Gene expression profiling of human mesenchymal stem cells derived from bone marrow during expansion and osteoblast differentiation - PubMed (original) (raw)

Gene expression profiling of human mesenchymal stem cells derived from bone marrow during expansion and osteoblast differentiation

Birgit Kulterer et al. BMC Genomics. 2007.

Abstract

Background: Human mesenchymal stem cells (MSC) with the capacity to differentiate into osteoblasts provide potential for the development of novel treatment strategies, such as improved healing of large bone defects. However, their low frequency in bone marrow necessitate ex vivo expansion for further clinical application. In this study we asked if MSC are developing in an aberrant or unwanted way during ex vivo long-term cultivation and if artificial cultivation conditions exert any influence on their stem cell maintenance. To address this question we first developed human oligonucleotide microarrays with 30.000 elements and then performed large-scale expression profiling of long-term expanded MSC and MSC during differentiation into osteoblasts.

Results: The results showed that MSC did not alter their osteogenic differentiation capacity, surface marker profile, and the expression profiles of MSC during expansion. Microarray analysis of MSC during osteogenic differentiation identified three candidate genes for further examination and functional analysis: ID4, CRYAB, and SORT1. Additionally, we were able to reconstruct the three developmental phases during osteoblast differentiation: proliferation, matrix maturation, and mineralization, and illustrate the activation of the SMAD signaling pathways by TGF-beta2 and BMPs.

Conclusion: With a variety of assays we could show that MSC represent a cell population which can be expanded for therapeutic applications.

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Figures

Figure 1

Figure 1

Upper panel: Osteogenic differentiation of MSC after passage 2 (A, B) and passage 10 (C, D). Alizarin Red S staining and Alkaline phosphatase assay were performed after 21 days of osteogenic induction (d21).100× magnification. Middle Panel: Morphological alterations during osteogenesis determined by microscopy on the day of induction (d0), day 4, 7, 14 and 21 after induction. All pictures are shown in 100× magnification. Lower Panel: Adipogenic differentiation of MSC in passage 10 characterized by Oil red O staining; (A) MSC not induced, undifferentiated; (B) MSC 21 days after induction with adipogenic medium 100× magnification.

Figure 2

Figure 2

Expression view of all clusters calculated by _k-_means clustering. _K_-means clustering was performed for 1108 selected genes/ESTs shown to be more than two fold up or down regulated in at least one time point during osteogenic differentiation. Genes were grouped in 12 clusters with distinct expression profiles. Relative expression levels (log2 ratios) are shown for each gene at different time points and for the mean expression values (magenta line).

Figure 3

Figure 3

Summary of osteogenic development; a) Graphical summary of the 3 phases of osteogenic development merged with the time scale; b) Chosen clusters after _k_-means clustering describing each developmental phase merged with the expression matrix of distinct key regulators.

Figure 4

Figure 4

Distribution of gene ontology (GO) terms regarding biological processes for genes in each cluster. The GO terms listed here are those present in at least 5% of the genes within the cluster. In brackets are the number of genes with associated GO terms and the number of genes within the cluster.

Figure 5

Figure 5

Illustration of the TGF-β signaling pathway derived from KEGG database with mapped genes according to their expression level (at least two fold up or down regulated); green = down-regulated genes, red = up-regulated genes and yellow = unregulated genes; mapped genes are at least two-fold differentially expressed.

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