Comparing the protein expression profiles of human mesenchymal stem cells and human osteoblasts using gene ontologies.

One of the hallmark events regulating the process of osteogenesis is the transition of undifferentiated human mesenchymal stem cells (hMSCs) found in the bone marrow into mineralized-matrix producing osteoblasts (hOSTs) through mechanisms that are not entirely understood. With recent developments in mass spectrometry and its potential application to the systematic definition of the stem cell proteome, proteins that govern cell fate decisions can be identified and tracked during this differentiation process. We hypothesize that protein profiling of hMSCs and hOSTs will identify potential osteogenic marker proteins associated with hMSC commitment and hOST differentiation. To identify markers for each cell population, we analyzed the expression of hMSC proteins and compared them to that of hOST by two-dimensional gel electrophoresis and two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS). The 2D LC-MS/MS data sets were analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Only 34% of the spots in 2D gels were found in both cell populations; of those that differed between populations, 65% were unique to hOST cells. Of the 755 different proteins identified by 2D LCMS/ MS in both cell populations, two sets of 247 and 158 proteins were found only in hMSCs and hOST cells, respectively. Differential expression of some of the identified proteins was further confirmed by Western blot analyses. Substantial differences in clusters of proteins responsible for calcium- based signaling and cell adhesion were found between the two cell types. Osteogenic differentiation is accompanied by a substantial change in the overall protein expression profile of hMSCs. This study, using gene ontology analysis, reveals that these changes occur in clusters of functionally related proteins. These proteins may serve as markers for identifying stem cell differentiation into osteogenic fates because they promote differentiation by mechanisms that remain to be defined.

Focusing of gene expression as the basis of stem cell differentiation.

In a prior report (Stem Cells Dev 14(4):354-366, 2005), we employed two-dimensional gel electrophoresis followed by advanced proteomics and the Database for Annotation, Visualization and Integrated Discovery (DAVID) to compare the protein expression profiles of mesenchymal stem cells to that of fully differentiated osteoblasts. These data were reported to advance technical approaches to define the basis of differentiation, but also led us to suggest that osteogenic differentiation of stem cells may result from the focusing of gene expression in functional clusters (e.g., calcium-regulated signaling proteins or adherence proteins) rather than simply from the induced expression of new genes, as many have assumed. Here, we have employed these analytical techniques to compare protein expression by mesenchymal stem cells directly with that of cells derived from them after induced osteogenic differentiation. Our results support the concept of gene focusing as the basis of differentiation. Specifically, induced differentiation results in a decrease in the number of mesenchymal cell markers and calcium-mediated signaling molecules expressed by their differentiated progeny. This effect was seen in parallel to increased expression of specific extracellular matrix (ECM) molecules and their receptors. These results strongly imply that changes in the ECM have a direct impact on stem cell differentiation, and that osteogenic differentiation of stem cells directed by matrix clues results from focusing of the expression of genes involved in Ca2+-dependent signaling pathways.