Correction: In Vivo Tumorigenesis Was Observed after Injection of In Vitro Expanded Neural Crest Stem Cells Isolated from Adult Bone Marrow.

[This corrects the article DOI: 10.1371/journal.pone.0046425.]

In vivo tumorigenesis was observed after injection of in vitro expanded neural crest stem cells isolated from adult bone marrow

Bone marrow stromal cells are adult multipotent cells that represent an attractive tool in cellular therapy strategies. Several studies have reported that in vitro passaging of mesenchymal stem cells alters the functional and biological properties of those cells, leading to the accumulation of genetic aberrations. Recent studies described bone marrow stromal cells (BMSC) as mixed populations of cells including mesenchymal (MSC) and neural crest stem cells (NCSC). Here, we report the transformation of NCSC into tumorigenic cells, after in vitro long-term passaging. Indeed, the characterization of 6 neural crest-derived clones revealed the presence of one tumorigenic clone. Transcriptomic analyses of this clone highlighted, among others, numerous cell cycle checkpoint modifications and chromosome 11q down-regulation (suggesting a deletion of chromosome 11q) compared with the other clones. Moreover, unsupervised analysis such as a dendrogram generated after agglomerative hierarchical clustering comparing several transcriptomic data showed important similarities between the tumorigenic neural crest-derived clone and mammary tumor cell lines. Altogether, it appeared that NCSC isolated from adult bone marrow represents a potential danger for cellular therapy, and consequently, we recommend that phenotypic, functional and genetic assays should be performed on bone marrow mesenchymal and neural crest stem cells before in vivo use, to demonstrate whether their biological properties, after ex vivo expansion, remain suitable for clinical application

Chromowave profile of <i>Asclepios</i>, displays the first eigenvector that explains 84% of the variance between <i>Asclepios</i> and NCSC mix.

<p>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 <i>Asclepios</i>.</p

Cancer is one of the main biological function hit of <i>Asclepios</i>.

<p>Microarray comparison between <i>Asclepios</i> and neural crest stem cell clones revealed 1,544 significant genes that were differentially expressed. Those genes were introduced in IPA for biological functions and pathway analyses. The results confirmed the tumor profile of <i>Asclepios</i> with cancer functions.</p

Dendrogram from agglomerative hierarchical clustering of Asclepios and several cell types, including tumor cell lines.

<p>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 (<a href="http://www.ncbi.nlm.nih.gov/gds" target="_blank">http://www.ncbi.nlm.nih.gov/gds</a>). 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 <i>p</i>-values (%). Red values are <b>AU </b><i>p</i>-values and green values are <b>BP</b> values. AU (Approximately Unbiased) <i>p</i>-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.</p

<i>In vivo</i> characterization of neural crest derived cells.

<p>To characterize neural crest-derived clones <i>in vivo</i>, we stereotaxically injected 50,000 cells of each NCSC clones (separetly) in mice striatum (<b>A</b>). <i>Asclepios</i> induced massive tumors after 4 weeks as attested by the beta-galactosidase expression of the tumor cells. (<b>B</b>). Immunological characterization of those tumors revealed that they were GFAP (<b>c</b>), beta-III-tubulin (<b>D</b>), nestin (<b>E</b>), N-cadherin (<b>F</b>) and NrCAM-positive (G). Lectin labeling (<b>H</b>) confirmed the presence of blood vessels in the tumors. Finally, no vimentin (<b>I</b>) or Sox2 (<b>J</b>) expressions were observed. Nuclei were counterstained with Dapi (blue). Scale bars = 50 µm.</p

Chromosomal distribution of <i>Asclepios</i> genes.

<p>Barplot comparing the chromosomal distribution of the differentially expressed genes (<i>p</i>-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 <i>Asclepios</i> and NCSCs, highlights the chromosome 11 enrichment after statistical univariate tests.</p

Clusters based on wavelet coefficients.

<p>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. <i>Asclepios</i> (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 <i>Asclepios</i> and NCSC represents only 5.9% of the variance, making NCSC the best reference to study <i>Asclepios</i> mRNA expression.</p