Browsing by Subject "induced pluripotent stem cells"

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    Characterizing Freezing and Thawing Responses of Multiple Types of Cells Cryopreserved in Both DMSO and non-DMSO Cryoprotectants
    (2018-09) Yu, Guanglin
    Cryopreservation is the technology used to stabilize cells at subzero temperature for a variety of applications including diagnosis and treatment of disease, and the production of therapeutic proteins. Current theories of cell damage during freezing were developed in the 1960’s, and little has changed since then. However, our understanding of cell biology as well as tools to interrogate cell responses during freezing has improved in the last 50 years. Low temperature Raman spectroscopy has been used to verify, for the first time using chemical spectra, the presence of ice inside the cell during freezing. With this tool, it is possible to internally observe frozen cells, and identify specific chemical and morphological changes that result in cell life or death. In this work, we propose to use this powerful tool to test two hypotheses to enhance our understanding of the mechanism of cell damage during freezing and thawing, and the manner by which the damage can be mitigated to improve cryopreservation outcome. For the first part of this work, we hypothesize that not all intracellular ice formation (IIF) is lethal and the conditions of cell membrane, cytoskeleton and mitochondria play an important role in determining IIF and cryopreservation outcome. Freezing responses of single cells as well as multi-cellular system cryopreserved and thawed in dimethyl sulfoxide (DMSO) solution will be examined to test this hypothesis. DMSO-free cryopreservation has attracted much recent interest due to the toxicity of DMSO. For the second part of this work, we hypothesis that non-DMSO multicomponent osmolyte solutions can be used to preserve cell viability and one component, disaccharide, acts to protect the cell through multiple interactions. Freezing responses of cells cryopreserved in a combination of non-DMSO cryoprotectants such as sugars, sugar alcohols, and amino acids will be examined. Interactions among sucrose (a typical disaccharide), water and cell membrane at low temperature will be also be investigated in order to test the second hypothesis. Enhancing our understanding of freezing damage and strategies to mitigate damage will improve the methods of preserving cell therapy products and therefore enable the treatment to reach the patients who could benefit from them.
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    Extracellular Matrix Guided Endothelial Differentiation
    (2022-08) Hall, Mikayla
    Cardiovascular disease is the leading cause of death worldwide. Due to recent advances including development of induced pluripotent stem cells, cardiac tissue engineering has emerged as a promising avenue for in vitro drug and device testing as well as eventual transplantation. Nutrient flow presents one of the major challenges to large scale engineered cardiac tissues which is necessary for many of the potential applications of engineered tissues. The lack of nutrient flow could be solved through tissue vascularization which requires endothelial cells lining vessels. The extracellular matrix (ECM) plays a vital role in tissue development and the majority of in vitro differentiation protocols rely on ECM substrates. Here we present two studies which investigate the role of the ECM in endothelial differentiation and the mechanisms activated by ECM engagement during differentiation. First, we investigate the role of individual ECM proteins in endothelial differentiation and elucidate pathways key to how ECM interactions promote differentiation. Second, a Design of Experiments approach was utilized to optimize the ECM composition for endothelial differentiation. The foundation of this work is a thorough knowledge of the role of the ECM during development, which guides protein selection and mechanistic investigation. An improved understanding of the role of ECM during in vitro differentiation will lead to better differentiation protocols and the potential for in situ differentiation. Ultimately, these studies will inform methods for engineered tissue vascularization to improve cell survival in large scale engineered tissues.
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    Identification of Extracellular Matrix Proteins Supportive of Endothelial Differentiation
    (2018-12) Ahmadova, Sevinj
    Efficient differentiation of pluripotent stem cells into endothelial cells represents one potential means of resolving vascularization issue in the field of tissue engineering. Recently, extracellular matrix proteins (ECM) have been shown potent stimulators of differentiation, including endothelial differentiation. Unclear is how the ECM can exhibit varying signaling stimuli over the course of a 14-day differentiation time scale. Here we test the possibility that an ECM deposited by cells over time could provide the temporal cues needed for endothelial differentiation. Mass spectrometry was employed as a means of measuring changes in deposition of ECM by cells and remodeling with ECM-triggered endothelial differentiation. As a start, the workflow for attaining accurate quantification was optimized. Then temporal dynamics of ECM expression were represented as a heat map and linear graphs of those ECM proteins with unique temporal shifts relative to control cultures. Collagen XVIII, Fibrilin 2, Fibulin 1, Galectin 9, Laminin subunit 5, Nidogen 2 fulfill these criteria and support the possibility that endothelial differentiation spurred by exogenously provided ECM is brought to completion by an evolving ECM composite.