Doctor of Philosophy, Case Western Reserve University, 2018, Genetics

The ability to culture stem cells in vitro has provided access to a wide variety of cellular states that are inaccessible or too rare to study otherwise. Here we demonstrate the use of stem cells to model two distinct developmental transitions, and examine the role that epigenetic regulation of transcription plays in controlling stem cell fate and function.First, we examine the transition from mouse embryonic stem cells (mESCs), representative of the pre-implantation epiblast, to mouse epiblast stem cells (mEpiSCs), representative of the post-implantation epiblast. These two states, while maintained by distinct signaling pathways, both retain the ability to re-integrate into their respective tissue of origin and contribute to the development of the entire organism. While the transcriptome of the two cell types is largely similar, they rely on dramatically different sets of enhancers to regulate that expression. 97% of the genes that are shared between the two states undergo a change in enhancer usage in the transition. The enhancers that arise in the mEpiSC state are present, but inactive, in the preceding mESC state and become enhancer clusters downstream in development.Second, we examine the development of oligodendrocytes from oligodendrocyte progenitors (OPCs). Oligodendrocytes wrap neural axons in a lipid-rich sheath known as myelin. This myelin is required for proper signal conduction, but is subject to immune attack in multiple sclerosis (MS). OPCs replace damaged oligodendrocytes early in the course of disease, but eventually fail. A chemical genetics screen reveals that BET bromodomain proteins are required for oligodendrocyte development. These proteins are epigenetic readers that integrate chromatin state and other signals to allow transcription elongation at many genes. Other inhibitors of elongation show similar effects on development, and treatment with BET bromodomain inhibitor (S)-JQ1 blocks activation of oligodendrocyte genes. Elongation genes are e (open full item for complete abstract)

Committee: Paul Tesar PhD (Advisor); Peter Scacheri PhD (Committee Chair); Helen Salz PhD (Committee Member); Ahmad Khalil PhD (Committee Member) Subjects: Bioinformatics; Biology; Developmental Biology; Genetics

Doctor of Philosophy, Case Western Reserve University, 2011, Cell Biology

Heart disease remains the leading cause of death in western society. Many clinicians and researchers have looked to stem cells (SC) for the next generation of heart failure therapies. To date, the majority of reports have focused on their effects when given exogenously. However, it is becoming increasingly understood that endogenous SC play an important role in myocardial repair responses. In contrast to early expectations, adult SC have little regenerative potential. Instead, their observed benefits are largely attributed to pro-survival, trophic, anti-inflammatory, and angiogenic paracrine effects. Little is known about the effects of age on SC function. Recent research suggests aging of the organism may be in part due to attenuation of the SC response to injury. Indeed, studies indicate exogenously given aged SC have decreased beneficial effects. In Chapter 2 of this dissertation, we demonstrate by a series of heterochronic bone marrow (BM) transplantations between young and old mice that aged BM is associated with a decreased compensatory response after transverse aortic constriction which causes pressure overload (PO). This diminished response is associated with decreased myocyte hypertrophy, increased myocyte dropout, increased fibrosis and interestingly, no change in vessel density. These findings suggest endogenous stem cells provide trophic and anti-apoptotic support to the myocardium. There was reduced BM cell migration to the heart and activated cardiac progenitor cells in mice with old BM although no difference in ex vivo migration between young and old BM to SC homing chemokines was observed. Additionally, intravenous mesenchymal stem cell injections failed to improve cardiac function after transverse aortic constriction (TAC). These results suggest a loss of a specific SC population and/or exhaustion of the SC response with age following PO. Chapter 3 investigates the possible involvement of deficient Notch1 signaling in age related declines in cardi (open full item for complete abstract)

Committee: Alan Levine PhD (Committee Chair); Marc Penn MD, PhD (Advisor); Roy Silverstein MD (Committee Member); Horst von Recum PhD (Committee Member) Subjects: Cellular Biology

Master of Sciences, Case Western Reserve University, 2017, Clinical Research

Anterior visual pathway disease affects >50% of multiple sclerosis (MS) patients. Optical coherence tomography (OCT) monitors MS-related retinal degeneration. Retinal nerve fiber layer (RNFL) and ganglion cell layer (GCL) thinning in MS correlates with clinical and visual disability, and brain atrophy. Mesenchymal stem cells (MSC) are immunomodulatory in MS animal models, targeting inflammatory CNS injury. A recent Cleveland Clinic phase 1 trial demonstrated feasibility/tolerability of autologous MSC transplant in MS patients. My study analyzed GCL and RNFL relationships with clinical, visual, and MRI measures, before and after MSC transplant. MSC transplant affected no differences from baseline to 6 months in GCL or RNFL thickness. Linear mixed effects models predicted significant, but modest GCL thickness increases following MSC transplant. Thus, MSC neuroprotection was indirectly modeled, but not directly measured clinically or radiographically. Future MSC trials in MS should utilize OCT, a sensitive biomarker for neurodegeneration and response to experimental neuroprotective agents.

