Browsing by Subject "Stem Cell"
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Item Automation of Stem Cell Protocols(2019-12) Johnson, BlakeHuman induced pluripotent stem cells (hiPSCs) have become a vital resource for researchers and industry due to their differentiation capacity, as well as providing access to the cell phenotypes and genotypes from any individual donor. Despite improvements in stem cell technology, maintaining iPS cell lines still requires a significant amount of time and technical skill from cell culture technicians. Such steps include consistent media changes, cell counting and confluence analyses, cell passaging, cryopreservation, and subsequent thawing and plating of those cells. For this research, these processes have been transitioned onto an automated cell culturing platform. It is shown here that the automated cell culturing platform is able to properly execute DMSO-free cryopreservation, thawing, plating, and cell maintenance. This demonstrated ability to perform these functions completely automated without a technician is a technical advancement in pluripotent stem cell culturing and may provide financial benefits within a cell culture laboratory.Item Characterization of Maturity Marker Cardiac Troponin I in Human-Induced Pluripotent Stem Cell-Derived Cardiac Myocytes(2018-12) Pelaez, AndresThe use of human-induced pluripotent stem cells (hiPSCs) have opened new possibilities when it comes to medical discovery and patient care. One avenue that has benefitted is the cardiovascular field. Attempts to increase patient life after a cardiovascular event have led to increasingly better results as time has gone on. Yet, one issue that arises is the maturity level of hiPSC-derived cardiac myocytes (hiPSC-CMs). Current guidelines state the importance of being 100% certain that the cell type in question has fully differentiated into another, meaning that they should contain the same phenotype, markers and function as the wildtype cells in question. Cardiac myocytes have thus been difficult to consider mature due to the lack of certain maturity markers available. In this study, the irreversible, stoichiometric isoform switch between cardiac troponin I (cTnI) and slow-skeletal troponin I (ssTnI) is used to increase cTnI expression levels in the hiPSC-CMs. Promising results were found in all cases, with the adenovirus transduction of cTnI and the transfection of antisense oligonucleotides (ASOs) yielding increased or decreased cTnI expression in hiPSC-CMs. This lays the framework for further experimentation to analyze cardiac myocyte maturity via the cTnI marker.Item Developing a Mouse Model of Metastatic Osteosarcoma for Immunotherapy(2022-12) SonamOsteosarcoma is a primary bone malignancy that primarily affects children and young adults. The 5-year survival rate for patients with localized osteosarcoma is 60-70% and only 20% for patients who present metastasis at the diagnosis. The standard of care treatment has not improved the prognosis in over 30 years and new approaches addressing tumor resistance and immune evasion are required. The long-term goals of this project are to address two objectives: i) To study the potential benefits and risks of immunotherapeutic drugs eBAT and ONIx on syngeneic mouse models of metastatic osteosarcoma, and ii) To study their mechanism of action. K7M2 is a well-characterized model of spontaneous pulmonary metastasis, but intravenous injection of these cells did not result in lung colonization, at any dosage, in the mice in our study. We inferred from the data that the tumor cells were rejected by the immune-competent host. To study the mechanism of action of ONIx, we have made progress in optimizing the protocol for the phagocytosis assay, and further analysis with a different target cell line is planned. Assays to verify the mechanism of action of eBAT are being planned. Overall, this project provided significant learnings on objective planning and designing of the experiments and provided opportunities to acquire expertise in several new methodologies.Item Duchenne muscular dystrophy and extraocular muscle: a potential sparing mechanism with therapeutic implications.