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Simultaneous detection of mRNA and protein stem cell markers in live cells.

Rhee WJ, Bao G - BMC Biotechnol. (2009)

Bottom Line: We found that the fluorescence signal from Oct-4-targeting molecular beacons provides a clear discrimination between undifferentiated and retinoic acid-induced differentiated cells.Using deconvolution fluorescence microscopy, Oct-4 mRNAs were found to reside on one side of the cytosol.We revealed that Oct-4 targeting molecular beacons do not seem to affect stem cell biology.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia 30332, USA.

ABSTRACT

Background: Biological studies and medical application of stem cells often require the isolation of stem cells from a mixed cell population, including the detection of cancer stem cells in tumor tissue, and isolation of induced pluripotent stem cells after eliciting the expression of specific genes in adult cells. Here we report the detection of Oct-4 mRNA and SSEA-1 protein in live carcinoma stem cells using respectively molecular beacon and dye-labeled antibody, aiming to establish a new method for stem cells detection and isolation.

Results: Quantification of Oct-4 mRNA and protein in P19 mouse carcinoma stem cells using respectively RT-PCR and immunocytochemistry confirmed that their levels drastically decreased after differentiation. To visualize Oct-4 mRNA in live stem cells, molecular beacons were designed, synthesized and validated, and the detection specificity was confirmed using control studies. We found that the fluorescence signal from Oct-4-targeting molecular beacons provides a clear discrimination between undifferentiated and retinoic acid-induced differentiated cells. Using deconvolution fluorescence microscopy, Oct-4 mRNAs were found to reside on one side of the cytosol. We demonstrated that, using a combination of Oct-4 mRNA-targeting molecular beacon with SSEA-1 antibody in flow cytometric analysis, undifferentiated stem cells can be clearly distinguished from differentiated cells. We revealed that Oct-4 targeting molecular beacons do not seem to affect stem cell biology.

Conclusion: Molecular beacons have the potential to provide a powerful tool for highly specific detection and isolation of stem cells, including cancer stem cells and induced pluripotent stem (iPS) cells without disturbing cell physiology. It is advantageous to perform simultaneous detection of intracellular (mRNA) and cell-surface (protein) stem cell markers in flow cytometric analysis, which may lead to high detection sensitivity and efficiency.

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The effect of Oct-4 targeting molecular beacon (MB4) on Oct-4 and IGF-2 mRNAs before and after differentiation. Shown in A and B are respectively the relative amount of Oct-4 and IGF-2 mRNAs in undifferentiated (UD) and RA-treated (RA) cells with (MB+) and without (MB-) Oct-4 targeting beacon MB4.
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Figure 6: The effect of Oct-4 targeting molecular beacon (MB4) on Oct-4 and IGF-2 mRNAs before and after differentiation. Shown in A and B are respectively the relative amount of Oct-4 and IGF-2 mRNAs in undifferentiated (UD) and RA-treated (RA) cells with (MB+) and without (MB-) Oct-4 targeting beacon MB4.

Mentions: In this work, we have demonstrated the use of molecular beacons to detect Oct-4 mRNA as a new method for the detection of cancer stem cells among normal cells or cancer cells. A particular issue is whether the biology of cancer stem cells is affected by the detection method, including the delivery approach and the probes inside the living cell. It is unlikely that unbound molecular beacon itself would affect cell biology such as gene expression and cell differentiation, since the oligonucleotide, fluorophore and quencher are not toxic. The effect of the delivery method using streptolysin O (SLO), however, needs to be determined, since SLO binds to cholesterol molecules on cell plasma membrane which may cause damages to cell membrane [28,29]. To address this issue, we delivered 1 μM of MB4 into undifferentiated P19 cells by SLO, and cultured these cells for 2 weeks. Further, undifferentiated cells with and without MB4 were cultured for 10 days and then treated with RA for 4 days (totally 2 weeks). The mRNA levels of Oct-4 and IGF-2 (Insulin-like Growth Factor-2) in these cells measured using RT-PCR are shown in Figure 6. Evidently, cells with and without MB4 had the same expression levels of Oct-4 (left panel) and IGF-2 (right panel) before and after differentiation, suggesting that SLO-based beacon delivery and probe/target hybridization did not have significant effects on cell physiology including gene expression, self-renewal, and differentiation. We also delivered Oct-4 targeting molecular beacons by SLO into mouse ESC, ES-D3 cells, for the isolation of ESCs from their differentiated cells. RT-PCR results and cell morphologies of differentiated cells with and without beacon indicated that there was no change in the ability of stem cell differentiation (unpublished data). To further establish the molecular beacon based method for detection and sorting of stem cells (including cancer stem cells and iPS cells), research is being conducted to demonstrate the sensitivity, specificity and safety of this technique. The results will be reported in a subsequent publication.


