Limits...
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.

Show MeSH

Related in: MedlinePlus

Changes of Oct-4 mRNA and protein levels 4 days after retinoic acid (RA) treatment in P19 embryonal carcinoma cells. A. Real-time PCR result of Oct-4 mRNA level in undifferentiated (UD) and differentiated (RA treated) P19 cells. B. Immunocytochemistry of Oct-4 protein (Red) before (UD) and after (RA) differentiation. Cell nuclei were stained with Hoechst 33342 (Blue).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2682800&req=5

Figure 1: Changes of Oct-4 mRNA and protein levels 4 days after retinoic acid (RA) treatment in P19 embryonal carcinoma cells. A. Real-time PCR result of Oct-4 mRNA level in undifferentiated (UD) and differentiated (RA treated) P19 cells. B. Immunocytochemistry of Oct-4 protein (Red) before (UD) and after (RA) differentiation. Cell nuclei were stained with Hoechst 33342 (Blue).

Mentions: As a positive control for molecular beacon based studies, we have quantified the changes in Oct-4 mRNA level 4 days after RA treatment in P19 cells. Cells were exposed to 500 nM of RA for 2 days followed by 2 days of incubation with medium absent of RA. As shown in Figure 1A, our real-time PCR results indicate that RA treatment decreased the Oct-4 mRNA level in differentiated cells to less than 1% of that in untreated EC cells. To check if decrease in Oct-4 mRNA level reflects changes in Oct-4 protein level, we carried out immunocytochemistry assays of Oct-4 protein before and after RA treatment using dye-labeled antibody for Oct-4. As demonstrated in Figure 1B, strong fluorescence signals were observed in the nuclei of undifferentiated cells, while there was essentially no signal after RA treatment. Since Oct-4 is a transcription factor, it is not surprising that the staining of Oct-4 protein is predominately in cell nucleus (Figure 1B).


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

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

Changes of Oct-4 mRNA and protein levels 4 days after retinoic acid (RA) treatment in P19 embryonal carcinoma cells. A. Real-time PCR result of Oct-4 mRNA level in undifferentiated (UD) and differentiated (RA treated) P19 cells. B. Immunocytochemistry of Oct-4 protein (Red) before (UD) and after (RA) differentiation. Cell nuclei were stained with Hoechst 33342 (Blue).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Changes of Oct-4 mRNA and protein levels 4 days after retinoic acid (RA) treatment in P19 embryonal carcinoma cells. A. Real-time PCR result of Oct-4 mRNA level in undifferentiated (UD) and differentiated (RA treated) P19 cells. B. Immunocytochemistry of Oct-4 protein (Red) before (UD) and after (RA) differentiation. Cell nuclei were stained with Hoechst 33342 (Blue).
Mentions: As a positive control for molecular beacon based studies, we have quantified the changes in Oct-4 mRNA level 4 days after RA treatment in P19 cells. Cells were exposed to 500 nM of RA for 2 days followed by 2 days of incubation with medium absent of RA. As shown in Figure 1A, our real-time PCR results indicate that RA treatment decreased the Oct-4 mRNA level in differentiated cells to less than 1% of that in untreated EC cells. To check if decrease in Oct-4 mRNA level reflects changes in Oct-4 protein level, we carried out immunocytochemistry assays of Oct-4 protein before and after RA treatment using dye-labeled antibody for Oct-4. As demonstrated in Figure 1B, strong fluorescence signals were observed in the nuclei of undifferentiated cells, while there was essentially no signal after RA treatment. Since Oct-4 is a transcription factor, it is not surprising that the staining of Oct-4 protein is predominately in cell nucleus (Figure 1B).

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