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Litster, Caroline Elizabeth. "Heart rate, heart rate variability, electrodermal activity and the differentiation-of-deception /." Title page, table of contents and abstract only, 2002. http://web4.library.adelaide.edu.au/theses/09SSPS/09sspsl7769.pdf.
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Hinds, Heather C. "Evaluating terminal differentiation of porcine valvular interstitial cells in vitro." Link to electronic thesis, 2006. http://www.wpi.edu/Pubs/ETD/Available/etd-050506-113014/.
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O'Brien, Meghan M. "A pilot proteomic analysis : the study of P19 cells in cardiac differentiation /." Connect to resource online, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1229374725.
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Buccini, Stephanie M. "Cardiogenic differentiation of induced pluripotent stem cells for regeneration of the ischemic heart." University of Cincinnati / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1382373160.
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Younce, Craig. "Zinc-Finger Protein MCPIP in Cell Death and Differentiation." Doctoral diss., University of Central Florida, 2009. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/2279.
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Monocyte chemotactic protein-1 (MCP-1) plays a critical role in the development of cardiovascular diseases. How MCP-1 contributes to the development of heart disease is not understood. We present evidence that MCP-1 causes death in cardiac myoblasts, H9c2 by inducing oxidative stress, ER stress and autophagy via a novel Zn-finger protein, MCP-1 induced protein (MCPIP). MCPIP expression caused cell death and knockdown of MCPIP, attenuated MCP-1 induced cell death. Expression of MCPIP resulted in induction of iNOS and production of reactive oxygen (ROS). It caused induction of NADPH oxidase subunit phox47 and its translocation to the cytoplasmic membrane. Oxidative stress led to the induction of ER stress markers HSP40, PDI, GRP78 and IRE1α. ER stress lead to autophagy as indicated by beclin-1 induction, cleavage of LC3 to LCII and autophagolysosome formation. Here, MCPIP-induced processes lead to apoptosis as indicated by caspase 3 activation and TUNEL assay. This cell death involved caspase 2 and caspase 12 as specific inhibitors of these caspases prevented MCPIP-induced cell death. Inhibitors of oxidative stress inhibited ER stress, and cell death. Specific inhibitors of ER stress inhibited autophagy and cell death. Inhibition of autophagy inhibited cell death. Microarray analysis showed that MCPIP expression caused induction of a variety of genes known to be involved in cell death. MCPIP caused activation of JNK and p38 and induction of p53 and PUMA. These results collectively suggest that MCPIP induces ROS/RNS production that causes ER stress which leads to autophagy and apoptosis through caspase 2/12 and IRE1α –JNK/p38-p53-PUMA pathway. These results provide the first molecular insights into the mechanism by which elevated MCP-1 levels associated with chronic inflammation may contribute to the development of heart failure.
A role for inflammation and MCP-1 in obesity and diabetes has been implicated. Adipogenesis is a key process involved in obesity and associated diseases such as type 2 diabetes. This process involves temporally regulated genes controlled by a set of transcription factors, C/EBPβ, C/EBPδ, C/EBPα, and PPARγ. Currently PPARγ is considered the master regulator of adipogenesis as no known factor can induce adipogenesis without PPARγ. We present evidence that a novel Zn-finger protein, MCPIP, can induce adipogenesis without PPARγ. Classical adipogenesis-inducing medium induces MCP-1 production and MCPIP expression in 3T3-L1 cells before the induction of the C/EBP family of transcription factors and PPARγ. Knockdown of MCPIP prevents their expression and adipogenesis. Treatment of 3T3-L1 cells with MCP-1 or forced expression of MCPIP induces expression of C/EBPβ, C/EBPδ, C/EBPα, PPARγ and adipogenesis without any other inducer. Forced expression of MCPIP induces adipogenesis in PPARγ-/- fibroblasts. Thus, MCPIP is a newly identified master controller that can induce adipogenesis without PPARγ.