Committee: Robert Fox (Committee Chair); Jeffrey Cohen (Committee Member); Robert Bermel (Committee Member); Daniel Ontaneda (Committee Member) Subjects: Medicine

Doctor of Philosophy, Case Western Reserve University, 2015, Biomedical Engineering

Existing imaging modalities for tracking cells in vivo have many limitations such as limited resolution or limited field of view. Cryo-imaging is the only imaging technology that enables cell tracking with single cell sensitivity throughout entire animals in small rodents. It provides cell detection anywhere in mice and determines cell densities far below that which can be observed with any other imaging technologies such as MRI, CT, PET, SPECT and BLI. Using the novel imaging and detection technologies, we explored therapeutic mechanisms of mesenchymal stem cell (MSC) in murine models of graft-versus-host disease (GVHD). Many investigators have found that intravascular infusion of exogenous MSCs improves outcomes of GVHD-induced animals. Although, at least 40 clinical trials are in progress, it is still unclear what the therapeutic mechanism is? In this study, the proposed working hypothesis is that the MSCs specifically go to secondary lymphoid organs (SLOs) in order to suppress alloreactive T-cells proliferation which eventually leads to the attenuation of GVHD. In this study, we show that the hypothesis is indeed the mechanism behind the scene and is testable using our proposed imaging technology and experiments. Firstly, we extended/optimized cryo-imaging technology to detect/analyze/visualize of fluorescently labeled cells (MSCs and T-cells). Secondly, we identified bio-distribution of MSCs in a GVHD mouse model. We found evidence showing that MSCs preferentially co-localize with the effector T-cells in the SLOs after intravenous infusion. Lastly, we observed that MSCs could suppress T-cells proliferation in vivo. Novel T-cell proliferation assays were established to study the effectiveness of the MSC therapy. The assays were developed based on SLO volume enlargement approach and CFSE dilution approach. This project is significant as we have developed a new, important technique for the study of stem cell bio-distribution with single cell sensitivity o (open full item for complete abstract)

Committee: David Wilson PhD (Committee Chair); Kenneth Cooke MD (Committee Member); Andrew Rollins PhD (Committee Member); Vira Chankong PhD (Committee Member); Wouter van't Hof PhD (Committee Member) Subjects: Biomedical Engineering; Biomedical Research; Computer Science; Experiments; Immunology; Medical Imaging

Doctor of Philosophy, Case Western Reserve University, 2013, Biomedical Engineering

Current stem cell and tissue engineering R&D aim to initiate and guide de novo tissue generation for the repair or replacement of native tissue compromised due to injury, disease, or other disorders. Biophysical cues present a safe and controllable means to modulate cell behavior and emergent cell fate. Scaling up from cellular to systems length scales, there is great future potential for prescriptive physical therapies that synergistically promote healing in conjunction with approaches including stem cell based regenerative tissue engineering and next generation implants cum delivery devices. This dissertation addresses key obstacles for biophysically-enhanced de novo tissue generation by (i) examining the potential of the femoral neck periosteum in discarded tissue from arthritic patients undergoing hip replacement surgery as a feasible source of adult stem cells for tissue engineering, (ii) unraveling the emergent spatial and temporal mechanoadaptive structure – function relationships of stem cells after exposure to volume and shape changing stresses, (iii) and implementing a coupled computational fluid dynamics and finite element method model to define future experiments to test the ability of mechanical cues to guide stem cell fate during healing. Addressing the first aim, an IRB approved study highlighted the potential of femoral neck periosteum as a new source for stem cell acquisition and banking of autologous multipotent cells for middle aged to aging individuals. The second aim, to unravel emergent anisotropy in model embryonic mesenchymal stem cell structure and nascent lineage commitment, showed that the actin and tubulin cytoskeleton exhibit emergent anisotropy in the apical-basal direction and that these changes correlate to regulation of transcription factors indicative of fate. Finally, the third aim addresses a parametric approach using a CFD-FEM model to predict the local normal and shear stresses at cell-fluid interfaces in a tissue template with (open full item for complete abstract)

Committee: Melissa Knothe Tate (Advisor); Edward Greenfield (Committee Member); Daniela Calvetti (Committee Member); Eben Alsberg (Committee Member) Subjects: Biomechanics; Biomedical Engineering; Biomedical Research; Cellular Biology; Developmental Biology; Engineering; Mechanical Engineering; Medicine; Molecular Biology; Scientific Imaging

