Browsing by Subject "Stem cell"
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Item Identification of novel anti-schistosomal therapies through studies of orthologous pathways during planarian regeneration(2015-02) Chan, JohnThe parasitic infection schistosomiasis afflicts over 200 million people and is clinically treated by a single drug - praziquantel (PZQ). Despite the fact that PZQ has served as a stalwart of anti-helminthic therapy for over three decades, it's molecular basis of action remains poorly understood. This roadblock prevents the rational design of alternative therapies and highlights the need for new approaches to study the parasitic flatworms that cause schistosomiasis. Towards this end, my thesis work has advanced the free living, non-parasitic planarian Dugesia japonica as an accessible laboratory model organism for studying flatworm gene function, identifying candidate druggable targets, and screening anti-parasitic lead compounds. The utility of this species derives in part from an unexpected phenology between regenerative outcomes in free living flatworms and lethality in parasitic species. These phenotypes were initially discovered through studies on the existing anti- schistosomal therapy, praziquantel, and I demonstrate that this phenology holds for a range of anti-schistosomal compounds with varying molecular targets and mechanisms of action. Capitalizing upon this phenology, experiments on the planarian D. japonica have yielded new insights into praziquantel's mechanism of action by linking the drug's efficacy to modulation of bioaminergic driven flatworm mobility. Expanding upon these initial findings in planarians, bioaminergic signaling was interrogated via pharmacological and genetic screens, revealing novel druggable targets (serotonin receptors) and lead compounds (ergot alkaloid derivatives) capable of subverting both planarian regeneration and parasite muscle function. Finally, given the predictive value of planarian regenerative outcomes in studying anti- parasitic therapies, I focused on understanding the regenerative patterning events that occur shortly after wounding. These studies demonstrate a crucial role for the CNS in determining regenerative outcomes, implicating a voltage operated Ca2+ channel isoform (Cav1B) and serotonin as key mediators of both "head" verses "tail" formation and flatworm muscle function. Collectively, the work presented in this thesis demonstrates the utility of a multi-species approach to resolve questions regarding the mechanism of action of the orphan drug praziquantel and to identify routes for novel anti-schistosomal drug development.Item Modeling and rescue of Duchenne muscular dystrophy cardiomyopathy using human induced pluripotent-derived cardiomyocytes(2021-01) Kamdar, ForumDuchenne muscular dystrophy (DMD) is the most common muscular dystrophy and affects 1:5000 boys born in the United States. DMD is a result of mutations in the dystrophin (DMD) gene that leads to the absence of the full length cytoskeletal protein dystrophin, which is expressed in skeletal muscle, brain, and heart. The absence of dystrophin leads to weakness of not only the skeletal muscle but also the heart. With advances in treatment for DMD, patients are living longer but a cardiomyopathic phenotype has been uncovered. DMD associated cardiomyopathy is nearly ubiquitous and is the leading cause of death with adults with DMD. There have been limited studies and therapies for dystrophic heart failure thus far, and there is a critical need to identify the pathophysiology and develop effective therapeutic regimens. In this thesis, I hypothesized that DMD cardiomyopathy could be modeled using DMD patient-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-derived CMs) to identify physiological changes and future drug therapies. To explore and define therapies for DMD cardiomyopathy, we used DMD patient-specific and dystrophin null isogenic hiPSC-derived CMs to examine the physiological response to adrenergic agonists and -blocker treatment. We further examined these agents in vivo using wildtype and the mdx mouse model. At baseline and following adrenergic stimulation, DMD hiPSC-derived CMs had a significant increase in arrhythmic calcium traces compared to isogenic controls. Further, these arrhythmias were significantly decreased with propranolol treatment. Using telemetric monitoring, we observed that mdx mice, which lack dystrophin, and were stimulated with isoproterenol had an arrhythmic death and the lethal arrhythmias were rescued, in part, by propranolol pretreatment. Using single cell and