Heart failure is a major cause of death in diabetic patients. Hyperglycemia is a major factor associated with diabetes that causes cardiomyocyte apoptosis that leads to diabetic cardiomyopathy. Cardiomyoycte apoptosis is a key event involved in the pathophysiological progression of diabetic cardiomyopathy. We have recently found that in ischemic hearts, MCP-1 can induce the zinc-finger protein, MCP-1 induced protein (MCPIP) that causes cardiomyocyte apoptosis. Although there is evidence that inflammation may play a role in diabetic cardiomyopathy, the underlying mechanisms are poorly understood. In this study, we show that treatment of H9c2 cardiomyoblasts and Neonatal Rat Ventricular Myocytes (NRVM) with 28mmol/L glucose concentration results in the induction of both transcript and protein levels of MCP-1 and MCPIP. Inhibition of MCP-1 interaction with CCR2 via specific antibody or with the G-coupled receptor inhibitors propagermanium and pertussis toxin attenuated glucose-induced cell death. Knockdown of MCPIP with specific siRNA yielded similar results. Treatment of cells with 28mmol/L glucose resulted in increased ROS production and phox47 activation. Knockdown of MCPIP attenuated these effects. The increased ROS production observed in H9c2 cardiomyoblasts and NRVM’s resulted in increased ER stress proteins GRP78 and PDI. Knockdown of MCPIP attenuated expression of both GRP78 and PDI. Inhibition of ER stress with TUDC and 4’PBA prevented high glucose-induced cell death death. Treatment of cells with 28mmol/l glucose resulted in autophagy as determined by an increase in expression of beclin-1 and through increased cleavage of LC3I to LC3II. Knockdown of MCPIP attenuated expression of beclin-1 and prevented cleavage of LC3. Addition of the autophagy inhibitors 3’methyladenine and LY294002 attenuated high glucose-induced H9c2 cardiomyoblast death. We conclude that high glucose-induced H9c2 cardiomyoblast death is mediated via MCP-1 induction of MCPIP that results in ROS that leads to ER stress that causes autophagy and eventual apoptosis.<br>Ph.D.<br>Department of Biomolecular Science<br>Burnett College of Biomedical Sciences<br>Biomedical Sciences PhD
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Wei, Wenjie, and 魏闻捷. "Calcium signaling in the cardiac differentiation of mouse embryonic stem cells." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hub.hku.hk/bib/B49617862.
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Intracellular Ca2+ mobilization via secondary messengers modulates multiple cell functions. Cyclic Adenosine 5’-Diphosphate-Ribose (cADPR) is one of the most well recognized endogenous Ca2+ mobilizing messengers. In mammalian, cADPR is mainly formed by CD38, a multi-functional enzyme, from nicotinamide adenine dinucleotide (NAD). It has previously been shown that the cADPR/CD38/Ca2+pathway mediates many cardiac functions, such as regulating the excitation-contraction coupling in cardiac myocytes and modulating the Ca2+ homeostasis during the ischemia injury of the heart. Thus it is reasonable to propose that the cADPR/CD38/Ca2+ pathway plays a role in cardiogenesis. The pluripotent mouse embryonic stem (mES) cells which can be induced to differentiate into all cell types provide an ideal model for studying cardiogenesis. The first part of this dissertation is to determine the role of CD38/cADPR/Ca2+pathwayin the cardiomyogenesis of mES cells. The data showed that CD38 expression was markedly up-regulated during the in vitro embryoid body (EB) differentiation of mouse ES cells, which indicated a regulatory role of CD38 in the differentiation process. Lentivirus mediated shRNA provides a convenient method to knockdown the expression of CD38 in mES cells. Surprisingly, beating clusters appeared earlier and more in CD38 knockdown EBs than that in control EBs. Likewise, the expressions of several cardiac markers were up regulated in CD38 knockdown EBs. In addition, more cardiomyocytes (CMs) existed in CD38 knockdown or 8-Br-cADPR, a cADPR antagonist, treated EBs than those in control EBs. On the other hand, over-expression of CD38 in mouse ES cells significantly inhibited CM differentiation. Moreover, we showed that CMs derived from the CD38 knock down mES cells possessed the functional properties characteristic of CMs derived fromnormal ES cells. Last, we showed that the CD38-cADPR pathway negatively modulated the FGF4-Erks1/2cascade during CM differentiation of mES cells, and transiently inhibition of Erk1/2 blocked the enhancive effects of CD38 knockdown on the differentiation of CM from mES cells. Taken together, our data indicate that the CD38/cADPR/Ca2+ signaling pathway suppresses the cardiac differentiation of mES cells.
One of the main goals of the researches on cardiac differentiation of ES cells is to enhance the production of CMs from ES cells, thereby providing sufficient amount of functional intact CMs for the treatment of severe heart disease. Nitric oxide (NO) has been found to be a powerful cardiogenesis inducer of mES cells, in that it can significantly increase the yield of ES-derived CM. The second objective of this dissertation is to explore the mechanism underlying the NO facilitated cardiomyogenesis of mES cells. We found that the NO did induce intracellular Ca2+ increases in mES cells, and this Ca2+ increase was due to internal Ca2+ release from ER through theIP3 pathway. Therefore, the expression of IP3 receptors (IP3Rs) in mES cells were knocked down by lentivirus-mediated shRNAs. Interestingly, only type 3 IP3R (IP3R3) knockdown significantly inhibited the NO induced Ca2+ release in mES cells. Moreover, NO facilitated cardiogensis of mES cells was abolished in IP3R3 knockdown EBs. In summary, our results indicate that the IP3R3-Ca2+ pathway is required for NO facilitated cardiomyogenesis of mES cells.<br>published_or_final_version<br>Physiology<br>Doctoral<br>Doctor of Philosophy
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Chau, Dinh Le Mary. "Role of Notch1 in Cardiac Cell Differentiation and Migration: A Dissertation." eScholarship@UMMS, 2007. https://escholarship.umassmed.edu/gsbs_diss/338.
