M.Sc Thesis

M.Sc StudentNovak-Petraro Atara
SubjectProteomic Profiling of Human Embryonic Stem Cells in Early
Differentiation Stages
DepartmentDepartment of Medicine
Supervisors PROFESSOR EMERITUS Joseph Itskovitz
DR. Michal Amit
DR. Tamar Ziv
Full Thesis textFull thesis text - English Version


Embryonic stem cells (ESCs) are cells derived from the inner cell mass of the blastocyst, and have dual abilities to differentiate into the three primary germ layers or to remain undifferentiated and proliferative over extended periods of time. The pathways involved in the maintenance of human ESCs as undifferentiated cells remain unknown. Since these pathways are governed by protein networks, determination of the proteome fingerprint of stem cells is a valuable prerequisite for future proteomic and metabolic investigation of expansion and differentiation processes. The molecular basis which enables ESCs to differentiate is a complex process that involves alteration of protein expression patterns resulting from changes in gene expression as well as changes in protein regulatory events associated with translation and post translation modifications. Proteomic approaches are therefore required to visualize and interpret the intricate cell profile changes associated with undifferentiated cells and with the process of differentiation.  In this study, we provide the first quantitative protein profile of human ESCs (hESCs) in undifferentiated state versus early differentiated state. iTRAQ labeling combined with two dimentional capillary chromatography coupled to MS was used to achieve a comprehensive quantitative proteomics of undifferentiated hESCs and of hESCs 6, 48 and 72 hours after initiation of differentiation. In addition 2-DE coupled with LC-MS/MS on QTOF was performed on undifferentiated hESCs and differentiated hESCs at eleven time points (0.5, 1, 1.5, 2, 3, 4, 5, 6, 24, 48 and 72 hours after initiating differentiation) to identify changes in the protein expression profile that occur in the early steps of differentiation. The results indicate that the time point of 48 hours is a turning point. About 70% of the proteins tested, and whose expression level had changed after the beginning of the differentiation process demonstrated a turning point at the 48hr time point. Out of the 164 differentially expressed proteins, 68% showed a tendancy of descent. Specific changes were observed in protein fingerprint of 2DE experiments between the tested time points, and twenty spots were identified by MS. Moreover, several selected proteins were confirmed to possess differential expression levels in hESCs and in differentiated hESCs using western blot analysis. The western blot analysis results further confirmed our findings.

At the first 48 hours after the differentiation initiation we could observe cellular processes such as: chromatin remodeling, cytoskeleton remodeling, a decrease in expression of glycolysis proteins, a decrease in transcription and translation and a low rate of mitosis.