Single-molecule approaches to study epiblast stem cell fate decision - PROJECT SUMMARY The goal of this proposal is to engineer viscoelastic substrates with embedded ‘Cell-zoo’ structures for efficient mesodermal differentiation and further elucidate the underlying mechanism using RNA sequencing. Tissue and organ failure, whether caused by accidents, sports injury, or aging, has become a significant health challenge in the US, accounting for half of the total annual healthcare costs. To tackle this issue, tissue engineering strategies can be employed by generating functional tissues in a laboratory environment. To provide an unlimited supply of cells for engineered tissues, pluripotent stem cells (mouse embryonic stem cells and mouse epiblast stem cells) can serve as outstanding models to determine how to efficiently direct cell fate for creating functional body cells. In addition to conventional chemical approaches, current mechanical approaches to direct stem cell differentiation struggle with poor control of cell lineage specification, which leads to a heterogeneous cell population at the end. To address this gap, this proposal aims to systematically integrate chemical and physical microenvironment-based approaches to drive lineage-specific cell differentiation. The preliminary data shows that mouse embryonic stem cells on viscoelastic substrates, engineered through additive manufacturing that mimic the living viscoelastic tissue properties, can direct lineage-specific mesodermal differentiation with excellent efficiency. The central hypothesis is that mimicking native tissue properties, especially viscoelasticity, via additive manufacturing can enable controlled stem cell differentiation. The long-term goal is to develop novel strategies for controlling the directed differentiation of pluripotent cells into all three germ-layers. To this end, the following three aims are proposed. The project's three aims include determining the effect of viscoelasticity and extracellular matrix contributions on mesoderm-specific lineage commitment (Aim 1), determining the effect of surface topology with geometric cues (“Cell-zoo”), using additive manufacturing, on mesoderm-specific lineage commitment (Aim 2), and identifying the mechanism of viscoelasticity-driven mesodermal differentiation using RNA sequencing (Aim 3). The project is innovative because it mimics the physiological microenvironment of native tissues using an additive manufacturing technique developed in PI’s lab, creates viscoelastic substrates with precisely controlled properties combined with geometric cues, synergistically integrates chemical signaling with the mechanical microenvironmental cues, and uses interdisciplinary approaches to determine the mechanism of cell differentiation. The expected outcomes include a clear mechanistic understanding of cell fate decisions and advancements in cell biomanufacturing. Additionally, three undergraduate and two graduate students will be exposed to meritorious research spanning stem cells, cell mechanics, additive manufacturing, and next-generation RNA sequencing. These students will actively conduct experiments, analyze data, and prepare manuscripts, contributing to both their academic growth and the advancement of the scientific agenda.