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The cardiac conduction system is responsible for maintaining and orchestrating the rhythmic contractions of the heart. Results from lineage tracing studies indicate that precursor cells in the ventricles give rise to both cardiac muscle and conduction cells. Using chick embryonic hearts, we have found that Notch signaling plays an important role in the differentiation of cardiac muscle and conduction cell lineages in the ventricles. Notch1 expression coincides with a conduction marker at early stages of conduction system development. Mis-expression of constitutively active Notch1 (NIC) in early heart tubes exhibited multiple effects on cardiac cell differentiation. Cells expressing NIC had a significant decrease in the expression of cardiac muscle markers, but an increase in the expression of conduction cell markers. Loss-of-function studies further support that Notch1 signaling is important for the differentiation of these cardiac cell types. Functional electrophysiology studies show that the expression of constitutively active Notch1 resulted in abnormalities in ventricular conduction pathway patterns.
During cardiogenesis, groups of myocardial cells become separated from each other, and migrate to form the trabeculae. These finger-like projections found within the ventricular chamber coalesce to generate the muscular portions of the interventricular septum, the thickened myocardium, and future sites of the conduction system. We have found that Notch signaling regulates the migration of cardiac cells during cardiogenesis. Over-expression of constitutively active Notch causes cells to localize more centrally within the heart, while loss-of-Notch function results in cells distributed within the periphery of the heart. Staining of heart sections shows that Notch signaling regulates the expression of N-cadherin, the predominant adhesion molecule in cardiomyocytes. We find that the effects of Notch on cell migration are two-fold: delamination and cell motility. Time-lapse studies demonstrate that Notch signaling increases cell motility, but does not affect speed or directionality of migration. Furthermore, we find that the effects of Notch on cell migration is independent of its effects on differentiation.
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Maliken, Bryan D. B. A. "Gata4-Dependent Differentiation of c-Kit+ Derived Endothelial Cells Underlies Artefactual Cardiomyocyte Regeneration in the Heart." University of Cincinnati / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1535375861364685.
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Huang, Tianfang. "Mechanism of Arsenical Toxicity on TGFβ Signaling and Genetic Regulation During Cardiac Progenitor Cell Differentiation". Diss., The University of Arizona, 2015. http://hdl.handle.net/10150/556428.
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Low to moderate level of chronic arsenic exposure contributes to cardiovascular ailments including heart disease and aneurysms. Current research on the etiology and progression of cardiovascular disease focuses mainly on adult which fails to capture the developmental origins of cardiovascular disease. Thus, disruption in morphogenetic events during early development may initiate and pattern the molecular programming of cardiovascular ailments in adulthood. A major contributor to ischemic heart pathologies is coronary artery disease, however the influences by environmental arsenic in this disease process are not known. Similarly, the impact of toxicants on blood vessel formation and function during development has not been studied. Coronary vessel development is initiated by precursor cells that are derived from the epicardium. Epicardial derived cells undergo proliferate, migrate, and differentiate into several cardiac cell types which are the cellular components of the coronary vessels. The key cellular event occurs in this process is the epithelial to mesenchymal transition (EMT), which can also be utilized by endocardial cushion cells to form aortic and pulmonary valves. The TGFβ family of ligands and receptors are essential for developmental cardiac EMT and coronary smooth muscle cell differentiation. Whether arsenic has any impact on TGFβ mediated cardiovasculogenesis is not known. Monomethylarsonous acid [MMA(III)] is the most potent metabolite of inorganic arsenic and has been shown to partly account for arsenic induced toxicity. The fetus is exposed to relatively higher levels of MMA (III) as compared to adults probably due to deficiency in methylation of transferred inorganic arsenic from the placenta. However, the developmental toxicity of MMA (III) has not yet been studied. In this study, we exploit a novel cardiac progenitor cell line to recapitulate epicardial EMT in vitro and to study developmental toxicity caused by arsenicals. We show that chronic exposure to low level of arsenite and MMA (III) disrupts developmental EMT programming in epicardial cells causing deficits in cardiac mesenchyme production. The expression of EMT program genes is also decreased in a dose-dependent manner following exposure to arsenite and MMA (III). Smad-dependent TGFβ2 canonical signaling and the non-canonical Erk signaling pathways are abrogated as detected by decreases in phosphorylated Smad2/3, Erk1/2 and Erk5 proteins. There is also loss of nuclear accumulation of p-Smad and p-Erk5 due to arsenical exposure. These observations coincide with a decrease in vimentin positive mesenchymal cells invading three-dimensional collagen gels. However, arsenicals do not block TGFβ2 stimulated p38 activation. Additionally, smooth muscle cell differentiation, which is proven to be governed by p38 signaling in epicardial cells, also remains intact with arsenical