In the body, cells grow in the extracellular matrix (ECM), which presents biochemical and mechanical signals to the cells inside. Hydrogel biomaterials are water-laden polymer networks that can mimic the properties of the ECM, allowing controlled study of cellular behavior in vitro. Many cells are mechanosensitive, but mechanical cues other than stiffness have not been fully investigated. This project aims to develop a platform in which degradability and strain can be activated by a researcher bio-orthogonally. We have synthesized a cyclic peptide crosslinker for a synthetic poly(ethylene glycol) hydrogel that acts as a Boolean AND-gate: one half is degradable by cell-secreted enzymes, and the other half is degradable by sortase, a bacterial enzyme, added by a researcher. We quantified the degradation of hydrogels made with this crosslink via fluorescence release and demonstrated that degradation only occurs after exposure to both enzymatic inputs. We further demonstrated that cells encapsulated in this material retain strong viability. We predict that cells will be unable to spread in this material until after a researcher adds sortase. After sortase addition, we expect that contractile cells will be able to locally degrade the material, spread, and generate strain. We intend to quantify spreading and strain with encapsulated fibroblasts. We also plan to use this platform to study development, by encapsulating immature cardiac stem cells and investigating the effect of fibroblast driven strain as a model; we predict that strain will trigger further specification of these immature cells. In addition to understanding the pathways for development, this research may help identify new therapeutic targets for disease, and it will also inform new strategies to grow tissue in vitro that more closely mimic the native environment.

Across a variety of signaling pathways, soluble factors in the extracellular matrix bind to protein receptors that span the cell wall, thereby triggering an information cascade that affects cell activity or function. It follows that by controlling the binding of signaling factors to these receptors, cell behavior and activity can be guided with substantial precision. In this project, we aim to design a system that allows de novo-developed protein agonists and antagonists, referred to as binders, to be activated with a high degree of temporal and spatial control within cell-encapsulating hydrogels. Towards this end, we employ methods derived from protein semisynthesis and click chemistry to tether binders to the hydrogel polymer network and then subsequently photo-release them from the network. We expect a difference in the functionality of binders when they are bound to the network compared to when they are released through light exposure and solubilized, thus achieving light-dependent control of the binder-receptor interaction and cell activity. This system will be the first to employ de novo developed agonist and antagonist biomolecules for the interrogation and control of cellular behavior. In so doing, it will expand the tool box of biomaterial engineering to include finer control over cells grown in 3D matrices, with direct implications in fields as diverse as therapeutic development, regenerative medicine, and organ-on-a-chip engineering.

Regenerative medicine compromises to repair and replace cells, tissues, and organs damaged by disease or aging. To control cell fate in regenerative medicine, methods enabling irreversible and spatiotemporally controlled protein activation would be beneficial, particularly to those that could be applied for both inter- and extracellular activation. Furthermore, an ideal strategy could be applied to virtually any protein and afford rapid activation. In my work, I have sought to develop and exploit such a method through protein photochemistry; in response to mild and cytocompatibile light exposure, user-specified proteins are irreversibly assembled into their bioactive form. I have optimized the processes for hydrogel formation and modifications to increase cell viability. Results further inform that I can biochemically customize the landscape both intra- and extra-cellularly with a photoactivatable mCherry construct. Moving forward, I intend to apply this technique to activate epidermal growth factors and other proteins in multiple physiological systems. Successful protein photoactivation provides a potential, less invasive mechanism for controlling cells in the extracellular matrix for tissue engineering and regenerative medicine.

The extra cellular matrix (ECM) is a complex, heterogenous environment that plays an important role in cellular functions such as proliferation, signaling, movement, and differentiation. The mechanical properties of the ECM vary spatially and temporally, across and within tissues, i.e., during development and disease progression. 3D biomaterial platforms, such as hydrogels – water-swollen polymeric networks—provide a greater understanding of matrix-cell interactions and can be used to study drug delivery and basic disease mechanisms. My research works to create a double network (DN) hydrogel system that allows for spatial control of ECM mechanics in 3D. Our system contains two different polymer networks, one of which uses light polymerization. I have optimized concentrations of multiple gel components and gel light exposure conditions to allow for accurately patterned stiffnesses within the gels. Currently, I am encapsulating live cells to study the amount of cell spreading and movement in the stiff and soft regions of the gels over the course of a week. I then fix, stain, and image each gel to quantify relative cellular spreading. Additionally, I have synthesized multiple components necessary for gel formation, cultured enzyme producing bacteria to degrade formed gels, and performed western blotting to analyze cellular protein concentrations. Imaging results have shown the DNs and the patterning process are cytocompatible. Current experiments have shown differences in fibroblast spreading between stiff and soft regions; future results are expected to show differences in protein expression within mechanosensitive pathways between patterning conditions. Using multiple, intertwined hydrogel networks, I have engineered a dynamic, heterogenous model of the ECM, enabling me to study cellular responses to mechanical stimuli. Accurate modeling of the ECM will allow for a better understanding of how diseases such as breast cancer progress based on differences in environmental stiffness and provide an in vitro platform for future cellular response research.