Doctor of Philosophy, University of Akron, 2018, Biomedical Engineering

Neurodegenerative diseases that are caused by deterioration of nerve cells in the brain and spinal cord affect more than 6 million Americans and cost nearly 0.8 trillion dollars annually in patient care. With a growing number of elderly population, the statistics are expected to worsen as there is currently no cure for these disorders. Modern medicines are at best palliative and only manage the symptoms. Therapeutic interventions to deliver functional neural cells to the ravaged tissue are essential to restore lost tissue functions. The use of stem cell-derived neural cells is a promising strategy for cell replacement therapies of neurodegenerative diseases. Embryonic stem cells (ESCs) are promising cell sources for therapeutic uses including cell replacement therapy of neural tissues. This is because ESCs have unlimited self-renewal and proliferation capabilities and the ability to differentiate into various neural cells. Nevertheless, despite significant investment and research, therapeutic uses of ESCs for neural cell replacement has been largely unsuccessful. Low and inconsistent yield of neural cells from ESCs and lack of a complete understanding of molecular mechanisms of neural differentiation of ESCs are major obstacles against clinical uses of ESC-based therapies. A cohort of cell surface bound and soluble factors, interactions of ESCs with their neighboring cells and extracellular matrix proteins, and various epigenetic factors may act synergistically to drive differentiation of stem cells. While most of current research is centered on functionalizing specific biomolecules on scaffolds and tuning the matrix stiffness, or altering media compositions to gain a better control over the neural differentiation of stem cells, the role of niche-mediated factors is less understood. In this study, we showed that intrinsic niche parameters such as stem cell colony size and interspacing between the two colonies can significantly impact the differentiation effic (open full item for complete abstract)

Committee: Hossein Tavana (Advisor); Marnie Saunders (Committee Member); Nic Leipzig (Committee Member); Yang Yun (Committee Member); Sailaja Paruchuri (Committee Member) Subjects: Biomedical Engineering; Biomedical Research; Engineering; Neurobiology

Doctor of Philosophy, University of Akron, 2018, Integrated Bioscience

Decellularized porcine myocardium has great potential in serving as a cardiac patch for heart repair after myocardial infarction (MI). However, using full thickness of decellularized porcine myocardium as a cardiac patch may lead to poor viability of the delivered cells due to perfusion limitation and may add an unwanted additional load to the infarcted heart. To address these issues we examined the feasibility of using a thin decellularized porcine myocardium slice (dPMS) as a cardiac patch to deliver therapeutic cells and assess whether this cardiac tissue engineering strategy would improve the function of injured myocardium caused by a MI. We started with obtaining thin dPMSs of various thicknesses by cryosectioning and then checked the effects of thickness on the mechanical properties of dPMS. Adipose derived Stem Cells (ASCs) from rats and pigs were isolated, characterized, and seeded on top of dPMSs with selected thicknesses. The viability, proliferation, infiltration, and differentiation of the seeded rat and pig ASCs were evaluated in vitro. The therapeutic outcomes of dPMS as a cardiac patch (with and without cells) were determined in vivo using a rat MI model. Our results confirmed that the fabricated dPMS can serve as a cell delivery platform to deliver a large amount of cells to injured myocardium with an increased cell retention rate as compared with direct cell injection. Furthermore, when used as a cardiac patch, dPMS integrates with host tissue, promotes host cell infiltration, increases vascularization in the infarcted area, and most importantly enhances cardiac function.

Committee: Ge Zhang Dr. (Advisor); Richard Londraville Dr. (Committee Member); Rouzbeh Amini Dr. (Committee Member); Liya Yin Dr. (Committee Member); Anand Ramamurthi Dr. (Committee Member) Subjects: Biomedical Engineering; Biomedical Research

Doctor of Nursing Practice Degree Program in Population Health Leadership DNP, Xavier University, 2018, Nursing

The effects of sleep loss are a population health issue for the 65.7 million family caregivers in the United States (U.S.). Between 60-95% of caregivers report poor sleep quality because of sleep disruption. The purpose of this Doctorate of Nursing Practice (DNP) scholarly project was to evaluate the effectiveness of a combination of interventions, referred to as a sleep bundle, to improve sleep quality for caregivers of children with chronic conditions such as bone marrow transplant (BMT) using the FADE (Focus Analyze Develop Execute Evaluate) quality improvement (QI) methodology. It was hypothesized that the sleep bundle will decrease sleep disruption, and ultimately improve the sleep quality of primary caregivers of BMT patients. Specific aims to test the hypothesis included: (1) Compare sleep efficiency and quality in caregivers with and without the sleep bundle; (2) Identify and mitigate barriers and obstacles to implement the sleep bundle. Although the sleep bundle was followed reliably the nights of sleep bundle intervention phase; the sleep bundle was not found to be statistically significant in improving the sleep efficiency and sleep quality of caregivers of BMT patients. Additional evaluation of the sleep bundle and its impact on improving the sleep efficiency and quality of caregivers is needed with a larger sample size. Possible alteration of specifics interventions combined in the sleep bundle should also be explored.