exposure. Overall these results show that arsenite and MMA (III) are strong and selective cardiac silencers. The molecular mechanisms of arsenical toxicity on TGFβ-Smad signaling in epicardial cells is further explored. A relatively high level of acute arsenical exposure rapidly depletes phosphorylated nuclear Smad2/3. Restoration of the nuclear accumulation of Smads can be achieved by inhibiting the expression or activation of Smad specific exportins suggesting that arsenicals augment Smad nuclear exportation. Abrogated Smad signaling caused by arsenicals is associated with severe deficits in EMT during mouse epicardium and chick endocardial cushion development. Thus progenitor cell outgrowth, migration, invasion and vimentin filament reorganization are significantly inhibited in response to arsenical exposure. Disrupted Smad nuclear shuffling is probably caused by zinc displacement on the MH-1 DNA binding domain of Smad2/3. Thus zinc supplementation restores both nuclear content and transcriptional activities of Smad2/3. Rescued TGFβ-Smad signaling by zinc also contributes to cellular transformation and mesenchyme production in embryonic heart explants. LINE1 (L1) retrotransposons are a group of mobile DNA elements that shape the genome via novel epigenetic controls. Although expression of L1 is required for early embryo implantation and development, abnormally elevated L1 is shown to inhibit embryonic cells from transforming and differentiating during organogenesis. Cellular redox signaling, which is regulated by antioxidant responsive elements (AREs), has been shown to play a key role in L1 activation and retrotransposition. However, whether L1 can be induced by the cellular oxidative stress caused by arsenic is not known. We provide evidence showing that L1 ORF-1 and ORF-2 mRNA levels are both up-regulated by arsenic. Nuclear accumulation of L1 ORF-2 is observed in response to 30 min arsenic exposure, which may lead to active retrotransposition events in the genome. Transcriptional activity of L1 is regulated by Nrf2 as mutations in ARE regions within the L1 promoter and Nrf2 silencing using siRNA both significantly inhibit L1 transcriptional activity. Nrf2 overexpression together with arsenic exposure creates synergic induction in L1 promoter activity suggesting that arsenic mediated L1 activation is partially Nrf2 dependent. Taken together, these findings reveal a molecular mechanism responsible for arsenic cardiac toxicity and define a novel genetic toxic effect of arsenic during embryonic heart development.
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Momtahan, Nima. "Extracellular Matrix from Whole Porcine Heart Decellularization for Cardiac Tissue Engineering." BYU ScholarsArchive, 2016. https://scholarsarchive.byu.edu/etd/6225.
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Heart failure is one of the leading causes of death in the United States. Every year in the United States, more than 800,000 people are diagnosed with heart failure and more than 375,000 people die from heart disease. Current therapies such as heart transplants and bioartificial hearts are helpful, but not optimal. Decellularization of porcine whole hearts followed by recellularization with patient-specific human cells may provide the ultimate solution for patients with heart failure. Great progress has been made in the development of efficient processes for decellularization, and the design of automated bioreactors. In this study, the decellularization of porcine hearts was accomplished in 24 h with only 6 h of sodium dodecyl sulfate (SDS) exposure and 98% DNA removal. Automatically controlling the pressure during decellularization reduced the detergent exposure time while still completely removing immunogenic cell debris. Stimulation of macrophages was greatly reduced when comparing native tissue samples to the processed ECM. Complete cell removal was confirmed by analysis of DNA content. General collagen and elastin preservation was demonstrated by SEM and histology. The compression elastic modulus of the ECM after decellularization was lower than native at low strains but there was no significant difference at high strains. Polyurethane casts of the vasculature of native and decellularized hearts demonstrated that the microvasculature network was preserved after decellularization. A static blood thrombosis assay using bovine blood was also developed. A perfusion bioreactor was designed and right ventricle of the decellularized hearts were recellularized with human endothelial cells and cardiac fibroblasts. An effective, reliable, and relatively inexpensive assay based on human blood hemolysis was developed for determining the remaining cytotoxicity of the cECM and the results were consistent with a standard live/dead assay using MS1 endothelial cells incubated with the cECM. Samples from the left ventricle of the hearts were prepared with 300 µm thickness, mounted on 10 mm round glass coverslips. Human induced pluripotent stem cells were differentiated into cardiomyocytes (CMs) and 4 days after differentiation, cardiac progenitors were seeded onto the decellularized cardiac slices. After 10 days, the tissues started to beat spontaneously. Immunofluorescence images showed confluent coverage of CMs on the decellularized slices and the effect of the scaffold was evident in the arrangement of the CMs in the direction of fibers. This study demonstrated the biocompatibility of decellularized porcine hearts with human CMs and the potential of these scaffolds for cardiac tissue engineering. Further studies can be directed toward 3D perfusion recellularization of the hearts and improving repopulation of the scaffolds with various cell types as well as adding mechanical and electrical stimulations to obtain more mature CMs.
