Browsing by Author "Bashor, Caleb J"

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    Implementation of Genetic Circuits for Engineered Mesenchymal Stem Cell Chondrogenic Differentiation
    (2023-04-20) Piepergerdes, Trenton C; Bashor, Caleb J
    The ability to culture stem cells and guide their differentiation has allowed myriad advancements in developmental biology and cell-based therapies. In vitro differentiation protocols have historically taken an “outside-in” approach where bioactive molecules (purified proteins, small molecules, etc.) are added exogenously to culture medium or functionalized onto culture surfaces (plates, scaffolds, etc.) to guide cell differentiation. Despite decades of protocol optimization, “outside-in” approaches result in heterogeneous populations where only a subset of cells matches the desired phenotype, and the presence of aberrant cell states makes the cells ineffective for therapeutic use. These shortcomings are particularly evident in cartilage tissue engineering, where the multipotency of mesenchymal stem cells (MSCs) is harnessed to regenerate cartilage tissue. In these therapies, MSCs produce areas of proper cartilage, but concurrently produce hypertrophic and fibrotic chondrocytes. These cells deposit bone and fibrous tissue, respectively, thus leading to suboptimal tissue properties and limiting clinical translation. The ability to encourage proper chondrogenic phenotypes while preventing undesired hypertrophic and fibrotic ones is thus of great interest to tissue engineers and developmental biologists alike. One promising strategy involves using synthetic gene circuits to precisely control the dose and timing of expression of genes critical to functional chondrogenic differentiation. This “inside-out” approach is inspired by natural cellular differentiation, where it has been demonstrated that precise timing and magnitude of expression of genes in key regulatory networks are responsible for driving differentiation to mature cell states. In this work, I developed a novel engineering platform that enabled this functionality with synthetic genetic circuits. The platform I created includes a novel framework for the quantitative design and implementation of genetic circuits in a variety of cell types paired with an in vitro model and assessment method using single cell RNA sequencing (scRNA-seq) that allows iterative circuit implementation and assessment of the effect of circuit function on cell phenotypes in a model of MSC chondrogenesis. This work represents a significant step forward for the fields of mammalian synthetic biology and tissue engineering by (1) allowing high throughput circuit design, creation, and implementation in mammalian cells and (2) providing an unprecedented description of chondrogenic differentiation trajectories and how to manipulate them.
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    pYtags enable spatiotemporal measurements of receptor tyrosine kinase signaling in living cells
    (eLife Sciences Publications Ltd., 2023) Farahani, Payam E; Yang, Xiaoyu; Mesev, Emily V; Fomby, Kaylan A; Brumbaugh-Reed, Ellen H; Bashor, Caleb J; Nelson, Celeste M; Toettcher, Jared E
    Receptor tyrosine kinases (RTKs) are major signaling hubs in metazoans, playing crucial roles in cell proliferation, migration, and differentiation. However, few tools are available to measure the activity of a specific RTK in individual living cells. Here, we present pYtags, a modular approach for monitoring the activity of a user-defined RTK by live-cell microscopy. pYtags consist of an RTK modified with a tyrosine activation motif that, when phosphorylated, recruits a fluorescently labeled tandem SH2 domain with high specificity. We show that pYtags enable the monitoring of a specific RTK on seconds-to-minutes time scales and across subcellular and multicellular length scales. Using a pYtag biosensor for epidermal growth factor receptor (EGFR), we quantitatively characterize how signaling dynamics vary with the identity and dose of activating ligand. We show that orthogonal pYtags can be used to monitor the dynamics of EGFR and ErbB2 activity in the same cell, revealing distinct phases of activation for each RTK. The specificity and modularity of pYtags open the door to robust biosensors of multiple tyrosine kinases and may enable engineering of synthetic receptors with orthogonal response programs.