Master's Theses

Date of Award

2011

Document Type

Thesis

Department

Biomedical Engineering

Keywords

Serial Subculture, Hmscs aging, Proteomics

Abstract

"Human mesenchymal stem cells (hMSCs) hold great potential for clinical applications in regenerative medicine. However, hMSCs occur at a very low frequency in bone marrow so the clinical use as cellular therapeutics requires suitable techniques for isolation and expansion. Furthermore, these cells can be expanded in vitro for only limited passage numbers before they reach a senescent state. Previous studies have shown that long term culture of hMSCs results in the decrease in proliferation rates, changes in genomic expressions, increase in cell sizes, and other morphological changes. Currently the mechanisms of hMSC aging and biomarkers associated with it have not been well either understood or established. The change of hMSC phenotype during serial sub-culture must be taken into account prior clinical applications. In this study, the protein expression profiles of serially sub-cultured hMSCs from passage 3 to passage 9 over 6 weeks were investigated using proteomics approaches. Human MSC proteins were separated using two-dimensional electrophoresis and differentially expressed proteins were identified with PDQuest 8.0 and MALDI-TOF Mass Spectroscopy. The significant differences of protein expression levels were investigated using ANOVA test. It was observed that the cell size also increases in late passages compared to early passages. There was more number of protein dots expressed on pH 4 to 7 gels compared to pH 6-11 gels. Differentially expressed hMSC proteins were normalized and clustered together and histograms were obtained to determine the proteins that follow similar trend in their expression. In addition, image analysis was performed to determine the overall intensity of proteins in each passage. Twenty-one differentially expressed hMSC proteins were successfully identified via Mass Spectroscopy and database searching. It was observed that Calreticulin, Glyceraldehyde-3-phosphate dehydrogenase, and alpha-crystallin B chain were up regulated in protein expression while Moesin (C-terminal frag), ii Poly (rc)-binding protein, Alpha-tropomyosin, Annexin A2, and Galectin 3 were down regulated. Similarly, it was found that Vimentin, Beta actin, Nucleophosmin, and Heat shock protein 70 (Nterminal frag) showed up regulation in protein expression at initial passages and down regulation at late passages while Transgelin, Prolyl 4-hydroxylase subunit alpha-2, Phosphatidylethanolamine-binding protein 1, Fructose-bisphosphate aldolase, and Alpha-enolase showed down regulation in early passages and up regulation in late passages. Such identification allows for the characterization and clarification of biological mechanisms, targets of drugs, and biomarkers that play vital role during cellular aging of hMSCs."

 
 

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