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Sargent, Carolyn Yeago. "Effects of hydrodynamic culture on embryonic stem cell differentiation: cardiogenic modulation." Diss., Georgia Institute of Technology, 2010. http://hdl.handle.net/1853/34710.
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Stem and progenitor cells are an attractive cell source for the treatment of degenerative diseases due to their potential to differentiate into multiple cell types and provide large cell yields. Thus far, however, clinical applications have been limited due to inefficient differentiation into desired cell types with sufficient yields for adequate tissue repair and regeneration. The ability to spontaneously aggregate in suspension makes embryonic stem cells (ESCs) amenable to large-scale culture techniques for the production of large yields of differentiating cell spheroids (termed embryoid bodies or EBs); however, the introduction of hydrodynamic conditions may alter differentiation profiles within EBs and should be methodically examined. The work presented here employs a novel, laboratory-scale hydrodynamic culture model to systematically interrogate the effects of ESC culture hydrodynamics on cardiomyocyte differentiation through the modulation of a developmentally-relevant signaling pathway. The fluidic environment was defined using computational fluid dynamic modeling, and the effects of hydrodynamic conditions on EB formation, morphology and structure were assessed. Additionally, EB differentiation was examined through gene and protein expression, and indicated that hydrodynamic conditions modulate differentiation patterns, particularly cardiogenic lineage development. This work illustrates that mixing conditions can modulate common signaling pathways active in ESC differentiation and suggests that differentiation may be regulated via bioprocessing parameters and bioreactor design.
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Maddali, Kamala Kalyani. "A mandatory requirement of PKC-[delta] in testosterone regulated coronary smooth muscle cell differentiation, proliferation and apoptosis /." Free to MU Campus, others may purchase, 2005. http://proquest.umi.com/pqdweb?did=1232392431&sid=1&Fmt=2&clientId=45247&RQT=309&VName=PQD.
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Henje, Blom Eva. "Anxiety and depression in adolescent females autonomic regulation and differentiation /." Stockholm, 2010. http://diss.kib.ki.se/2010/978-91-7409-807-5/.
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SHELTON, ELAINE L. "Thr roles of Twist1 and Tbx20 in endocardial cell proliferation, migration, and differentiation during endocardial cushion development." University of Cincinnati / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1212062811.
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Farouz, Yohan. "Designing biomaterials for controlled cardiac stem cell differentiation and enhanced cell therapy in the treatment of congestive heart failure." Thesis, Sorbonne Paris Cité, 2015. http://www.theses.fr/2015USPCB114/document.
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La thérapie cellulaire se positionne comme une stratégie prometteuse pour inciter le cœur infarci à se régénérer. A cet effet, des études récentes placent des espoirs considérables dans l’utilisation des cellules souches embryonnaires et notre laboratoire a déjà démontré comment les différencier en progéniteurs cardiovasculaires, un type de précurseurs cellulaires qui ne peut aboutir qu’à la formation de cardiomyocytes, de cellules endothéliales ou de cellules de muscles lisses. Cet engagement précoce réduit leur capacité de prolifération anarchique et en même temps leur permet de rester suffisamment plastiques pour éventuellement s’intégrer plus facilement avec le tissue hôte. Cependant, les études précliniques et cliniques d’injection de ces cellules s’avérèrent décevantes. Malgré de légères améliorations de la fonction cardiaque, on observa une trop faible survie cellulaire ainsi qu’un taux de rétention des cellules dans le myocarde remarquablement bas. Afin d’étudier ce problème, mes travaux de thèse ont porté non seulement sur la conception de nouveaux biomatériaux pouvant servir de moyen de transport et d’intégration des cellules dans la zone infarcie, mais aussi sur la conception de biomatériaux permettant de contrôler précisément l’environnement cellulaire au cours du processus de différenciation de cellules souches pluripotentes humaines en cardiomyocytes. Grâce aux importantes interactions entre nos laboratoires de recherche fondamentale et de recherche clinique, nous avons tout d’abord développé de nouvelles techniques de fabrication et de caractérisation de patches de fibrine cellularisés qui sont récemment entrés dans un essai clinique