Browsing by Subject "Extracellular Matrix"

Now showing 1 - 5 of 5
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    3D Printing to Recapitulate Cardiac Tissue Development, Structure, and Function
    (2019-09) Kupfer, Molly
    Heart disease is the leading cause of death worldwide, due in large part to the low regenerative capacity of the heart. With recent advances in stem cell biology, cardiac tissue engineering with human cells has emerged as an avenue to replace lost muscle after a cardiac event and to produce in vitro, human models for drug and medical device testing. However, efforts in this realm are still limited in their ability to recapitulate the complex, microscale interactions that enable macroscale function of cardiac muscle. 3D printing is a technology that is poised to meet this challenge, as it enables precise deposition of cues that are critical for cells to connect with each other and engage with their environment. Here we present three studies that capitalize on the replicative power of 3D printing as tool to advance the functionality of engineered cardiac tissues by promoting connections between cardiomyocytes, supporting cells of the heart, and the extracellular matrix. The foundation of this work lies in our view that the generation of physiologically relevant tissue mimics requires a robust mechanistic understanding of how these systems develop in vivo, and how the vital interactions that occur between differentiating cells and their environment can be recapitulated in vitro. Doing so will enable us to address critical gaps in field of cardiac tissue engineering while advancing clinical models and therapeutics.
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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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    Fibronectin and Laminin, extracellular matrix proteins, have an origin in mesoderm tissue and promote anterior neural tube closure in zebrafish
    (2023-05) Olson, Kirsta
    The neural tube, the precursor to the vertebrate central nervous system, forms through the process of neurulation. During neurulation, a flat neural plate folds and the lateral edges bind to form a closed tube. Failure of neurulation results in neural tube defects (NTD). In humans, NTD causes life-threatening birth defects such as anencephaly, spina bifida, and craniorachischisis. Work from several laboratories has demonstrated that mesoderm is required for anterior neural tube closure in mice and zebrafish. In all vertebrates, there is an extracellular matrix between the mesoderm and the developing neural tube. I hypothesized Fibronectin (Fn) and Laminin (Lam), extracellular matrix proteins, have an origin in mesoderm tissue and promote anterior neural tube closure. Supporting my hypothesis, the mRNA for extracellular matrix genes lama1, lama2, lamb1a, lamc1, fn1a, and fn1b were all expressed in mesoderm tissue adjacent to the developing neural tube. Further, expression of these lam and fn genes in the mesoderm occurred during neurulation. This suggests that one role of mesoderm in neural tube closure is to contribute to the extracellular matrix that surrounds the developing neural tube. Consistent with this and supporting my hypothesis, an extracellular matrix is required for neural tube closure in zebrafish. Loss of either lamc1 or fn1a alone resulted in a closed anterior neural tube. In contrast, approximately 50% of lamc1 ; fn1a double mutants had an open forebrain neural tube. In the midbrain, some of the embryos deficient in lamc1 ; fn1a had a double roof plate phenotype. In the hindbrain of wildtype embryos, ephrin receptor A4a (ephA4a) is expressed in rhombomeres 1, 3, and 5. Interestingly, most lamc1 ; fn1a double mutants had ephA4a expression in all three rhombomeres, but a small subset displayed a “twisted brain” phenotype that is also present in embryos with severe loss of mesoderm and an open anterior neural tube. This project provides additional connections among the mesoderm, the extracellular matrix, and neurulation.
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    High-Throughput Method for Microfluidic Placement of Cells in Micropatterned Tissues
    (2013-04-20) Sevcik, Emily
    Recent studies have shown that cell shape and tissue structure can dictate functional behavior in engineered tissues (1). One method for controlling tissue structure in vitro is microcontact printing, where extracellular matrix proteins are patterned on a substrate to construct arrays of single cells or multicellular tissues. This technique is used to create tissues that mimic in vivo architecture which can be used to study tissue properties and disease mechanisms (2). Traditional seeding of cells on the substrate is imprecise, but our group has developed a microfluidic device for spatial control of cell seeding, which creates more replicable high-fidelity tissues. However, the current method is low-throughput and labor intensive. Here, we present a scalable system of multiple microfluidic devices for parallel cell seeding. This high-throughput, precise approach reduces experimental variation, making biochemical assays on single cell arrays possible in future work. We will use this system to create large arrays of single cells of various shapes for phenotypic studies and to create arrays of tissues with varying cellular organization. 1)Alford, P. W., Nesmith, A. P., Seywerd, J. N., Grosberg, A., & Parker, K. K. (2011). Vascular smooth muscle contractility depends on cell shape. Integrative Biology, 3(11), 1063-1070. 2)Ruiz, S. A., & Chen, C. S. (2007). Microcontact printing: A tool to pattern. Soft Matter, 3(2), 168-177.
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    Lipocalin 2 Deficiency Influences Transforming Growth Factor-Beta Effect On Inflammation And Extracellular Matrix Remodeling In Inguinal Adipocytes
    (2015-09) Pfarr, Ambria
    Chronic low-grade inflammation present in hypertrophic obesity has the ability to cause remodeling of the adipose tissue due to the increased presence of macrophages and pro-inflammatory cytokines. Transforming Growth Factor-β (TGF- β) is a cytokine released from macrophages that is increased in obesity and plays a major role in extracellular matrix (ECM) remodeling of tissues. Lipocalin 2 (Lcn2), a cytokine expressed in adipose tissue, is related to inflammation and ECM remodeling. Due to the qualities that both adipokines possess the role in extracellular matrix remodeling and inflammation, we looked into the TGF- β effect on the regulation of ECM and inflammatory cytokines in Lcn2 deficient adipocytes to acquire more information about the function of Lcn2 under inflammatory stimuli and the association with metabolic diseases. Therefore, we performed experiments to address how Lcn2 knock out (KO) influences the response of adipocytes to TGF- β in the production of proinflammatory and anti-inflammatory cytokines and ECM proteins. Our results demonstrate that TGF- β down-regulates Lcn2 expression at both the mRNA and protein levels in inguinal adipocytes. Lcn2 KO adipocytes have lower levels of TGF- β expression, but normal levels of p70S6K phosphorylation and normal response to TGF- β stimulation in mammalian target of rapamycin complex 1 (mTORC1) signaling activation. However, the inhibitory effect of rapamycin on TGF- β expression is attenuated in Lcn2 KO adipocytes. Moreover, Lcn2 KO impairs the rapamycin potentiation of TGF- β effect on the expression of ECM proteins, but shows little effect on dysregulation of cytokines.