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Multiplex Detection of the Stem Cell Pluripotency Markers c-Myc, Nanog, Oct 3/4 and Sox2: MAP Human

n Stem Cell Pluripotency Magnetic Bead Panel 1 MILLIPLEX


EMD Millipore, Billerica, MA 01821 USA Ralf Reiners, Nikolai Stankiewicz, Susanne Wendland, Rick Wiese, Helen Sabzevari, Michael Wolf and Anita Seshire
Abstract
The transcription factors Oct 3/4, Sox2 and Nanog play a critical role in the maintenance of pluripotency in stem cells. Additionally, overexpression of any one or a combination of these three factors has been reported in solid tumors from a variety of tissues. The presence of pseudogenes and differentially expressed protein isoforms, complicates the accurate detection of the physiologically relevant form of these proteins in stem cell research. Here, we report the development of a MILLIPLEX MAP multiplex assay for the simultaneous, specific and sensitive detection of the pluripotency markers Oct 3/4, Sox2 and Nanog, as well as of c-Myc protein levels in cellular lysates. The sensitivity of the MILLIPLEX MAP Human Stem Cell Pluripotency Magnetic Bead Panel 1 (Cat. No. 48617MAG) assay is 100-fold greater when compared to Western Blot analysis. This sensitivity is of special importance for the detection of rare cancer stem cell populations. Application of the assay to a panel of human cancer cell lines from breast, lung and colon origins revealed Sox2 to be the most often expressed factor among these lines. Of the cell lines assayed, only one breast cancer cell line expressed detectable levels of all three transcription factors, suggesting a role for Oct 4, Sox2 and Nanog in cancer stem cells of this particular cell line. These experiments demonstrate the utility of this pluripotent stem cell panel for the sensitive analysis of stem cells from a variety of lineages. The expression of the analytes Oct 3/4, Sox2, Nanog and c-Myc was also examined in the model cell lines Ntera-2cl.D1 and HeLa and compared with application data for induced pluripotent stem cells (iPS).

Results

Methods
Luminex 200 system. This is a compact unit consisting of an analyzer, a computer and software (Luminex Corporation, Austin, TX). Microspheres. Magnetic microsphere beads were purchased from Luminex Corporation. Each set of beads is distinguished by different ratios of two internal dyes yielding a unique fluorescent signature to each bead set. Capture antibodies were covalently coupled to the carboxylate-modified magnetic microsphere beads. Figure 1. Comparison of the sensitivity of MILLIPLEX MAP assay and Western Blot. (A) Oct 4, Sox2 and Nanog were assayed either alone (singleplex) or simultaneously (triplex) in NT2 lysates. The graphs represent mean MFI values of triplicate measures +/- SD. (B) Detection of Oct 4, Sox2 and Nanog in NT2 lysates by Western Blot analysis. Anti-Cofilin antibody was used to detect Cofilin as a loading control. MFI = Median Fluorescence Intensity. SD = standard deviation.

Figure 2. Sox2 detection is isotype-specific. Minimal crossreactivity to Sox1 is observed as Sox1 shares the greatest homology with Sox2. (A) Subgroups of the Sox protein family, grouped according to their homology grade (*adapted from Schepers et al. 2002). (B) Mean PEMFI values +/-SD from Sox2 singleplex in lysates from Sox-positive NT2 and MCF7, and the Sox2-negative cell lines MDA-MB 231 and HCC38. Besides the presence/absence of Sox2, the cell lines express other Sox proteins as indicated. (C) Mean PE-MFI values +/SD from triplicates of NT2 cells and transfected HCT116. HCT116 were transfected with either pCMV6-XL5 control-vector, pCMV6-XL5SOX1 expression vector or pc3-huSOX2 expression vector. MFI = Median Fluorescence Intensity, SD = standard deviation.

Figure 5. Level of the proteins Oct 4, Sox2, Nanog and c-Myc in human foreskin fibroblasts, human STEMCCA-LoxP pre-excised iPS and post-excised iPS cells compared to control cell lines. The induced pluripotent stem cells were generated from HFF, P6, (Cat. No. SCC058) by using the STEMCCA Cre-Excisable Constitutive Polycistronic (OKSM) Lentivirus Reprogramming Kit (Cat. No. SCR511). Mean MFI values measured in HFF and iPS cells generated thereof, as well as in control cell lines Ntera-2cl.D1 & HeLa and Assay Buffer 1 (blank).