de phase I. A partir de cette formulation clinique approuvée par les autorités de régulation, nous avons élaboré toute une gamme de matériaux composites uniquement à base de matières premières pertinentes dans ce cadre clinique, dans le but d’améliorer la maturation des progéniteurs cardiovasculaires une fois greffés sur le cœur défaillant. Dans cette optique, nous avons également développé un modèle in vitro permettant d’étudier précisément l’influence combinée de la rigidité du substrat et du confinement spatial sur la différenciation des cellules souches en cardiomyocytes. Grâce à des techniques de microfabrication sur substrat mou, il a été possible de positionner précisément les cellules souches pluripotentes dans des espaces restreints d’élasticité variable. Ainsi, nous avons pu observer que même en utilisant des protocoles chimiques éprouvés basés sur la modulation de cascades de signalisation impliquées dans le développement cardiaque, une très forte hétérogénéité pouvait apparaître en fonction de l’environnement physique des cellules. Nous avons ainsi pu extraire les caractéristiques principales permettant une différenciation cardiaque efficace, reproductible et standardisée et les avons appliquées à la fabrication d’une nouvelle génération de patches composés de matériaux cliniques et de couches multiples de bandes synchrones de cardiomyocytes. De fait, ces travaux ouvrent de nouvelles voies dans l’utilisation de biomatériaux pour la production industrielle de cardiomyocytes et pour la fabrication de patches cliniques, cellularisés ou non, dans le traitement de l’insuffisance cardiaque<br>Cell therapy is a promising strategy to help regenerate the damaged heart. Recent studies have placed a lot of hopes in embryonic stem cells and our lab had previously found a way to differentiate them into cardiac progenitors, cells that can only differentiate into cardiomyocyte, endothelial cells or smooth muscle cells. This early commitment decreases their proliferative capabilities, yet maintains their plasticity for better integration inside the host tissue. However, clinical and pre-clinical injection studies did not really meet the expectations. Even though slight improvements in cardiac function were demonstrated, very low cell viability has been observed, as well as a very low retention of the cells inside the myocardium. To address this problem, my PhD projects not only focus on the design of new biomaterials to act as a vehicle for cell delivery and retention in the infarcted area, but also on the design of biomaterials that control the cellular environment during the differentiation of pluripotent stem cells into cardiomyocytes. Going back and forth between the labs and the clinics, we first developed new techniques for the fabrication and the characterization of a cell-laden fibrin patch that is now undergoing phase I clinical trial. From the approved clinical formulation, we then propose new blends of clinical materials that will eventually improve the maturation of the cardiac progenitors once grafted onto the failing heart. In this perspective, we developed an in vitro model to investigate the combined influence of matrix elasticity and topographical confinement on stem cell differentiation into cardiomyocytes. By using microfabrication techniques to pattern pluripotent stem cells on substrates of controlled stiffness, we demonstrate that even using a widely recognized chemical-based protocol to modulate signaling cascades during differentiation, much heterogeneity emerges depending on the cellular physical environment. We thus extracted the main features that led to controlled and reproducible cardiac differentiation and applied it to the fabrication of next generation of multi-layered anisotropic cardiac patches in compliances with clinical requirements. This work opens new routes to high-scale production of cardiomyocytes and the fabrication of cell-laden or cell-free clinical patches
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Olson, Erik Ryan. "Signaling mechanisms controlling the proliferation and differentiation of cardiac fibroblasts." [Kent, Ohio] : Kent State University, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=kent1162230649.
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Thesis (Ph.D.)--Kent State University, 2006.<br>Title from PDF t.p. (viewed Jan. 11, 2007 ) Advisor: J Gary Meszaros. Keywords: cardiac fibroblast, angiotensin II, fibrosis, MAPK Includes bibliographical references (p. 150-168).
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Swinarski, Marie. "PCP-driven cardiac remodeling couples changes in actomyosin tension with myocyte differentiation." Doctoral thesis, Humboldt-Universität zu Berlin, Lebenswissenschaftliche Fakultät, 2017. http://dx.doi.org/10.18452/17775.
