Mechanisms of Biophysical Effects on Differentiation of Mesenchymal Stem Cells and Real-Time Evaluation of Biophysical Effects with Surface Plasmon Resonance Technique

During differentiation, linage-determining transcription factors are up-regulated to initiate differentiation at the very early stage of induction. However, besides transcriptional regulation, biophysical effects through cytoskeleton reorganization, cell-cell interaction or cell-matrix interaction also participate in the direction of cell fate commitment and lineage determination. During early osteogenic differentiation of human MSCs, remarkable changes of biophysical cues were observed including cytoskeleton reorganization, integrin redistribution and change of membrane tether length.Among all the components of cytoskeleton, actin filament (F-actin) plays the central role to modulate biophysical cues at the early stage of osteogenic differentiation. In our previous study, we found that thymosin beta-4 (T4), a G-actin sequestering peptide, altered cell fate determination of MSCs through biophysical effects without direct regulation of linage-determining transcription factor. T4 facilitated adipogenic differentiation accompanied with upregulation of adipocyte adhesion molecule and inhibited osteogenic differentiation through actin filament reorganization, indicating that cell fate choice can be initiated via biophysical effect rather than transcriptional regulation. So far, it is not clear how actin filament reorganization results in regulation of lineage-determining transcription factors. The aim of this study is to elucidate mechanisms of osteogenic and adipogenic fate determination induced by actin filament reorganization on mesenchymal stem cells. In this study, actin polymerization enhancer and inhibitor will be used to explore the mechanisms of actin filament reorganization on MSC differentiation. F-actin/G-actin ratio at different time point of induction will be measured. Bone morphogenetic protein (BMP) pathway, WNT pathway, extracellular signal-regulated kinases (ERK) pathway, Rho/Rho kinase (ROCK) and matrix metalloproteinase (MMP) pathway will be thoroughly investigated. Moreover, during differentiation, real-time image will be recorded by a biosensor using surface plasmon resonance (SPR) technique to trace the actin filament reorganization. SPR is a non-invasive, time-saving and fluorescence-staining free real-time image system with high sensitivity. In this study, modified SPR technique which possesses extra-high sensitivity and specificity will be used; it enables us to record the change of membranous and peripheral cytosolic biophysical effect induced by cytoskeleton reorganization on MSC differentiation non-invasively in a real-time fashion. Similar experiments have not yet been found in the literature and the originality of the study is justified. Importantly, besides mechanisms of biophysical effects on differentiation of mesenchymal stem cells, the platform of SPR technique will serve as an important platform technology to enable non-invasive, real-time, quantitative measurement of the effects of physical and biological stimuli on cell fate determination of MSCs.