Committee: Elizabeth Bragg PhD, RN (Committee Chair); Robin Saxon DNP, RN (Committee Co-Chair) Subjects: Health Care; Health Sciences; Medicine; Nursing; Psychology

Master of Sciences, Case Western Reserve University, 2017, Clinical Research

Gliomas are primary brain tumors and are challenging to treat. Traditionally, gliomas are characterized by their histologic appearance and grade. Recently, molecular data have complemented histopathologic data and provided new insights on glioma biology and behavior. Vasorin is a transmembrane protein that is commonly overexpressed in glioblastoma, the most aggressive type of glioma. The function of Vasorin is not well understood but involves regulation of apoptosis induced by inflammation or hypoxia and regulation of transforming growth factor-beta (TGF-beta) signaling. The purpose of this study was to use a bioinformatics approach to discern potential roles for Vasorin in glioma biology. Vasorin expression correlated strongly with tumor grade, glioblastoma, IDH wild type gliomas, TERT promoter mutation and unmethylated MGMT promoter. Proteomic analyses revealed potential interactions between Vasorin and developmental programs, including Notch, TGF-beta and homeobox proteins, and IDH1. Additional studies are needed to validate these findings.

Committee: Jeremy Rich (Committee Chair) Subjects: Biology; Medicine

Master of Science in Biological Sciences, Youngstown State University, 2015, Department of Biological Sciences and Chemistry

The use of biological products such as platelet-rich plasma (PRP) and mesenchymal stem cells (MSCs) has become an incredibly promising and advantageous product in modern regenerative therapeutics. The occurrence of incisional herniation following invasive abdominal surgeries remains a particularly complicating issue in post-surgery recovery with about 10% of patients experiencing herniation following laparotomy (Bucknall et al., 1982). This study aims to provide a cell-based therapy for the treatment of surgical wounds and enhance healing. Using a rat model, bone marrow-derived MSCs (BM-MSCs), PRP, and a collagen scaffold (CollaTape) are applied to the surgical site following an abdominal laparotomy. The rats were allowed to heal for 4 weeks before tissue samples of the wound site were harvested to analyze wound characteristics. Histological examination of wound sites treated with BM-MSCs indicates important information for differences in specific events involved in the wound healing process including collagen deposition, connective tissue organization, and muscle regeneration. It was found that applying PRP and CollaTape to the abdominal incisions was able to increase collagen abundance and stimulate new muscle growth. The application of MSCs to the injury was able to further enhance the deposition of organized collagen fibers in a dose-dependent manner. Additionally, characteristics of the BM-MSCs, including their ability to differentiate into chondrogenic, adipogenic, and osteogenic cell lineages was performed in order to exhibit their stem potential.

Committee: Diana Fagan PhD (Advisor); Mark Womble PhD (Committee Member); Joanna Krontiris-Litowitz PhD (Committee Member) Subjects: Biomedical Research; Cellular Biology; Medicine; Surgery

Doctor of Philosophy, Case Western Reserve University, 2014, Molecular Biology and Microbiology

Glioblastoma Multiforme (GBM) and other cancers are challenging to treat due to their intertumoral and intratumoral heterogeneity. The hierarchical model of intratumoral heterogeneity describes the existence of a cellular hierarchy in glioblastoma with a cancer stem cell (CSC) population at the apex. These CSCs can initiate tumors and are resistant to therapies, suggesting that they are responsible for tumor recurrence. CSCs give rise to differentiated cells, which have limited tumor initiation abilities but provide a supportive CSC niche. Here, I examine the cellular cues that maintain both CSCs and the niche by tuning the relative levels of differentiation and self-renewal in GBM. I suggest Bone Morphogenetic Proteins (BMPs) as a driver of differentiation, as BMPs have been shown to promote CSC differentiation and are strongly expressed in GBM. Extrapolating from existing paradigms in development and in cancer, I then hypothesize that extracellular BMP antagonists could be the parallel drivers of CSC self-renewal.I subsequently demonstrate that a BMP antagonist, Gremlin1, is secreted specifically by CSCs, supporting CSCs in the context of the endogenous differentiation signals provided by BMP. I promote self-renewal and increase growth of non-CSCs by overexpression of Gremlin1, and drive differentiation and slow growth of CSCs by Gremlin1 knockdown. Finally, I examine the mechanisms downstream of Gremlin1 that drive its effects on growth and self-renewal, as well as the mechanisms upstream of Gremlin1 that promote its differential secretion by CSCs. Downstream of Gremlin1, cell proliferation effects are mediated by p21 inhibition, and self-renewal is partly mediated by activation of Wnt signaling. Upstream of Gremlin1, I identify XBP1, a pro-survival factor in the unfolded protein response, as a CSC-specific signal that might be promoting Gremlin1 expression. In the course of this thesis, I identify a novel molecular target, Gremlin1, as well as severa (open full item for complete abstract)