bulk RNA-seq, we compared DMD and control hiPSC-derived CMs, mdx mice and control mice (in the presence or absence of propranolol and isoproterenol) and defined pathways that were perturbed under baseline conditions and pathways that were normalized following propranolol treatment in the mdx model. We also undertook transcriptome analysis of human DMD left ventricle samples and found that DMD hiPSC-derived CMs have similar dysregulated pathways as the human DMD heart. We further determined that relatively few DMD patients see a cardiovascular specialist or receive β-blocker therapy. The results of these experiments highlight important mechanisms and therapeutic interventions from human to animal and back to human in the dystrophic heart. Importantly, these results may serve as a platform to elucidate further mechanisms of DMD cardiomyopathy and serve as a platform for testing novel therapies. Our results also provide a rationale for an adequately powered clinical study that examines the impact of β-blocker therapy in patients with dystrophinopathies.Item The role of cellular calcium channels in planarian biology(2011-11) Zhang, DanIdentification of signaling pathways and therein drugable targets, to manipulate stem cell behavior in vivo is a major focus of regenerative medicine. This dissertation focuses on the role of Ca2+ channels in stem cell differentiation and regeneration in a simple in vivo model, the planarian flatworm. These animals maintain a totipotent population of stem cells that give rise to all the cell types in the worm. Previously, we discovered that the isoquinoline drug praziquantel (PZQ) caused a robust (100%) and complete duplication of the entire anterior-posterior (AP) axis during flatworm regeneration to yield two-headed (bipolar) organisms. My studies mechanistically dissect these observations to show that PZQ subverted regeneration via activation of a specific neuronal voltage-gated Ca2+ channel (VGCC) isoform (Cav1A). Surprisingly, another isoform Cav1B was found to play opposing roles in axis formation to promote tail regeneration, suggesting a delicate interplay between Ca2+ signals critical for nervous system regeneration. Further dissection of the downstream pathway showed that RNAi of Cav1A blocked PZQ-evoked bipolar regeneration, Ca2+ entry and decreases in Wnt levels, the output of Hedgehog signaling. Thus, these data demonstrated that calcium signaling regulated regeneration through modulating Hedgehog signaling, a pathway that has been shown to regulate neuronal stem cell behavior, patterning and growth in diverse development processes. Taken together, these findings add new insights into the mechanisms that govern planarian regeneration. Additionally, my work on intracellular Ca2+ release channels in this system led to the identification of the planarian inositol 1, 4, 5-trisphosphate receptor (IP3R). Studies designed to elucidate the biological significance of this protein by in vivo RNAi knockdown led to the discovery that sexual planarians underwent severe defects of laying eggs in the absence of IP3R, although it failed to produce an obvious phenotype in asexual worms. Thus, these data provided genetic evidence that IP3R plays an important role in regulating reproductive physiology in planarian flatworms. In summary, the data obtained in this thesis have revealed essential roles of Ca2+ signaling in regulating planarian stem cell differentiation and reproductive physiology.Item Scalable culture systems for expansion and directed differentiation of rat multipotent adult progenitor cells.(2010-05) Park, YonsilAdvances in stem cell science have stimulated the prospect of stem cells as therapeutics. For the translation of stem cell research to technology, robust and efficient expansion and differentiation is essential. Rat multipotent adult progenitor cells (rMAPCs) are a type of adult stem cells isolated from the rodent bone marrow. MAPCs can be expanded in vitro without obvious senescence, and are capable of differentiating into cell types of mesoderm, endoderm, and ectoderm in vitro. They are typically maintained surface adherent at low cell densities of 100-300 cells/cm2 in order to maintain their broad potency, which would make scale-up for further clinical applications cumbersome. In this study, we explored different cultivation methods to investigate the feasibility of scalable culture systems for rMAPCs: (1) high density 2D culture, stirred bioreactor culture as (2) 3D aggregates and (3) on microcarriers. Culturing cells under hypoxic condition (5% O2) during the isolation, has yielded rMAPC expressing