Research Project
10291544
PROJECT SUMMARY Tissue and organ failure, either due to injury or aging, are becoming a major health problem worldwide with an estimated cost of one-half of the total annual healthcare expenses. To address this issue, tissue engineering approaches can be leveraged by utilizing functional body cells created in a laboratory setting. Pluripotent Epiblast Stem Cells (EpiSCs) can serve as an excellent model to determine how to direct cell fate for creating functional body cells. However, even with the best chemically-defined differentiation protocol of pluripotent stem cells, the control of cell-lineage specification remains poor. Besides chemical signaling, it is now widely accepted that physical signals from the extracellular matrix (ECM) play a crucial role in cell fate determination. Nevertheless, control of cell-lineage specification by such mechanical forces alone could not be improved possibly due to the lack of precise control of forces at the single-molecule level and the lack of synergy between chemical signaling and mechanical pathways. To address this gap, the proposed study aims to provide a mechanistic framework of single EpiSC fate decisions (self-renewal and differentiation) based on chemical and single-molecule force based approaches. The central hypothesis is that the synergistic effect of chemical and single-molecule force cues via cell-ECM and cell-cell interactions can control fate decisions far more effectively than previously possible. The long-term goal is to develop novel approaches to control the directed differentiation of pluripotent cells into all three germ-layers. To this end, the following three aims are proposed. Specifically, Aim 1 will focus on understanding the mechanism of single-molecule force mediated differentiation of EpiSCs into the mesoderm lineage. The force transmission into single cells via single ?v?3 integrins will be controlled by tension gauge tethers. These DNA-based rupturable tethers can precisely limit the amount of force at the single-molecule level. Together with chemical signaling, such precise control and specific targeting of mechanical pathways may lead to superior control of cell differentiation into the mesoderm. In Aim 2, the mechanism of self-renewal of single EpiSCs will be identified by defining a microenvironment composed of self-renewal promoting ligands such as E-cadherin. In Aim 3, differentiation of single EpiSCs will be defined via the Notch pathway by engineered low- tolerance tension gauge tether called ?nano-yoyo? to activate force-dependent Notch signaling. The proposed work will elucidate detailed molecular, chemical, and mechanical pathways that contribute to specific lineage commitments. Finally, three undergraduate and two graduate students will gain research experience in rigorous and intensive research in the areas of stem cells, cell mechanics, and biophysics. Students will conduct experiments, analyze and summarize data, and prepare manuscripts simultaneously advancing the proposed scientific agenda.