Committee: Jeremy Rich (Advisor); Thomas Egelhoff (Committee Chair); Eric Arts (Committee Member); William Schiemann (Committee Member); Paul Tesar (Committee Chair) Subjects: Biology; Biomedical Research; Cellular Biology

Master of Sciences, Case Western Reserve University, 2011, Biology

This thesis provides a commercialization strategy for stem cell-derived human cardiomyocytes (SC-hCM) with an overview of pharmacological and electrophysiological profiles of SC-hCM and non-human Purkinje Fiber (PF) action potential (AP) assays. PF AP assays are commonly used to assess cardiac risk of pharmaceuticals in preclinical development but they often provide false negative or false positive results. The SC-hCM AP assay offers the potential advantages of increased pharmacological sensitivity, reduced compound consumption, higher throughput and cost effectiveness compared to conventional PF assays. The electrophysiological and pharmacological profiles of SC-hCMs were compared to canine and rabbit PFs. The effects of reference compounds were measured in ventricular-type SC-hCMs by perforated-patch, current clamp recording and compared with results obtained in PF AP assays. In conclusion, SC-hCMs provide an attractive alternative to PF AP assays; since this assay accurately predicts cardiac risk of known torsadogenic compounds and demonstrates an overall pharmacological sensitivity that is greater than conventional rabbit or canine PF assays

Committee: Roy Ritzmann Ph.D (Committee Chair); Christopher Cullis Ph.D (Advisor); Andrew Bruening-Wright Ph.D (Committee Member); Jean Welter Ph.D (Committee Member) Subjects: Biology; Entrepreneurship; Health Care; Health Care Management; Health Sciences; Marketing; Pharmaceuticals; Pharmacology; Physiology; Technology; Toxicology

Doctor of Philosophy, Case Western Reserve University, 2010, Biomedical Engineering

The long term goal of this research is to use cryo-imaging to detect and spatially map nearly every stem cell in a mouse and quantify tissue specific, cell therapy PK. With this enabling technology, one will be able to quantitatively assess specificity of homing, quiescent survival in specific niches, emerging homing factors, delivery processes, cell engraftment dose response, etc., analyses which have, at best, been done qualitatively. To this end, we developed a cryo-imaging system, which alternates between sectioning (10-40 µm) and imaging bright field and fluorescence block-face image volumes with micron-scale-resolution. For applications requiring single-cell detection of fluorescently labeled cells anywhere in a mouse, we developed an algorithm, next-image processing, for reduction of subsurface fluorescence. Next-image processing greatly improves axial-resolution, enabling high quality 3D volume renderings, and improved automated enumeration of single cells by up to 24%. To answer many of the pressing questions in stem cell therapies, automated methods for quantification and detection of stem cells are required. We developed algorithms for the automated detection and quantification of fluorescently labeled cells. Our model based quantification algorithm was performed on low resolution images of fluorescently labeled stem cells and the results were compared to visual quantification of stem cells in high resolution images. In all cases but two, the algorithm was within ± 1 cells of the actual number present in a cluster. The total number of cells counted by the expert was 393 compared to 386 by the algorithm, giving an error rate of 1.7%. To estimate homing of stem cells to damaged tissues, quantification of stem cells in infracted mice hearts was compared to non-infarcted control mice hearts. The number of MSC's detected in the heart was significantly higher for infarcted mice. The delivery ratio for infarcted mice was 8.0 ± 0.8 times larger than the deliver (open full item for complete abstract)

Committee: David Wilson PhD (Committee Chair); Marc Penn MD, PhD (Committee Member); Andrew Rollins PhD (Committee Member); James Basilion PhD (Committee Member) Subjects: Biomedical Research

Doctor of Philosophy, Case Western Reserve University, 2013, Biomedical Engineering