Simultaneous detection of mRNA and protein stem cell markers in live cells.

Rhee WJ, Bao G - BMC Biotechnol. (2009)

The effect of Oct-4 targeting molecular beacon (MB4) on Oct-4 and IGF-2 mRNAs before and after differentiation. Shown in A and B are respectively the relative amount of Oct-4 and IGF-2 mRNAs in undifferentiated (UD) and RA-treated (RA) cells with (MB+) and without (MB-) Oct-4 targeting beacon MB4.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2682800&req=5

Figure 6: The effect of Oct-4 targeting molecular beacon (MB4) on Oct-4 and IGF-2 mRNAs before and after differentiation. Shown in A and B are respectively the relative amount of Oct-4 and IGF-2 mRNAs in undifferentiated (UD) and RA-treated (RA) cells with (MB+) and without (MB-) Oct-4 targeting beacon MB4.
Mentions: In this work, we have demonstrated the use of molecular beacons to detect Oct-4 mRNA as a new method for the detection of cancer stem cells among normal cells or cancer cells. A particular issue is whether the biology of cancer stem cells is affected by the detection method, including the delivery approach and the probes inside the living cell. It is unlikely that unbound molecular beacon itself would affect cell biology such as gene expression and cell differentiation, since the oligonucleotide, fluorophore and quencher are not toxic. The effect of the delivery method using streptolysin O (SLO), however, needs to be determined, since SLO binds to cholesterol molecules on cell plasma membrane which may cause damages to cell membrane [28,29]. To address this issue, we delivered 1 μM of MB4 into undifferentiated P19 cells by SLO, and cultured these cells for 2 weeks. Further, undifferentiated cells with and without MB4 were cultured for 10 days and then treated with RA for 4 days (totally 2 weeks). The mRNA levels of Oct-4 and IGF-2 (Insulin-like Growth Factor-2) in these cells measured using RT-PCR are shown in Figure 6. Evidently, cells with and without MB4 had the same expression levels of Oct-4 (left panel) and IGF-2 (right panel) before and after differentiation, suggesting that SLO-based beacon delivery and probe/target hybridization did not have significant effects on cell physiology including gene expression, self-renewal, and differentiation. We also delivered Oct-4 targeting molecular beacons by SLO into mouse ESC, ES-D3 cells, for the isolation of ESCs from their differentiated cells. RT-PCR results and cell morphologies of differentiated cells with and without beacon indicated that there was no change in the ability of stem cell differentiation (unpublished data). To further establish the molecular beacon based method for detection and sorting of stem cells (including cancer stem cells and iPS cells), research is being conducted to demonstrate the sensitivity, specificity and safety of this technique. The results will be reported in a subsequent publication.

Bottom Line: We found that the fluorescence signal from Oct-4-targeting molecular beacons provides a clear discrimination between undifferentiated and retinoic acid-induced differentiated cells.Using deconvolution fluorescence microscopy, Oct-4 mRNAs were found to reside on one side of the cytosol.We revealed that Oct-4 targeting molecular beacons do not seem to affect stem cell biology.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia 30332, USA.

ABSTRACT

Background: Biological studies and medical application of stem cells often require the isolation of stem cells from a mixed cell population, including the detection of cancer stem cells in tumor tissue, and isolation of induced pluripotent stem cells after eliciting the expression of specific genes in adult cells. Here we report the detection of Oct-4 mRNA and SSEA-1 protein in live carcinoma stem cells using respectively molecular beacon and dye-labeled antibody, aiming to establish a new method for stem cells detection and isolation.

Results: Quantification of Oct-4 mRNA and protein in P19 mouse carcinoma stem cells using respectively RT-PCR and immunocytochemistry confirmed that their levels drastically decreased after differentiation. To visualize Oct-4 mRNA in live stem cells, molecular beacons were designed, synthesized and validated, and the detection specificity was confirmed using control studies. We found that the fluorescence signal from Oct-4-targeting molecular beacons provides a clear discrimination between undifferentiated and retinoic acid-induced differentiated cells. Using deconvolution fluorescence microscopy, Oct-4 mRNAs were found to reside on one side of the cytosol. We demonstrated that, using a combination of Oct-4 mRNA-targeting molecular beacon with SSEA-1 antibody in flow cytometric analysis, undifferentiated stem cells can be clearly distinguished from differentiated cells. We revealed that Oct-4 targeting molecular beacons do not seem to affect stem cell biology.

Conclusion: Molecular beacons have the potential to provide a powerful tool for highly specific detection and isolation of stem cells, including cancer stem cells and induced pluripotent stem (iPS) cells without disturbing cell physiology. It is advantageous to perform simultaneous detection of intracellular (mRNA) and cell-surface (protein) stem cell markers in flow cytometric analysis, which may lead to high detection sensitivity and efficiency.

Show MeSH
Related in: MedlinePlus