Comparing the Protein Expression Profiles of Human Mesenchymal Stem Cells and Human Osteoblasts Using Gene Ontologies

Abstract

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.

Laminin-5 activates extracellular matrix production and osteogenic gene focusing in human mesenchymal stem cells

“…This focusing is characterized by two patterns: a generalized drop in the number of different proteins/genes expressed during hMSC differentiation to form osteoblasts, and a gain of expression in a subset of functionally related proteins/genes (Salasznyk et al, 2005a(Salasznyk et al, , 2005b. These patterns are reflected in Figure 2: first, the length of the hMSC bars is generally longer than either Ln-5 hMSC or hOST, especially at the top of each panel.…”

“…Cryopreserved hMSC and hOST were routinely passaged as previously described (Salasznyk et al, 2005b). Ln-5 was obtained by growing 804G rat bladder carcinoma cells as previously described (Gospodarowicz, 1984).…”

Focal adhesion kinase signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells

“…Considerable work has focused on the role of integrin-mediated signaling during progression of osteoblast differentiation [15,17,18,32,33], but our work focuses on a much earlier step, when hMSC are still capable of differentiating into multiple cell types. Clearly, a different set of decisions are being made by hMSC at this earlier step of osteogenesis, in that down-regulation of adipogenic, chondrogenic, neural, and muscular genes is occurring at the same time that osteogenic genes are being upregulated [34,35].…”

Protein Expression Profiles during Osteogenic Differentiation of Mesenchymal Stem Cells Derived from Human Umbilical Cord Blood

“…In our study, expression of hnRNP H, which is associated with adhesion (de Hoog et al 2004), increased 37-fold during the proliferation phase. Salasznyk et al (2005) used 2-D PAGE and 2-D LC/ MS/MS to characterize protein expression patterns in MSCs and mineralized-matrix-producing osteoblasts (OSTs). Among 737 spots identified by 2-D PAGE, only 257 (34%) were observed in both cells, 312 spots were observed in OSTs only, and 168 in MSCs only.…”