Open Access Research

Transcriptomic and phenotypic analysis of murine embryonic stem cell derived BMP2+ lineage cells: an insight into mesodermal patterning

Michael Xavier Doss1, Shuhua Chen1, Johannes Winkler1, Rita Hippler-Altenburg1, Margareta Odenthal2, Claudia Wickenhauser2, Sridevi Balaraman2, Herbert Schulz3, Oliver Hummel3, Norbert Hübner3, Nandini Ghosh-Choudhury4, Isaia Sotiriadou1, Jürgen Hescheler1 and Agapios Sachinidis1*

Author Affiliations

1 Institute of Neurophysiology, University of Cologne, Robert-Koch Str. 39, 50931 Cologne, Germany

2 Institute of Pathology, University of Cologne, Joseph-Stelzmann-Str. 9, 50931 Cologne, Germany

3 Max-Delbrueck-Center for Molecular Medicine - MDC, Robert-Rössle Str. 10, 13092 Berlin, Germany

4 Department of Pathology, The University of Texas Health Science Center at San Antonio, TX 78229, USA

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Genome Biology 2007, 8:R184  doi:10.1186/gb-2007-8-9-r184

Published: 4 September 2007

Additional files

Additional data file 1:

EBs were generated using the conventional hanging drop protocol (see Materials and methods and Figure 5b) and the expression of the T-bra, flk1, smooth muscle α-actin, cardiac Troponin T (c-Troponin T), NF-H, NF-M and AFP was detected using RT-PCR (for the conditions, see Additional data file 14).

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Additional data file 2:

The fold change of the expression of the genes was calculated by using the formula: fold-change = <a onClick="popup('http://genomebiology.com/2007/8/9/R184/mathml/M2','MathML',630,470);return false;" target="_blank" href="http://genomebiology.com/2007/8/9/R184/mathml/M2">View MathML</a>. The ΔCt of a gene in the sample in which it is expressed at the lowest level is taken as ΔCt gene2 to calculate the fold change using the above formula. The resulting fold change is expressed as the percentage of the maximum fold change (100%) for that particular gene in every assay. Values are expressed as mean ± standard deviation (n = 3, technical replicates).

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Additional data file 3:

Transcripts belonging to the GO category 'transcription' that are upregulated at least two-fold (t-test p value < 0.01) in the BMP2+ cells compared to the control cells in the seven-day-old EBs, and those upregulated compared to the control cells in the seven-day-old EBs and the undifferentiated BMP2 ES cells.

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Additional data file 4:

Transcripts belonging to the GO category 'apoptosis' that are upregulated at least two-fold (t-test p value < 0.01) in the BMP2+ cells compared to the control cells in the seven-day-old EBs, and those upregulated compared to the control cells in the seven-day-old EBs and the undifferentiated BMP2 ES cells.

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Additional data file 5:

Transcripts belonging to the MAPK signaling pathway that are upregulated at least two-fold (t-test p value < 0.01) in the BMP2+ cells compared to the control cells in the seven-day-old EBs, and those upregulated compared to the control cells in the seven-day-old EBs and the undifferentiated BMP2 ES cells. The schematic is of the KEGG MAP kinase signaling pathway, indicating the upregulated genes (labelled with red background) in the BMP2+ cells compared to the control cells in the seven-day-old EBs.

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Additional data file 6:

Transcripts belonging to the GO category 'development' that are downregulated at least two-fold (t-test p value < 0.01) in the BMP2+ cells compared to the control cells in the seven-day-old EBs.

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Additional data file 7:

Transcripts belonging to the TGFβ signaling pathway that are specifically upregulated at least two-fold (t-test p value < 0.01) in the BMP2+ cells compared to the control cells in the seven-day-old EBs as well as a schematic of the KEGG TGFβ signaling pathway indicating the upregulated genes (labelled with red background and white letters).

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Additional data file 8:

Transcripts belonging to the GO category 'M phase' that are specifically upregulated at least two-fold (t-test p value < 0.01) in the BMP2+ cells compared to the control cells in the seven-day-old EBs and to the undifferentiated BMP2 ES cells.

