Figure 1.
Phenotypic characterization of neural-crest derived cells isolated from adult bone marrow.
Neural crest stem cells were isolated from double transgenic Wnt1/Cre-R26R/LacZ mice and cultured under clonal conditions. A–B. Neural crest derived clones were morphologically similar to classical BMSC. As clones have been isolated from double transgenic mice Wnt1-CRE/R26R-LacZ, neural crest-derived cells are expressing beta-galactosidase, visualized after an X-gal staining (A). C–L. Immunological characterization revealed that neural crest derived cells were nestin (C), P75NTR (D), Sox10 (E), CD9 (F), MMP12 (G), CDH13 (H), CD82 (I) positives, but CD24 (J), CD38 (K) and MMP13 (L) negatives. M–N. A percentage of neural crest stem cells were able to differentiate into beta-III-tubulin-positive cells when co-cultivated with GFP-positive cerebellar granule neurons (M), however, Asclepios showed a higher percentage of positive cells as 50.25%±1.70% of cells were beta-III-tubulin-positive, when around 15% of cells were observed with the other clones (N) (mean ± SEM, n = 3, p<0.001, ANOVA followed by Bonferroni post hoc test). Nuclei were counterstained with Dapi (blue) on panels C to N. Scale bars = 30 µm.
Figure 2.
In vivo characterization of neural crest derived cells.
To characterize neural crest-derived clones in vivo, we stereotaxically injected 50,000 cells of each NCSC clones (separetly) in mice striatum (A). Asclepios induced massive tumors after 4 weeks as attested by the beta-galactosidase expression of the tumor cells. (B). Immunological characterization of those tumors revealed that they were GFAP (c), beta-III-tubulin (D), nestin (E), N-cadherin (F) and NrCAM-positive (G). Lectin labeling (H) confirmed the presence of blood vessels in the tumors. Finally, no vimentin (I) or Sox2 (J) expressions were observed. Nuclei were counterstained with Dapi (blue). Scale bars = 50 µm.
Figure 3.
Clusters based on wavelet coefficients.
The first, second and third components are represented in figure A, B and C respectively and summarized on figure D. These components respectively explain 61.2, 22.7 and 5.9% of the variance of the dataset. For each component, samples in the same orientation over the y-axis are clustered together. Asclepios (Asc); Neural Crest Stem Cells mix (NCSC); Tumor cell lines 67NR, 66cl4 and 4T1 (TCL, GSE11259); Neural Precursor From Embryonic Stem Cells (NPFES, GSE8024) are represented in these clusters. The difference between Asclepios and NCSC represents only 5.9% of the variance, making NCSC the best reference to study Asclepios mRNA expression.
Figure 4.
Chromosomal distribution of Asclepios genes.
Barplot comparing the chromosomal distribution of the differentially expressed genes (p-value<0.001–1,544 probesets) in blue to the overall background filtered dataset (19,667 probesets) in red. This barplot, based on the comparison between Asclepios and NCSCs, highlights the chromosome 11 enrichment after statistical univariate tests.
Figure 5.
Chromowave profile of Asclepios, displays the first eigenvector that explains 84% of the variance between Asclepios and NCSC mix.
Each chromosomal signal has been wavelet transformed. Under and over expressions are respectively below or above 0 on the y-axis. The x-axis displays the chromosomal position labeled with cytoband names. A large part of the chromosome 11 is under expressed in Asclepios.
Table 1.
Gene type expression on chromosome 11.
Figure 6.
G1/S checkpoints regulation pathway highlights regulated genes in the comparison between Asclepios and NCSC mix. Red genes are over-expressed and Green genes are down-expressed in Asclepios. Fold changes from the comparison are written for single (non complex) genes. This pathway was build with Ingenuity Pathway Analysis software. Major tumor suppressors are down regulated suggesting a high proliferation of Asclepios cells.
Figure 7.
Cell proliferation assay was performed using tetrazolium compound based CellTiter 96® AQueous One Solution Cell Proliferation (MTS) assay (Promega). 5×103 cells of each NCSC clone were seeded into wells of a 96-well plate. After 24 and 48 hours of culture under regular growth conditions (Mesencult medium), MTS assay was performed according to the manufacturer's instructions. Each experience was performed in triplicate and repeated 3 times (n = 3). No difference was observed after 24 hours of culture, however, a highly significant increase in absorbance was detected for Asclepios in comparison with the other clones (p<0,001; repeated measures ANOVA, followed by Tuckey post-test), reflecting a higher proliferation rate in an interval of 48 hours.
Table 2.
Cancer is one of the main biological function hit of Asclepios.
Figure 8.
Dendrogram from agglomerative hierarchical clustering of Asclepios and several cell types, including tumor cell lines.
Dendrogram generated after agglomerative hierarchical clustering using Euclidean distance, complete linkage and multiscale bootstrap resampling. 61 expression arrays were included in an unsupervised analysis with hierarchical clustering of samples. Spontaneous epithelial mammary tumor cell lines (SEMTCL - GSE13259); Tumor cell lines 67NR, 66cl4 and 4T1 (TCL67NR, TCL66cl4 and TCL4T1 - GSE11259); Embryonal tumor deriving from neural crest cells (NCCE85, NCCE135, NCCP90 - GSE11356); Multipotent adult progenitor cells (MAPC - GSE6291); Developing Heart (DH - GSE7196); Neural precursors obtained from embryonic stem cells (NPFES - GSE8024); White and brown adipose (WAA, BAA - GSE8044); Head Neck Neural Crest Stem Cells (E115FAKCtle1 - GSE11149); Murine acute myeloid leukemia (UAML - GSE30747). Datasets are accessible on GEO datasets/NCBI (http://www.ncbi.nlm.nih.gov/gds). The dendrogram was built with the Euclidean distance as dissimilarity metric and the complete linkage method for definition of the structure. Values on the edges of the clustering are p-values (%). Red values are AU p-values and green values are BP values. AU (Approximately Unbiased) p-values were computed by multiscale bootstrap resampling. BP (Bootstrap Probability) values were computed by normal bootstrap resampling. R-cran “pvclust” package was used for assessing the uncertainty of this hierarchical cluster analysis for 10,000 permutations of genes. Those values indicated how strongly the cluster was supported by the data.