A. James Clark School of Engineering
Permanent URI for this communityhttp://hdl.handle.net/1903/1654
The collections in this community comprise faculty research works, as well as graduate theses and dissertations.
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Item Combinatorial microRNA Loading into Extracellular Vesicles for Increased Anti-Inflammatory Efficacy(MDPI, 2022-10-21) Pottash, Alex Eli; Levy, Daniel; Jeyaram, Anjana; Kuo, Leo; Kronstadt, Stephanie M.; Chao, Wei; Jay, Steven M.Extracellular vesicles (EVs) have emerged as promising therapeutic entities in part due to their potential to regulate multiple signaling pathways in target cells. This potential is derived from the broad array of constituent and/or cargo molecules associated with EVs. Among these, microRNAs (miRNAs) are commonly implicated as important and have been associated with a wide variety of EV-induced biological phenomena. While controlled loading of single miRNAs is a well-documented approach for enhancing EV bioactivity, loading of multiple miRNAs has not been fully leveraged to maximize the potential of EV-based therapies. Here, an established approach to extrinsic nucleic acid loading of EVs, sonication, was utilized to load multiple miRNAs in HEK293T EVs. Combinations of miRNAs were compared to single miRNAs with respect to anti-inflammatory outcomes in assays of increasing stringency, with the combination of miR-146a, miR-155, and miR-223 found to have the most potential amongst the tested groups.Item Induced Pluripotent Stem Cell-Derived Extracellular Vesicles Promote Wound Repair in a Diabetic Mouse Model via an Anti-Inflammatory Immunomodulatory Mechanism(Wiley, 2023-06-19) Levy, Daniel; Abadchi, Sanaz Nourhammadi; Shababi, Niloufar; Ravari, Mohsen Rouhani; Pirolli, Nicholas H.; Bergeron, Cade; Obiorah, Angel; Mokhtari-Esbuie, Farzad; Gheshlaghi, Shayan; Abraham, John M.; Smith, Ian M.; Powsner, Emily H.; Solomon, Talia J.; Harmon, John W.; Jay, Steven M.Extracellular vesicles (EVs) derived from mesenchymal stem/stromal cells (MSCs) have recently been explored in clinical trials for treatment of diseases with complex pathophysiologies. However, production of MSC EVs is currently hampered by donor-specific characteristics and limited ex vivo expansion capabilities before decreased potency, thus restricting their potential as a scalable and reproducible therapeutic. Induced pluripotent stem cells (iPSCs) represent a self-renewing source for obtaining differentiated iPSC-derived MSCs (iMSCs), circumventing both scalability and donor variability concerns for therapeutic EV production. Thus, it is initially sought to evaluate the therapeutic potential of iMSC EVs. Interestingly, while utilizing undifferentiated iPSC EVs as a control, it is found that their vascularization bioactivity is similar and their anti-inflammatory bioactivity is superior to donor-matched iMSC EVs in cell-based assays. To supplement this initial in vitro bioactivity screen, a diabetic wound healing mouse model where both the pro-vascularization and anti-inflammatory activity of these EVs would be beneficial is employed. In this in vivo model, iPSC EVs more effectively mediate inflammation resolution within the wound bed. Combined with the lack of additional differentiation steps required for iMSC generation, these results support the use of undifferentiated iPSCs as a source for therapeutic EV production with respect to both scalability and efficacy.Item Assessment of Mechanical Cues to Enhance the Clinical Translation of Extracellular Vesicles(2022) Kronstadt, Stephanie Marie; Jay, Steven M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Mesenchymal stem cells (MSCs) are a common source for cell-based therapies due to their innate regenerative properties. However, these cells often die shortly after injection and, if they do survive, run the risk of forming tumors. Cell-secreted nanoparticles known as extracellular vesicles (EVs) have been identified as having therapeutic effects similar to those of their parental cells without the safety risks. Specifically, MSC EVs have emerged as a promising therapeutic modality in a multitude of applications, including autoimmune and cardiovascular diseases, cancer, and wound healing. Despite this promise, low levels of naturally occurring EV cargo may necessitate repeated doses to achieve clinical benefit, countering the advantages of EVs over MSCs. The current techniques to combat low EV potency (e.g., loading external molecules or using chemicals) are not agreeable to large-scale manufacturing techniques and would substantially increase the regulatory burden associated with EV translation. Fortunately, mechanical cues within the microenvironment have potential to overcome these translational barriers as they can alter EV therapeutic effects but are also cost-effective and can be precisely manipulated in a reproducible manner. The goal of this project is to understand how these cues impact MSC EV secretion and physiological effects. We showed that flow-derived shear stress applied to MSCs seeded within a 3D-printed scaffold (i.e., the bioreactor) can significantly upregulate EV production (EVs/cell) while maintaining the in vitro pro-angiogenic effects of MSC EVs. Interestingly, we demonstrated that MSC EVs generated using the bioreactor system significantly improved wound healing in a diabetic mouse model, with increased CD31+ staining in wound bed tissue compared to animals treated with flask cell culture-generated MSC EVs. Furthermore, for the first time, we showed that mechanical confinement of MSCs within micropillars could augment MSC EV production and bioactivity. Lastly, we demonstrated that soft substrates composed of various polydimethylsiloxane (PDMS) formulations could increase MSC EV production and activity as well. Through the work performed here, we have laid the groundwork to elucidate the relationship between cell mechanobiology and EV activity that will ultimately enable an adaptable and scalable EV therapeutic platform.