Conclusion
The MILLIPLEX MAP Human Stem Cell Pluripotency Panel 1 (Cat. No. 48-617MAG) enables the simultaneous analysis of the pluripotency markers Oct 3/4, Sox2, Nanog and c-Myc protein in cellular lysates. The sensitivity of the assay exceeds the Western Blot analyses commonly used for the quantification of intracellular protein markers by a factor of 100. The antibodies used for Sox2 detection specifically detect Sox2 and no other Sox proteins from groups CDEF. A low cross-reacitivity to Sox1 was observed, but can be neglected due to low signal intensity. The results for the tested breast, lung and colon cancer cell lines revealed that Sox2 is the most often expressed factor among the four analytes. Only in the T47D breast cancer cell line were Oct 4, Sox2 and Nanog all expressed at detectable levels. The detection of all analytes in Ntera-2cl.D1 and the iPS cells shows the potential of this assay in the analysis of a variety of cell lines. The addition of the Human Stem Cell Pluripotency Panel 1 to the MILLIPLEX MAP family of kits opens up the opportunity of multiplex protein detection in stem cell research using Luminex xMAP technology.

Tissue Culture. Cells were cultured according to ATCC guidelines in recommended media. Cells were grown to approximately 85% confluence. Sample Preparation. Cells were lysed and samples collected according to MILLIPLEX MAP Cell Signaling Buffer and Detection Kit (Cat. No. 48-602) instructions or lysed using a NP40-based lysis buffer. Immunoassay Protocol. The multiplex assay was performed in a 96-well plate using MILLIPLEX MAP Assay Buffer 1 (Cat. No. 43010). The detailed procedure is as follows: rinse the plate with 100 L assay buffer Add 25 L samples, 25 L beads to each well incubate overnight wash the beads two times with assay buffer add biotinylated detection antibody cocktail, 1h RT remove detection and add 25 L MILLIPLEX MAP Streptavidin-Phycoerythrin (Cat. No. 45-001D), 30 min RT remove SAPE and resuspend beads in 150 L assay buffer read on Luminex instrumentation.

Corresponding Author: Nikolai.Stankiewicz@merckgroup.com


MILLIPLEX is a registered trademark of Merck KGaA. Stemcca, EMD Millipore and the M logo are trademarks of Merck KGaA, Darmstadt, Germany. Luminex is a trademark of Luminex Corp, Austin, TX. ATCC is a registered trademark of the American Type Culture Collection Corporation. 2012 Millipore Corporation. All rights reserved. Lit. No. PS1092EN00 , rev. A. 08/12

Figure 4. Expression levels of the Oct 3/4, Sox2, Nanog and c-Myc in the model cell lines Ntera-2cl.D1 and HeLa. The Ntera-2cl cell line served as a positive control for the analytes Oct 3/4, Sox2 and Nanog, and as negative control for c-Myc. Conversely, HeLa was the positive control for c-Myc and the negative control for the other analytes. The measured MFI values demonstrate the utility of these cell lines for use as control lysates.

Figure 3. Simultaneous detection of Oct 4, Sox2 and Nanog in breast, colon and lung cancer cell lines. (A) The three TFs Oct 4, Sox2 and Nanog were simultaneously detected in 5-6 cell lines for each of the three main cancer entities (breast, colon and lung) using the multiplex (triplex) assay described here. PE-MFI values were normalized against the expression of the housekeeping protein GAPDH. Means of triplicates are shown +/-SD. Cell lysates were derived from three independent experiments. (B) Protein expression intensities in the tested cell lines. Expression levels were compared to protein expression levels in NT2 cells. > 50 % of expression compared to NT2 was considered high, < 50% medium and < 10% low. Pos Ctrl or PC = positive control NT2, Neg Ctrl or NC = negative control HUVEC, MFI = Median Fluorescence Intensity, SD = standard deviation.

Summary
We have developed the MILLIPLEX MAP Human Stem Cell Pluripotency Panel 1 (Cat. No. 48-617MAG) on magnetic beads to measure total cellular pluripotency protein markers in cell lysates. Assays require approximately 25 g cellular lysate per sample (duplicate assay wells) and are specific, sensitive and reproducible. The MILLIPLEX MAP Human Stem Cell Pluripotency Panel 1 contains 3 basic pluripotency markers Oct 3/4, Sox2 and Nanog commonly used in stem cell research and c-Myc as a internal control.

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