Mesenchymal stem cells (MSCs) are tissue culture plastic adherent cells that are able to differentiate into multiple cell types, modulate the immune system, and aid in wound repair. These versatile cells are thus being investigated to treat a wide variety of different diseases such as graft vs. host disease, rheumatoid arthritis, myocardial infarction, and damaged cartilage. In addition, with genetic modification, MSCs can be used as drug delivery vehicles to treat cancer or as a cell replacement therapy to treat osteogenesis imperfecta. However, the delivery of MSCs to a target organ, and the delivery of stem cells in general, remains a major challenge. This thesis investigates the hypothesis that when MSCs are efficiently delivered into the circulation, they will home to sites of injury. First, MSCs were genetically modified by lentiviral transduction with a dual reporter gene vector containing monomeric red fluorescent protein and luciferase in order to track the cells in vivo in real time. It was found that polybrene, an additive commonly used during transduction, severely inhibited MSC proliferation. A new method was developed for efficient lentiviral transduction using protamine sulfate, and other techniques. MSC engraftment to sites of injury was investigated by delivering cells into mice irradiated on one leg, with the non-irradiated leg serving as an internal negative control. Delivery of the cells into the aortic arch resulted in initial cell distribution throughout the entire body and engraftment in only the irradiated leg as detected by bioluminescent imaging (BLI). The biolumescence increased over time, indicating that the engrafted MSCs proliferated in vivo. In contrast, cells delivered intravenously were trapped in the lungs with no engraftment. This homing is specific to MSCs, since it was not observed with kidney cells. Furthermore, aortic arch delivery was efficient, as engraftment was consistently observed with a dose of only 2.5 x 105 cells/mouse (open full item for complete abstract)

Committee: Roger E. Marchant PhD (Committee Chair); Arnold I. Caplan PhD (Advisor); Horst von Recum PhD (Committee Member); James M. Anderson MD, PhD (Committee Member); Alison Hall PhD (Committee Member) Subjects: Biology; Biomedical Engineering; Medicine

PHD, Kent State University, 2025, College of Arts and Sciences / School of Biomedical Sciences

Aortic valve stenosis (AS) is a progressive, life-threatening condition characterized by fibro-calcific remodeling of the aortic valve, leading to obstruction of blood flow and impaired cardiac function. Despite its increasing prevalence, no pharmacological therapy currently exists to halt or reverse the disease, highlighting a critical need for novel, non-invasive interventions. This dissertation investigates the therapeutic potential of exosomes derived from NAMPT-overexpressing mesenchymal stem cells (NAMPT-Exo) in mitigating AS and elucidates the underlying mechanisms of action. Using our newly developed endothelial cell-specific CXCR4 knockout (EC CXCR4 KO) induced AS mouse model, we demonstrate the therapeutic potential of NAMPT-Exo in mitigating AS. NAMPT-Exo treatment in EC CXCR4 KO mice significantly attenuated AS progression, reduced valvular calcification and thickening, and improved cardiac function, as shown by echocardiography and histological analyses. Mechanistic studies further revealed that NAMPT-Exo treatment modulates endothelial-to-mesenchymal transition (EndMT), a key contributor to AS pathogenesis. Additionally, we found that miR-146a-3P expression was significantly elevated in cardiac endothelial cells of AS mice, while exosome treatment effectively reduced its levels. These findings provide strong evidence that NAMPT-enriched MSC-derived exosomes represent a novel, cell-free therapeutic strategy for aortic valve stenosis and miR146a-3P may play an essential role in the disease development and progression.

Committee: William Chilian (Committee Member); Feng Dong (Advisor) Subjects: Biomedical Research; Pharmacology