Oral tolerance to antigens is a mechanism by which immune responses are inhibited to prevent chronic inflammation and tissue damage in response to common exposures, such as dietary antigens or commensal bacteria. Regulatory T cells (Tregs) are an important cell type in such regulation of immune responses, especially in the intestines. There is, however, heterogeneity within Tregs, including a subset that expresses the transcription factor RORgt. However, the mechanisms by which RORgt+ Tregs carry out their suppressive function are currently unknown. Intestinal Tregs are induced in the mesenteric lymph nodes (MLNs) by antigen-presenting dendritic cells (DCs) that migrate from the gut. Antigen transfer from DCs to Tregs is crucial for the development of oral tolerance and DCs are largely regarded as being upstream of Tregs. However, it has also been shown that Tregs play a role in DC migration via a CTLA-4-mediated mechanism. Because this DC-Treg relationship is not fully understood with regard to Treg heterogeneity, I have examined the changes in DC populations in a mouse model where the RORgt+ Treg population alone has been ablated. To do this, I developed a new panel of antibodies to use in flow cytometry in order to characterize the subpopulations of DCs in the intestines and related organs. I harvested and processed murine spleens, MLNs, Peyer's Patches, and small intestine lamina propria in order to compare the populations systemically and locally. I anticipate seeing fewer DCs in the mice lacking RORgt+ Tregs and more DCs in the small intestine and Peyer's patches. This work furthers our understanding of the intricacies of the intestinal immune system. This knowledge can be applied to vaccine research, as RORgt+ Tregs have been implicated as suppressors of immune response to oral vaccines. Intestinal immunity is also of interest in allergy and gut inflammation research.

The health of Sockeye salmon (Oncorhyncus Nerka) stocks are of high importance to the cultural well-being and sovereignty of Coast Salish tribes. There are multiple ecotypes of Sockeye that include anadromous, potamodromous Kokanee, and resident Sockeye that all carry distinct and significant cultural value for Indigenous communities, as well as distinct ecological roles within the ecosystems they inhabit. Sockeye are impacted by anthropogenic stressors such as habitat degradation and pollution, which can cause shifts in food web dynamics, population declines, and impact commercial and traditional fisheries. To more effectively manage these diverse life histories for conservation, it is crucial to understand the distinct ecological functions that may cause ecotypes to differ in their vulnerability to anthropogenic stressors, thus requiring different management strategies. We will analyze carbon and nitrogen isotopic signatures of Sockeye from Lake Washington to differentiate their ecological niches. Muscle samples were collected from 46 frozen sockeye samples, freeze dried, and then analyzed using mass spectrometry. Preliminary results suggest that there are significant differences between the isotopic signatures of anadromous and potamodromous ecotypes. We hypothesize further interpretation of the results coupled with genetic analysis will identify differing ecological roles in accordance with the diverse life history strategies Lake Washington Sockeye display. This study has been conducted as part of ongoing collaborative efforts with partners at the Snoqualmie Tribe, and the Kokanee Work Group, which aims to restore the Kokanee and Sockeye populations of Lake Sammamish. The results of this work will directly inform management actions taken by our partners to conserve the native Sockeye populations of King County, Washington.

Risk variants of apolipoprotein L1 gene (APOL1) increase the risk of chronic kidney diseases in populations of African ancestry. We seek to study this disease, which is unique to humans, in human kidney organoids derived from induced pluripotent stem (iPS) cells. However, APOL1 is not expressed in organoids at baseline. While interferon (IFN)-gamma is a potent inducer of APOL1 expression, our prior experiments suggested that IFN-gamma itself disrupts organoid structures, limiting the degree to which the specific effects of APOL1 can be assessed. To improve the kidney organoid system as a better platform to model APOL1-associated nephropathy without IFN-gamma stimulation, I am establishing an APOL1 inducible expression system in kidney organoids. I hypothesize that cell lines with the risk variants will demonstrate an accelerated rate of degradation compared to the non-risk variant, modeling risk variant-dependent cytotoxicity. The inducible expression system will be established by generating iPS cell lines encoding Tet-On system sequences, enabling both tunable and temporal control of APOL1 expression using doxycycline. Plasmids were constructed by PCR amplifying and inserting the targeted sequences into a homology-dependent repair template with a doxycycline-inducible promoter. Ligation had to be done multiple times due to the high prevalence of self-ligation of the backbone vector and backward insert orientation. However, we found that adding alkaline phosphatase to dephosphorylate 5’ ends of the backbone vector significantly improved the integration rate and led to the successful construction of plasmids. Next, CRISPR-Cas9 gene editing will be utilized to introduce the APOL1 gene variants into the AAVS1 safe harbor locus in iPS cell lines, which can be differentiated into kidney organoids. This project aids in isolating the phenotype of APOL1 on human kidney organoids with various cell types, which will be a valuable tool in developing an in-vitro pathophysiological assay such as use in therapeutic drug discovery.