Proteomic Profiling of Stem Cell Differentiation

点击翻译按钮获取中文摘要

DESCRIPTION (provided by applicant): Bone marrow mesenchymal stem cell (BMMSC) has potential to differentiate into different cell types. The long-term goal of this research is to control BMMSC differentiation in vitro and use BMMSC-derived smooth muscle cell (SMC) to construct tissue-engineered vascular grafts. The cyclic mechanical strain in the vessel wall and transforming growth factor 13(TGF-B) play important roles in SMC differentiation and vascular remodeling. The investigator has shown that both mechanical strain and TGF-I31 increase the expression of SMC markers in BMMSC. However, the underlying mechanisms and the proteomic changes of BMMSCs during differentiation are not well understood. Proteomic profiling provides a systematic and powerful approach to studying stem cell functions. The investigator hypothesizes: (1) BMMSC and its differentiation can be characterized by the expression of specific protein markers, and (2) mechanical strain and TGF-B] regulate BMMSC differentiation through distinct mechanisms and synergize the differentiation of BMMSCs into SMCs. Two specific aims are proposed in this exploratory/development study. In Aim 1, the investigator will determine the proteomic profile of BMMSC and identify potential markers of BMMSC. TGF-B] and other differentiation factors will be used to induce the proteome changes and differentiation of BMMSC. A comprehensive strategy using state-of-the-art technologies will be employed for proteome analysis. 2D gel electrophoresis and multidimensional liquid chromatography will be used for protein/peptide separation. Mass spectrometry will be used for protein identification and characterization. A reference map of BMMSC will be generated. The proteins only expressed in undifferentiated BMMSC will be identified. In Aim 2, the investigator will determine the proteome changes in BMMSC in response to mechanical strain and TGF-B. The proteins with changes in expression and post-translational modifications will be identified. Immobilized metal affinity capture will be employed for phosphopeptide enrichment. The global mechano-chemical signal transduction during BMMSC differentiation will be determined, and a map will be generated for the signaling pathways differentially regulated, shared, and synergized by mechanical strain and TGF-B]. Categorization and cluster analysis will be performed to correlate the proteins and signaling pathways. This study will advance our knowledge on BMMSC proteome and differentiation, and lead to more focused and in-depth biological studies in the future. The successful accomplishment of the goals will have high impact on stem cell engineering, and provide a rational basis for engineering BMMSC for vascular tissue repair.