PhD, University of Cincinnati, 2024, Medicine: Cancer and Cell Biology

Congenital heart disease (CHD) stands as the most prevalent congenital anomaly, impacting approximately 4 per 1000 live births. Single ventricle (SV) CHD, constituting 7.7% of CHDs, represents a severe and intricate form of CHD where patients typically exhibit a single dominant left or right ventricle at birth, insufficient to sustain normal pulmonary and systemic circulation. SV CHDs encompass diverse cardiac malformations, including semilunar (SL) or atrioventricular (AV) valve hypoplasia, stenosis/atresia, and malalignment of the pulmonary artery and aorta. The varied manifestations of SV abnormalities imply a complex disease etiology intertwined with various cardiac developmental anomalies. Despite substantial progress in understanding the molecular mechanisms of various SV diseases, intrinsic defects in human cardiac endothelium and related valvular structures remain elusive. The advent of single-cell RNA sequencing (scRNA-seq) has revolutionized our ability to precisely dissect cellular identity, functions, and interactions during cardiogenesis and CHD pathogenesis. Leveraging human induced pluripotent stem cells (iPSCs) expands our experimental repertoire, allowing the generation of various cardiac cell types to functionally validate genomic and transcriptomic data in cellular models. Herein, we combined scRNA-seq analysis of human tissues with patient-derived iPSCs to unravel the endothelial/valvular pathobiology in SV diseases, such as hypoplastic left heart syndrome (HLHS) and pulmonary stenosis (PS). The effort extended to specify iPSC-derived valve endothelial cells (VECs) and valve interstitial cells (VICs) representing the specificity of all four valves. Despite the previous knowledge of myocardial defects in HLHS pathology, we demonstrated the intrinsic defect in disease vascular arterial endothelium, including KMT2D-NOTCH mediated proliferation, angiogenesis, and EC-smooth muscle cell interactions. Our results suggested the importance o (open full item for complete abstract)

Committee: Mingxia Gu M.D. Ph.D. (Committee Chair); Susanne Wells Ph.D. (Committee Member); Mei Xin Ph.D. (Committee Member); Katherine Yutzey Ph.D. (Committee Member); Yanbo Fan Ph.D. (Committee Member) Subjects: Biology

Doctor of Philosophy, Case Western Reserve University, 2024, Genetics

Epilepsy is a neurological disorder characterized by repeated seizures. It affects 1% of the US population. Despite the recent progress in antiepileptic drug discovery and development, approximately 30% of the patients do not respond to current therapies. A major pitfall of the current antiepileptic drug discovery pipeline is current animal seizure models. The most widely used animal models are mechanically or chemically induced models, and they fail to recapitulate drug-resistant seizures, which are largely genetic epilepsies. Induced pluripotent stem cells (iPSCs) are promising alternative preclinical epilepsy models. iPSCs preserve the genetic information of epilepsy patients and can be an unlimited source of neuronal cell types of interest. Many studies have shown that iPSC disease models successfully reproduce epilepsy phenotypes. Herein, we successfully leveraged stem cell reprogramming technologies and CRISPR/Cas9 gene editing tools to generate iPSCs models of three drug-resistant epilepsies with monogenic cause: Early infantile epileptic encephalopathy, subtype 76 (EIEE-76), Microcephaly, Epilepsy, Diabetes Syndrome (MEDS), and GRIN2A-related epilepsy. Using these models, we uncovered pathophysiological mechanisms underlying the epilepsy phenotype of these rare genetic epilepsies that were previously unknown. We demonstrated EIEE-76 patient variant of ACTL6B dysregulates genes essential for neuronal development and that EIEE-76 patient-derived neurons demonstrated elevated electrophysiological activity than control, consistent with the patient phenotype. Also, we elucidated MEDS patient-derived neural progenitor cells (NPCs) have defects in secretory protein trafficking. Impaired protein trafficking during neurogenesis resulted in abnormal neuronal differentiation and electrophysiologically less active neuronal population. Next, we took a step further and developed a disease-relevant, high-throughput, phenotypic screen platform to identify potential an (open full item for complete abstract)

Committee: Ashleigh Schaffer (Advisor); Yan Li (Committee Member); Drew Adams (Committee Chair); Anthony Wynshaw-Boris (Committee Member); Andrew Pieper (Committee Member) Subjects: Genetics

PhD, University of Cincinnati, 2023, Medicine: Immunology

Hematopoiesis is a complex and tightly regulated biological system in which the stem cells and progenitors balance between mature lineage outputs and self-renewal. Environmental stimuli, genetic predispositions, and host responses together control and alter underlying transcriptional network. Various animal models and single-cell multi-omics approaches have been developed to unravel the network and mechanisms associated with it. This dissertation characterizes relationship between gut microbiome (environmental stimuli) and host; describes human and murine hematopoietic stem cells and progenitors; and demonstrates methods to incorporate with biological models to derive insightful biological readouts. Microbiome models have informed our understanding of microbiome-dependent metabolites, their role in homeostatic hematopoiesis, and nominated some metabolites as potential therapeutics. However, conclusions about gut microbiota-induced biological functions have been called into question after recent results from metabolite clinical trials and conflicting findings across studies. To systematically evaluate the impact of durably different gut microbiome on homeostatic hematopoiesis, we analyzed three C57BL/6 mouse microbiome models with defined flora, and contrasted with germ-free and standard-pathogen-free C57BL/6 mice. Metagenomics and metabolomics confirmed distinct microbial species present, their relative diversity, and associated changes in physiology and serum metabolites. We find that a minimal microbiome composition restores the majority of metabolites absent in germ-free mice, but community complexity controls metabolite abundance and perturbs metabolite pathways and transcriptional signatures. However, immunophenotyping reveals comparable innate and adaptive immune populations across models. Bone marrow transplant reveals normal homeostatic HSC function, and in vitro assays reveal similar neutrophil innate immune function across models. Thus, defined flo (open full item for complete abstract)

