SUMMARY

Due to their characteristic properties of self-renewal and differentiation, stem cells hold the capacity to serve as phenotype-specific cell factories for various regenerative medicine and tissue engineering applications. However, current phenotyping techniques, which typically employ multiple surface protein-specific antibodies, are often insufficient to identify or enrich cells of a target phenotype. An improved technique that could select target cells could be used to purify starting cell populations for directed differentiation protocols or to enrich specific terminally differentiated phenotypes for tissue engineering. The goal of this project is to investigate cellular mechanical parameters as stem cell phenotype markers to complement the currently available biomolecular markers. This objective was accomplished through 1) the establishment of cell stiffness as a single-cell marker of potency in both mesenchymal stem cells, which give rise to cells of the connective tissue, and limbal stem cells, which replenish the cornea, 2) the development of a method to compare cell mechanics and gene expression at the single-cell level, which will enable more detailed studies of the relationships between cell phenotype, mechanics, and structure, and 3) the determination that pluripotent embryonic stem cells, which are softer than their differentiated progeny, can be enriched using a cell stiffness-based microfluidic sorting device, as assessed by potency-related morphological and genetic factors. Ultimately, this project established cell stiffness as a marker of stem cell differentiation in various cell systems, with applications to label-free selection of target cell phenotypes.