(2009-10) Kallestad, Kristen MarieThis project investigates the role of extraocular muscle (EOM) progenitor cells in sparing the muscles from pathology associated with Duchenne Muscular Dystrophy (DMD). Mouse models of muscular dystrophy and wild type mice were analyzed by flow cytometry and cell culture for the size, heterogeneity and functional characteristics of stem and satellite cell populations of EOM and limb muscles. EOM have a 5-fold increase in progenitor cells compared with limb muscles. Additionally, an enriched population of cells expressing the stem cell marker CD34 but no other typical stem or differentiation markers (Sca-1, CD45, CD31, pax-7, m-cadherin) exists in the EOM. We refer to this population as EOMCD34 cells. The EOMCD34 cells are present in developing muscle, but only maintained in adult EOM, surviving in very aged animals. The EOMCD34 cells are also present in EOM of DMD model animals, but not their limb muscles. EOMCD34 cells are resistant to apoptosis and proliferate in vivo. Finally, these cells are capable of forming myotubes in vitro. The EOMCD34 cells may represent a primitive stem cell population, which is capable of maintaining life-long pools of myogenic precursor cells. Since EOM continuously remodel throughout life, unlike other skeletal muscle, it is logical that the proliferative potential of their precursor cells is enhanced. Since one proposed mechanism of DMD muscle destruction is exhaustion of the reparative progenitor cells, the EOMCD34 cells might prove useful for myoblast transplant therapies for DMD.Item The Effects Of Different Wheat Types On Colon Cancer Risk(2020-08) Thyne, VanessaColon cancer is the third most common cancer and the second leading cause of cancer-related deaths in the United States (1). A variety of risk factors play a role in the etiology of this disease, including environmental risk factors (2, 3). Because of this, considerable research has gone into finding ways to increase consumption of foods that have an inverse association with colon cancer (4). While several epidemiological studies have shown an association between a diet high in whole grains and a lower risk of colon cancer, experimental studies have been inconsistent. This study looks at the effects of wheat class on colon carcinogenesis during the post-initiation stage of colon cancer development in rats. It also examined whether intermediate wheatgrass (IWG) commercially known as Kernza TM, a perennial grass being developed as an alternate to wheat, modifies colon cancer risk in a way similar to red wheat. A major endpoint of this study was enumeration of colonic phenotypic markers known as aberrant crypt foci (ACF), an early pre- cancerous lesion. Additionally, based on previous findings indicating that the type and amount of mucin production is a marker for dysplasia (5), changes in mucin production was examined as well. Finally, CD44, a putative marker of cancer stem cells, was determined immunohistochemically as an additional indicator of colon cancer risk. It was found that there was a significantly greater number of sialomucin-stained ACF (SIM_ACF), and mucin-depleted ACF (MDF) staining in white wheat and vs. red wheat, indicating a higher degree of dysplasia in white wheat. This shows a greater protective effect of red wheat vs. white wheat in the ACF of the colon. Moreover, staining for CD44 was found to be higher in ACFs of white wheat and IWG vs. red wheat. The correlation between the two, indicated by a dysplasia score, 0.7029 (p<0.0001) demonstrates a positive relationship between CD44 and dysplasia. The reduced number of dysplastic markers along with a higher dysplasia score in white wheat and IWG vs. red wheat supports a protective effect of red wheat.Item Examination of Post-Thaw Behavior between DMSO and Non-DMSO Cyopreserved Bone Marrow Mesenchymal Stem Cells(2016-12) Stumbras, AronMesenchymal stem cells (MSCs) provide great potential for off the shelf therapeutics because of their immunomodulatory paracrine effects. Clinical trials have utilized MSCs to treat autoimmune diseases but low efficacy, possibly due to cryopreservation methods, has limited trial progression. MSCs cryopreserved in DMSO survive well but have been shown to exhibit functional differences compared to fresh cells. This has created a need for DMSO-free cryoprotectants and for defined assays to test their functionality. To test functional aspects of MSCs post thaw we utilized recovery, viability, attachment and proliferation assays as well as pSTAT1 activity in an attempt to highlight the effects of DMSO cryopreservation on freshly thawed cells. Additionally, we provide evidence through the addition of a DMSO-free cryopreservation solutions that there may be alternatives to freezing cells than the industry standard DMSO. One solution specifically, SGI showed similar behavior to DMSO frozen samples in all metrics. The discovery and definition of DMSO-free cryoprotectants may help increase efficacy in clinical trials and help move current MSCs treatments closer to off the shelf therapies.Item Human iPSC-Derived Cardiac Myocytes: Toward an In Vitro Model of Cardiac Physiology(2017-05) Wheelwright, MatthewCardiovascular Disease is a growing public health issue in the modern world, with a high incidence rate that continues to