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Additional data file 9:

(A,B) Pairwise comparison (BMP2 versus control EBs): 2,258 probeset IDs that were differentially expressed in BMP2+ cells compared to control EBs (two-condition comparison) were converted to GenBank accession numbers and redundancies were removed (1,833 unique transcripts). Stanford Source was used to obtain GO biological process (BP) annotations. Genesis 1.7.0 was used to visualize and identify GO BP categories of interest and extract corresponding lists of transcripts. For the categories adhesion, cell cycle, cell death, cell-cell signaling, cellular metabolism, development, stress response, signal transduction, transcription and transport, 1,541 annotations were established for 1,172 transcripts. The pie chart (A) shows the distribution of these annotations. The bar chart (B) shows the number of genes in the categories adhesion, cell cycle, cell death, cell-cell signaling, cellular metabolism, development, stress response, signal transduction, transcription and transport separately for up- and downregulated transcripts. (C,D) Three-condition comparison (BMP2 versus control EBs and BMP2 ES cells). For the three-condition comparison, 551 unique transcripts were obtained from 672 probeset IDs that were differentially expressed in BMP2 versus control EBs and versus undifferentiated BMP2 ES cells using the approach described above. For the categories adhesion, cell cycle, cell death, cell-cell signaling, cellular metabolism, development, stress response, signal transduction, transcription and transport, 430 annotations were established for 268 transcripts. The pie chart (C) shows the distribution of these annotations. The bar chart (D) shows the number of genes in the categories adhesion, cell cycle, cell death, cell-cell signaling, cellular metabolism, development, stress response, signal transduction, transcription and transport separately for up- and downregulated transcripts. (E) Clustering analysis of the probesets identified as differentially expressed in the two-condition comparison (see A) and assigned to the GO category 'development'. Expression data were normalized using the RMA algorithm and hierarchical clustering was done using Cluster 2.11 as described. Visualization of the hierarchical clustering of probe sets identified as differentially expressed in the pairwise comparison (see A) and assigned to the GO category 'development'. Each probe set is represented by a single column of colored boxes; each array is represented by a single row. Cells with unchanged probe sets are colored black, upregulation is indicated with reds of increasing intensity, and downregulation with greens of increasing intensity. The dendrogram on the top of the figure represents the similarity matrix of probe sets, the dendrogram to the right the similarity matrix of arrays.

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Additional data file 10:

A video clip of β-actin CGR8 cardiomyocytes.

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Additional data file 11:

A video clip of BMP2+ cell-derived cardiomyocytes without green filter.

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Additional data file 12:

A video clip of BMP2+ cell-derived cardiomyocytes with green filter.

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Additional data file 13:

(A) Anti-SMA staining on mouse liver tissue. (B) MyoD1 on mouse embryonic sections. (C) α-Actinin on embryonic cardiac tissue of mouse. (D) F4/80 staining on mouse skin sections. (E) Pan cytokeratin on small intestine of mouse. (F) Ksp-Cadherin on mouse kidney sections. (G) GFAP on rat brain sections. (H) E-cadherin on mouse kidney sections. (I) Alizarin Red staining on human limb bone sections. (H) Sudan Red stainings on cytospin leukocytes.

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Additional data file 14:

Primers used for RT-PCR analysis.

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Additional data file 15:

Complete Affymetrix dataset for all experimental conditions, representing three independent experiments. Data were RMA-normalized as described in Materials and methods. For each probe set, the average expression value and standard deviations are given (columns B-G) as well as the change fold between conditions (columns H-J) and the results of Student's t-test between the experimental conditions (K-M). Expression values for the individual experimental replicates are provided in columns N-V.

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Additional data file 16:

Normalized Affymetrix data set for the probe sets identified to be differentially downregulated in BMP2+ cells compared to control EBs (higher than two-fold downregulation, t-test p value < 0.01).

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Additional data file 17:

Normalized Affymetrix dataset for the probe sets identified to be differentially downregulated in BMP2+ cells compared to control EBs and undifferentiated BMP2 ES cells (intersection of downregulation in BMP2+ cells compared to control EBs (higher than two-fold downregulation, t-test p value < 0.01) and compared to undifferentiated BMP2 ES cells (higher than two-fold downregulation, t-test p value < 0.01)).

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Additional data file 18:

Normalized Affymetrix dataset for the probe sets identified to be differentially upregulated in BMP2+ cells compared to control EBs (higher than two-fold upregulation, t-test p value < 0.01).

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Additional data file 19:

Normalized Affymetrix dataset for the probe sets identified to be differentially upregulated in BMP2+ cells compared to control EBs and compared to undifferentiated BMP2 ES cells (intersection of upregulation in BMP2+ cells compared to control EBs (higher than two-fold upregulation, t-test p value < 0.01) and compared to undifferentiated BMP2 ES cells (higher than two-fold upregulation, t-test p value < 0.01)).

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