates mesenchymal differentiation in malignant glioma

The Effect of Telomerase Template Antagonist GRN163L on Bone-Marrow-Derived Rat Mesenchymal Stem Cells is Reversible and Associated with Altered Expression of Cyclin d1, cdk4 and cdk6

Six-colour fluorescent imaging of lymphoid tissue based on colour addition theory

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