increase, and poor mortality rates. Recent technological advances have made it possible to efficiently derive cardiac myocytes from human induced pluripotent stem cells (hiPSC-CMs). These have been seen as a model for human heart disease, as well as a potential source for cellular transplantation into failing diseased heart tissue. Many laboratories have devoted substantial effort to examining the functional properties of hiPSC-CMs, including electrophysiology, intracellular calcium handling, and gene/protein expression and force. In the first part of this thesis, we utilize traction force microscopy (TFM) to determine the maximum force production of isolated hiPSC-CMs under varied culture and assay conditions. We elucidate here the relationship between cell morphology and force production, and find a significant relationship between cell size and force. HiPSC-CMs developing in culture for two weeks produce significantly less force than cells cultured from one to three months and hiPSC-CMs cultured for three months resemble the cell morphology of neonatal rat ventricular myocytes. Unexpectedly, hiPSC-CMs produce less force when assayed on increasingly stiff substrates, and generate less strain energy. Finally, hiPSC-CMs cultured in conditions of physiologic calcium concentrations are larger and produce more force than cells cultured in standard media. In the second part of this thesis, we address the concept of immaturity in hiPSC-CMs, and attempt to accelerate maturation. We use genome editing to engineer hiPSC-CMs that contain an inducible gene expression cassette, in order to overexpress two proteins associated with maturity: SERCA2a and cardiac troponin I (cTnI). We find that we are able to overexpress both proteins in differentiated hiPSC-CMs after two weeks of treatment with doxycycline. SERCA2a-overexpressing cells showed significant alterations in physiologic function, including increased chronotropy and decreased time to peak in calcium transients following treatment with isoproterenol, a β-adrenergic agonist. Furthermore, using an impedance-measuring system to track contractility kinetics, we found that SERCA2a-overexpressing cells had shortened time to peak and time to baseline after gene induction, with continued response to isoproterenol. As a sign of maturation, SERCA cells also expressed increased cTnI, a key marker of maturity. Using RNAseq, we found that cTnI-overexpressing cells had marked, global changes in their gene expression profile. Key findings include upregulation of genes associated with cardiac contractility and development, such as cardiac myomesin and tropomyosin and ryanodine receptor, and downregulation of genes associated with pacemaker and ventricular cell types, such as HCN and GREM2, and genes associated with skeletal myocytes, such as skeletal muscle actin. Overall, our findings show that hiPSC-CMs have physiologic function similar to that of immature cardiac myocytes, but that we are able to induce maturation by overexpression of genes associated with maturity.Item Utilization of A 3D Culture System of Collagen-Mimic Peptide Gfoger-Based Hydrogel to Model Osteosarcoma from Engineered Ipsc(2021-05) Thueson, HannaModeling the early stages of human osteosarcoma development remains a significant challenge. Most existing human models are derived from patient tumor tissue which is used to establish tumor cell lines or xenograft models in immunodeficient mice. These models are largely derived from late or end stage disease and do not allow the study of the early events of transformation. Further, 2D cell lines are largely homogenous and do not replicate the heterogeneity of primary tumors. Xenografted models more closely replicate the primary tumor but can have low engraftment rates and are logistically challenging to maintain. The immunocompromised nature of xenografted mice limits the potential for immunotherapy studies. The work presented here establishes a 3D culture system to model early-stage osteosarcoma development from engineered human iPSC. When cultured as aggregates in a GFOGER (integrin-specific glycine-phenylalanine-hydroxyproline-glycine-glutamate-arginine) based hydrogel known to promote osteoblastic differentiation, osteoblasts engineered with osteosarcoma-associated mutations readily form 3D organoids. Histological analysis supports that 3D culture of iPSC-derived osteoblasts promotes a more tissue-like phenotype with increased mineralization and ECM development within the tissue construct. In addition, preliminary functional studies suggest that 3D culture promotes transformative properties and an osteosarcoma phenotype. This novel approach has potential for future applications in disease modeling, in vivo studies, and drug discovery.