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Im Zuge der frühen embryonalen Herzentwicklung entstehen ausgehend von einem einfachen Herzschlauch zwei deutlich voneinander getrennte Herzkammern. Die Kardiomyozyten des Atriums und Ventrikels weisen spezifische Eigenschaften auf, die sich morphologisch wie auch funktionell auf das Herz auswirken. Veränderungen in der Gewebsarchitektur werden hauptsächlich durch Zellinterkalation und kollektive Zellmigration erreicht. Viele Studien zeigen, dass der Wnt/PCP-Signalweg eine essentielle Rolle in der Regulation dieser Bewegungen einnimmt. Die Daten dieser Studie belegen, dass die nicht-kanonischen Liganden Wnt11 und Wnt5b sowie die Kernkomponenten des PCP Signalweges Fzd7, Vangl2, Dvl2 und Pk1 an der Steuerung der Reorganisation der Kardiomyozyten während der Kammerbildung beteiligt sind, was Einfluss auf die Architektur des frühen Myokardiums nimmt. Effektoren des PCP Signalweges umfassen das Zytoskelett sowie Adhäsions- und Migrationsprozesse. In dieser Studie wird gezeigt, dass die Komponenten dieses Signalweges im Myokardium hauptsächlich Prozesse der Actomyosin Modulation regulieren und damit unter anderem die Morphologie der Kardiomyozyten beeinflussen. Zusätzlich ist die frühe Kardiogenese durch eine Relokalisierung der phosphorylierten Form der Myosin Regulatory Light Chain (MRLC) vom Kern zur Membran gekennzeichnet. Hier wird gezeigt, dass die Phosphorylierung von MRLC sowie die Relokalisation von den Kernkomponenten des PCP Signalweges kontrolliert werden sowie dass es im Verlauf der frühen Herzentwicklung u.a. durch die Relokalisierung von pMRLC zu Änderungen in der Gewebespannung kommt, welche sich auf die nukleäre Spannung auswirken und damit Veränderungen in der Genregulation hervorrufen. Diese Veränderungen werden hauptsächlich durch Effekte auf die Lokalisation und Aktivität des Serum Response Factors (SRF) vermittelt, welche in diesem Kontext durch die PCP Kernkomponente Pk1 reguliert sind.<br>Formation of a complex multiple-chambered heart from the simple linear heart tube does not only require orchestrated morphogenesis of the myocardium, but also cardiac muscle differentiation and changes in intercellular electrical coupling. To date, the processes that lead to the formation of a functional syncytium are incompletely understood. One of the major pathways controlling multiple aspects of organogenesis and tissue morphogenesis is the planar cell polarity (PCP) pathway. Changes in tissue architecture are controlled by cell intercalation and collective cell migration. It is widely accepted that Wnt/PCP signaling plays a crucial role in guiding these cellular processes. This study provides evidence that morphogenesis of the heart is controlled by the non-canonical ligands Wnt11 and Wnt5b and the PCP core components Fzd7, Vangl2, Dvl2, and Pk1 through regulation of cell rearrangements during embryonic cardiac remodeling. Downstream effectors of the PCP pathway target adhesion processes, cytoskeleton, and migration. Here, it is revealed that PCP signaling in the heart affects cardiomyocyte morphology and actomyosin organization. Specifically, changes in the subcellular localization of the phosphorylated non-muscle myosin II regulatory light chain (pMRLC) at LHT stage are targeted by the PCP pathway core components. Furthermore, actomyosin relocalization concurs with changes in nuclear tension and SRF signal transduction within the myocardium. This study unravels a novel function of the PCP core component Pk1 in regulation of SRF translocation and target gene expression that is critical to cardiac maturation. Taken together, this study provides evidence that the PCP pathway is a major regulator of cardiac remodeling and organ maturation by modulating mechanosensitive SRF signal transduction involved in muscle differentiation.
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Self, Magdalen Romany Rowen. "Repair of a wounded heart : an investigation into the effects of aggregation on the differentiation of stem cells to cardiomyocytes." Thesis, University of Nottingham, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.523583.
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Di, Guglielmo Claudia. "Biotechnological approaches to cardiac differentiation of human induced pluripotent stem cells." Doctoral thesis, Universitat de Barcelona, 2016. http://hdl.handle.net/10803/385921.
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The heart can be considered the most important organ of our body, as it supplies nutrients to all the cells. When affected from injuries or diseases, the heart function is hampered, as the damaged area is substituted by a fibrotic scar instead of functional tissue. Understanding the mechanisms leading to heart failure and finding a cure for cardiac diseases represents a major challenge of modern medicine, since they are the leading cause of death and disability in Western world. Being the heart a vital organ it is difficult to have access to its cells, especially in humans. In order to model it or find therapeutic strategies many approaches and cell sources have been studied. For example cardiac stem cells, skeletal myoblasts, bone marrow-derived cells and peripheral blood mononuclear cells have been tested in pre-clinical and clinical trials, without significant tissue regeneration. Human pluripotent stem cells (hPSC) are thought to be the most promising cell type in the field, thanks to their unlimited capacity of self-renewal and retention of differentiation potency. Induced pluripotent stem cells (iPSC) are pluripotent cells derived through reprogramming from adult cells, easily accessible from patients, like keratinocytes. iPSC can be differentiated to cardiac cells, through stage-specific protocols that reproduce embryonic development, offering a very useful platform for modelling diseases of patients with heart failure, for testing new drugs, and for cellular therapy in the future. However, properly mimicking cardiac tissue is very complex, since not only the correct cardiac cell type has to be reproduced, but also its overall cellular composition, architecture and biophysical functions.
