Implementation of Genetic Circuits for Engineered Mesenchymal Stem Cell Chondrogenic Differentiation

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dc.contributor.advisorBashor, Caleb Jen_US
dc.creatorPiepergerdes, Trenton Cen_US
dc.date.accessioned2023-08-09T18:20:16Zen_US
dc.date.created2023-05en_US
dc.date.issued2023-04-20en_US
dc.date.submittedMay 2023en_US
dc.date.updated2023-08-09T18:20:16Zen_US
dc.description.abstractThe 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.en_US
dc.embargo.lift2024-05-01en_US
dc.embargo.terms2024-05-01en_US
dc.format.mimetypeapplication/pdfen_US
dc.identifier.citationPiepergerdes, Trenton C. "Implementation of Genetic Circuits for Engineered Mesenchymal Stem Cell Chondrogenic Differentiation." (2023) Diss., Rice University. <a href="https://hdl.handle.net/1911/115134">https://hdl.handle.net/1911/115134</a>.en_US
dc.identifier.urihttps://hdl.handle.net/1911/115134en_US
dc.language.isoengen_US
dc.rightsCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.en_US
dc.subjectsynthetic biologyen_US
dc.subjectbioengineeringen_US
dc.subjecttissue engineeringen_US
dc.subjecten_US
dc.titleImplementation of Genetic Circuits for Engineered Mesenchymal Stem Cell Chondrogenic Differentiationen_US
dc.typeThesisen_US
dc.type.materialTexten_US
thesis.degree.departmentBioengineeringen_US
thesis.degree.disciplineEngineeringen_US
thesis.degree.grantorRice Universityen_US
thesis.degree.levelDoctoralen_US
thesis.degree.nameDoctor of Philosophyen_US
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