Wnt1 and BMP2: two factors recruiting multipotent neural crest progenitors isolated from adult bone marrow

Abstract

Recent studies have shown that neural crest-derived progenitor cells can be found in diverse mammalian tissues including tissues that were not previously shown to contain neural crest derivatives, such as bone marrow. The identification of those "new" neural crest-derived progenitor cells opens new strategies for developing autologous cell replacement therapies in regenerative medicine. However, their potential use is still a challenge as only few neural crest-derived progenitor cells were found in those new accessible locations. In this study, we developed a protocol, based on wnt1 and BMP2 effects, to enrich neural crest-derived cells from adult bone marrow. Those two factors are known to maintain and stimulate the proliferation of embryonic neural crest stem cells, however, their effects have never been characterized on neural crest cells isolated from adult tissues. Using multiple strategies from microarray to 2D-DIGE proteomic analyses, we characterized those recruited neural crest-derived cells, defining their identity and their differentiating abilities.

Fig. 1

BMP2 and Wnt1 increase the number of nestin-positive cells in cultured adult bone marrow cells. a–c Immunofluorescent labeling for nestin (green) and dapi (blue) of passage 1 Wnt1- and BMP2-stimulated cells (a) and untreated cells (b). c Comparison of the percentage of nestin-positive cells in presence or absence of Wnt1 and Bmp2. Stromal cells isolated from bone marrow have been cultivated in the presence of 10 ng/ml Wnt1 and BMP2 factors. After 1 passage, the number of nestin-positive cells significantly increased compared to the control condition (without Wnt1 and BMP2). Student’s t test, p < 0.0001, n = 3. This effect disappeared after four passages, as no significant differences could then be observed. Student’s t test, p > 0.05, n = 3. d–g Stimulated and unstimulated cells were co-cultured for 5 days with GFP-positive CGN (green). GFAP (red) was equally expressed under both conditions (d untreated; e treated), while Tuj1-positive cells (red) could only be observed in Wnt1 and BMP2 treated cells (f untreated; g treated). Absence of GFP expression confirmed the bone marrow origin of some Tuj1-expressing cells in g. Nuclei were counterstained with Dapi (blue). Blue arrows show neural expression markers by presumptive neural crest-derived cells (Tuj1- or GFAP-positive, but GFP-negative cells), while yellow arrows show undifferentiated stromal cells (Tuj1- or GFAP-, and GFP-negative). Scale bars 30 μM. Significant differences are indicated as *p < 0.05, **p < 0.01 and ***p < 0.001

Fig. 2

Clonal screening of nestin-positive bone marrow stromal cells. BMSC were diluted at clonal density (0.7 cell/well in 96-wells plates), in the presence of Wnt1 and BMP2. In those culture conditions, 1.2% of the cells proliferated as clonal culture. All clones were nestin-positive (a), p75^NTR-positive (b) and Sox10-positive (c). Four clones were selected and co-cultured for 5 days with GFP-positive CGN (green). Tuj1 (red, d) and GFAP (red, e)-positive, GFP-negative cells were quantified. All clones were able to differentiate into GFAP- and Tuj1-positive cells, but the percentages of expression of these two markers varied from one clone to another. Nuclei were counterstained with Dapi (blue). Arrows show neural expression markers by clonally generated cells. Scale bars 30 μM

Fig. 3

Functional characterization of selected Wnt1/BMP2 treated clones of BMSC. The differentiation potential of clone 1 and 4 was analyzed. Those clones were able to differentiate into adipocytes (oil red O coloration, a), melanocytes (l-Dopa colorimetric test, b), smooth muscle (SMA green, nuclei counterstained with Dapi-blue, c) and osteocytes (alkaline phosphatase activity, d) Scale bars 30 μm (a, c), 50 μM (b)

Fig. 4

Quantitative RT-PCR characterization of selected Wnt1/BMP2 treated clones of BMSC. Quantitative RT-PCR was performed on total RNA extracted from clone 1 and clone 4, as well as from neural stem cells (NSC-positive control for neural lineage) and fibroblasts (3T3-positive control for mesenchymal lineage) that were used as controls. The results of quantitative RT-PCR measurements are expressed as percent of each gene expression in clone 4 (arbitrarily set as 100%) after normalization with the house-keeping GAPDH gene expression. Three samples of each clone or control have been processed and for each experiment, measurements are made in triplicate. Student’s t test was performed to compare clone 1 and clone 4. Significant differences are indicated as *p < 0.05; **p < 0.01 and ***p < 0.001

Fig. 5

Dendrogram generated after agglomerative hierarchical clustering using Euclidean distance, average linkage and multiscale bootstrap resampling. 118 expression array data sets were included in an unsupervised analysis with hierarchical clustering of samples. All replicates were merged under the same denomination when possible. The dendrogram was build with the Euclidean distance as dissimilarity metric and the average 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 1,000 permutations of genes. Those values indicated how strongly the cluster was supported by the data and consequently strongly confirmed the same embryonic origin of clone 1, clone 4 and NCSC clone isolated from Wnt1-CRE/R26R double transgenic mice

Fig. 6

Proteomic analysis of clone 1 and clone 4 using comparative 2D-DIGE technology. a, b A representative two-dimensional gel electrophoresis protein separation used for determining differentially expressed proteins in clone 1 and 4. Contoured spots correspond to proteins of interest that were picked for mass spectrometry identification. Master numbers of identified proteins from b are indicated in yellow boxes. b Lists the proteins overexpressed in clone 1 and clone 4 with their master number, Uniprot ID, name, calculated t test value for differential expression, calculated fold-expression ratio, theoretical isoelectric point and molecular mass. c Over-expression of glycolytic and carbohydrate metabolism enzymes has been confirmed by Western blotting