Proteomics reveals multiple routes to the osteogenic phenotype in mesenchymal stem cells

Abstract

Background: Recently, we demonstrated that human mesenchymal stem cells (hMSC) stimulated with dexamethazone undergo gene focusing during osteogenic differentiation (Stem Cells Dev 14(6): 1608-20, 2005). Here, we examine the protein expression profiles of three additional populations of hMSC stimulated to undergo osteogenic differentiation via either contact with pro-osteogenic extracellular matrix (ECM) proteins (collagen I, vitronectin, or laminin-5) or osteogenic media supplements (OS media). Specifically, we annotate these four protein expression profiles, as well as profiles from naïve hMSC and differentiated human osteoblasts (hOST), with known gene ontologies and analyze them as a tensor with modes for the expressed proteins, gene ontologies, and stimulants.

Results: Direct component analysis in the gene ontology space identifies three components that account for 90% of the variance between hMSC, osteoblasts, and the four stimulated hMSC populations. The directed component maps the differentiation stages of the stimulated stem cell populations along the differentiation axis created by the difference in the expression profiles of hMSC and hOST. Surprisingly, hMSC treated with ECM proteins lie closer to osteoblasts than do hMSC treated with OS media. Additionally, the second component demonstrates that proteomic profiles of collagen I- and vitronectin-stimulated hMSC are distinct from those of OS-stimulated cells. A three-mode tensor analysis reveals additional focus proteins critical for characterizing the phenotypic variations between naïve hMSC, partially differentiated hMSC, and hOST.

Conclusion: The differences between the proteomic profiles of OS-stimulated hMSC and ECM-hMSC characterize different transitional phenotypes en route to becoming osteoblasts. This conclusion is arrived at via a three-mode tensor analysis validated using hMSC plated on laminin-5.

Figure 1

Directed Component scores. The directed component score ranks the samples (experiments) from stem cells to osteoblasts.

Figure 2

Directed Component plots of hMSC, hOST, and stimulated stem cells. Directed Component plots of hMSC, hOST, and stimulated stem cells demonstrate distinct lineages of ECM-hMSC vs. OS-hMSC.

Figure 3

Weights on the GOs for the directed and second components. Weights on the GOs are plotted for the directed and second components. GOs falling outside the circle of standard deviation are critical for the components.

Figure 4

Scatter plot of GeneIDs. 361 GeneIDs are projected onto the space spanned by the first and third components in GeneID mode. Red color and diamond shape are used to indicate the GeneIDs considered significant and examined closely. The rest of the GeneIDs have comparatively low loading coefficients and areconsidered insignificant.

Figure 5

Three-way Proteomics Array and Tucker3 Model. Proteomics Array with GeneID, Category, and Sample modes is modeled with [P Q R] – Tucker3 model. A, B, and C are component matrices corresponding to GeneID, Category, and Sample modes, respectively. G is the core array and E contains the residuals for each entry in X.