Committee: H. Leighton Grimes Ph.D. (Committee Chair); Daniel Lucas Ph.D. (Committee Member); Jose Cancelas-Perez M.D. Ph.D. (Committee Member); Nathan Salomonis Ph.D. (Committee Chair) Subjects: Immunology

Doctor of Philosophy (Ph.D.), University of Dayton, 2022, Biology

Hippo pathway is an organ size regulating pathway that has implications in organogenesis, cell competition, compensatory proliferation, and regeneration. Previous studies have identified the role of impaired Hippo pathway in cancer and Alzheimer's. Down-regulation of pathway causes over proliferation by upregulation of target gene expression that promotes proliferation and prevents apoptosis. On the contrary, pathway hyper-activation causes cell death by upregulating a pro-apoptotic protein Hid and without affecting the expression of Diap-1, a target protein that prevents apoptosis. However, down-regulation of Hid fails to significantly rescue Hippo pathway mediated cell death. Work from a previous graduate student in our lab has shown that Hippo signaling regulates a key cell death gene dronc which is a homolog of the initiator caspase-9 in mammals. When Hippo levels are high, Yki is sequestered in the cytoplasm and dronc levels are high and cell death is the result. In contrast to that when Hippo is downregulated, Yki is free to move to the nucleus and cause changes in gene expression and dronc is also downregulated and the result is proliferation of cells. But the phenomenon between Yki and dronc are poorly studied. The aim of my work was to thus identify how Yki that interacts with Hippo pathway to regulate cell death via dronc regulation. Dronc is also a common target to the Ecdysone signaling which is an important steroid hormone in insects that allows spatio-temporal of gene expression to regulate events of cell death and growth during molting and metamorphosis. But the relationship between Hippo Signaling and Ecdysone signaling to regulate a common target such as dronc is underexplored. To attain this goal, we tested for genetic interaction between known Ecr pathway components and the Hippo pathway components to determine if there is any genetic epistasis between the components. Our experiments led to the identification of a feedback loop in which the downst (open full item for complete abstract)

Committee: Madhuri Kango-Singh (Advisor); Pitychoutis Pothitos (Committee Member); Andreas Bergmann (Committee Member); Amit Singh (Committee Member); Mark Nielsen (Committee Member) Subjects: Biology

Doctor of Philosophy, Case Western Reserve University, 2021, Genetics

The pancreatic islet contains multiple endocrine (hormone-producing) cells that play critical roles in glucose homeostasis. Failure of the islet cells, especially the insulin-secreting β cells, is central to the development of diabetes. However, the molecular landscapes of the human pancreatic islet in normal and disease states remain unclear. Here we performed large-scale single-cell RNA-Seq for both adult human islets and stem-cell-derived β-like cells in differentiation. We first analyzed the transcriptome of 39,905 single islet cells from 9 human donors and observed distinct β cell heterogeneity trajectories associated with obesity or type II diabetes (T2D). We therefore developed RePACT, a sensitive single-cell analysis algorithm to identify both common and specific signature genes for obesity and T2D. We mapped both β cell-specific genes and disease signature genes to the insulin regulatory network identified from a genome-wide CRISPR screen. Our integrative analysis discovered the previously unrecognized roles of the cohesin loading complex and the NuA4/Tip60 histone acetyltransferase complex in regulating insulin transcription and release. Second, we generated 95,308 single-cell transcriptomes for the entire differentiation process from hESCs to β-like cells and reconstructed a lineage tree to study temporally 10 regulated genes. We identified so-called `switch genes' at the branch points of endocrine/non-endocrine cell fate choices, revealing insights into the mechanisms of differentiation-promoting reagents, such as NOTCH and ROCKII inhibitors, thus providing the framework for improved differentiation protocols. Over 20% of all detectable genes are activated multiple times during differentiation, even though their enhancer activation is usually unimodal, indicating extensive gene reuse driven by different enhancers. We also identified a stage-specific enhancer in the TCF7L2 diabetes GWAS locus that drives a transient wave of gene expression in pancreatic (open full item for complete abstract)

Committee: Fulai Jin (Advisor); Li Yan (Advisor); Ann Harris (Committee Chair); Peter Scacheri (Committee Member); Jonathan Haines (Committee Member) Subjects: Genetics