high levels of the embryonic stem cell specific transcription factor Oct4, which is associated with their greater potency. First, the effect of oxygen tension and cell density on the growth rate and potency of MAPC were examined. MAPC exhibited an increased growth rate at hypoxic conditions (5%) than at normoxic conditions (21%). Furthermore, when inoculated at a cell density of 1000 cells/cm2, MAPC exhibited a small but significant increase in growth rate compared to cells seeded at 300 cells/cm2 at both oxygen levels, though the difference was more pronounced under hypoxic conditions. The Oct4 mRNA or protein expression level and the ability of MAPC to differentiate towards endothelium- and hepatocyte-, and neuroectoderm-like cells were shown to be unaffected by cultivation at a higher cell density and/or oxygen tension for 48 days (1000 cells/cm2; 21% O2; with subculturing every 48 hr). The results provide evidence that MAPC isolated under hypoxic conditions and expressing high levels of Oct4 can be readily cultured at a higher cell density without any apparent loss of potency. Encouraged by MAPCs ability to grow at high density, we explored aggregate formation of MAPC for cell expansion as well as differentiation. Culture in aggregates may be an ideal method to allow large scale expansion, if combined with bioreactor cultures. Time lapse microscopy revealed three stages during the initial period of aggregate formation: agglomeration, compaction, and expansion. Compared to cells from adherent culture, significantly more cells from 3D culture are in G0/G1 phase and fewer in S phase suggesting a partial restriction in cell proliferation possibly due to spatial restriction in aggregates. There was no significant difference in Oct4 level and aggregate size when aggregation was at 5% or 21% O2 after 4 day culture. However, aggregation at 21% O2 increased the percent of cells in G0/G1 and increased expression of early differentiation markers such as Flk1 and Afp. Cultivation of MAPC aggregates in stirred bioreactor lead to a 70-fold expansion in six days with final cell densities of about 106 cells/ml. Importantly, the MAPC aggregates recovered from stirred bioreactors could be differentiated to hepatocyte-like cells that expressed Albumin, Aat, Tat transcripts and also secreted albumin and urea.The cells expressed several mature hepatocyte-lineage genes and asialoglycoprotein receptor-1 (ASGPR-1) surface protein, and secreted albumin and urea. Lastly, the experience with MAPC microcarrier culture was extended to human embryonic stem cell (hESC) microcarrier culture. hESCs as small clumps were attached to Matrigel-coated microcarriers and expanded 10-fold during 4 day static culture. The level of pluripotency-related genes, OCT4 and SOX2, were maintained compared to day 0 cells. The cells expanded on microcarriers underwent hepatic differentiation to increase hepatic genes such as AFP and ALBUMIN. Both aggregate and microcarrier cultivation methods for scalable expansion combined with differentiation can potentially be used to generate large numbers of MAPC and MAPC-derived differentiated cells. These culture systems thus offer the potential of large-scale expansion and differentiation of stem cells in a more controlled bioreactor environment.Item A study of primary and secondary heart field populations in iPS cell-derived embryoid bodies(2013-12) Johnson, Caitlin M.As an embryo develops, the heart is the first functional organ to form. There are four cell populations that are the primary contributors to the developing heart during embryogenesis. The populations which comprise the majority of the organ are the primary and secondary heart fields. The primary heart field commits to the cardiac lineage slightly before the secondary heart field in development. During cardiac lineage commitment, cells pass through a series of stages before becoming terminally differentiated. Prior to differentiation, cells express the transcription factor Oct4 which functions to maintain the pluripotent state of the cell. Oct4 has been also proposed to play a direct role in early stages of cardiac lineage commitment. We used an Oct4-CreER reporter iPS cell line with embryoid body differentiation to determine presence of Oct4 transcript at a given time to study the cells of primary and secondary heart fields. Use of this system allowed us to separate cells based on Oct4 expression, and further characterize the gene expression in these populations. We probed for the gene expression patterns of the heart fields to test the role of Oct4 expression in the individual fields.