In order to study these aspects, we applied biotechnological strategies such as the use of transgenic cell lines for obtaining pure and scalable differentiated cells to be cultured in a 3D scaffold with a perfusion bioreactor. Although it is well known that iPSC can give rise to cardiomyocytes in vitro, not every cell line can be efficiently differentiated. Thus, a cell line-specific differentiation protocol has to be identified and optimized. We finally identified a fast and efficient stage-specific differentiation protocol suitable for the iPSC lines used in this work, derived from human keratinocytes. With this protocol, we can reproducibly obtain close to 50% cardiomyocytes after 15 days of differentiation. One important feature of currently available differentiation protocols is that the target cell type is obtained among a heterogeneous cell population. To track the cardiac population of interest we generated transgenic cell lines where the reporter protein GFP follows the expression of different genes specific for stages of differentiation, such as T (Brachyury) for mesoderm; NKX2.5 for cardiac progenitors; and MHC for cardiomyocytes. Moreover, cardiomyocytes obtained from hPSC using currently available differentiation protocols are typically immature, mostly resembling embryonic or fetal cardiomyocytes, arguably because of the lack of mechanical and electrical stimuli that only a 3D environment can provide. In order to create a piece of tissue in 3D we used a collagen and elastin-based scaffold, to mimic the structural proteins of endogenous extracellular matrix. We also built a perfusion bioreactor to culture the construct. After initial validation with primary cultures of rat neonatal cardiomyocytes, we tested iPSC-derived cardiac cells at different stages of differentiation. While early mesoderm or cardiac progenitors could not survive in our system, iPSC differentiated to cardiomyocytes, could be retained and maintained alive within the scaffold for at least 4 days.
In conclusion, in this work we combined biotechnological tools in order to obtain a test platform for studying the mechanisms underlying cardiac differentiation, maturation, as well as providing valuable in vitro systems for disease modelling, drug screening of patient-specific heart muscle cells and cell therapy.<br>El corazón es el órgano más importante del cuerpo: impulsando la sangre, aporta oxigeno y nutrientes a cada célula del organismo. En caso de fallo cardiaco la función del corazón no puede recuperarse, ya que los cardiomiocitos son reemplazados por una cicatriz fibrosa no funcional. Las enfermedades cardiacas representan la mayor causa de muerte y enfermedad en el mundo occidental y entender los mecanismos de las patologías cardiacas, así como encontrar curas para ellas, es un desafío de primaria importancia para la medicina moderna. Siendo el corazón un órgano vital y difícilmente accesible, resulta imprescindible encontrar una fuente celular alternativa. Las células madre humanas con pluripotencia inducida (iPSC – induced pluripotent stem cells) parecen óptimas, porque se derivan de simples biopsias de piel de pacientes y se pueden diferenciar a cualquier tipo celular, cardiomiocitos incluidos. Aún así, diferenciar el tejido cardiaco es muy complejo: no solamente se debe de reproducir el tipo celular, sino también su composición celular, su arquitectura y sus funciones biofísicas. Para estudiar estos aspectos, por un lado obtuvimos tres líneas celulares de iPSC reporteras de genes específicos de diferentes estadios de diferenciación cardiaca (T para mesodermo, NKX2.5 para progenitores cardiacos y alpha-MHC para cardiomiocitos), y por otro desarrollamos un biorreactor adecuado para el cultivo de células cardiacas en 3D. Utilizamos las líneas transgénicas como herramienta para seleccionar células en diferentes estadios de diferenciación y las co-cultivamos con fibroblastos en un andamio compuesto de colágeno y elastina (imitando la matriz extracelular cardiaca y la composición celular del corazón). En conjunto, este estudio revela que las iPSC pueden ser retenidas y cultivadas en nuestro sistema 3D. Mientras células de mesodermo temprano y progenitores cardiacos no completaron la diferenciación cardiaca, los cardiomiocitos derivados de iPSC con cultivo convencional y cultivados en el biorreactor pudieron ser mantenidos viables en el mismo al menos 4 días. La aproximación experimental aquí presentada representa una base para desarrollar plataformas de estudio in vitro paciente-especificas para modelar enfermedades cardiacas humanas y estudios de fármacos, así como ofrecer una herramienta de estudio de los mecanismos de la diferenciación y maduración cardiacas.
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Moncada, Diaz Silvia Juliana. "Repopulation and Stimulation of Porcine Cardiac Extracellular Matrix to Create Engineered Heart Patches." BYU ScholarsArchive, 2018. https://scholarsarchive.byu.edu/etd/8806.
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Heart failure is the main cause of death for both men and women in the United States. The only proven treatment for patients with heart failure is heart transplantation. The goal of this research is to create patches of tissue that could mimic the function of the native heart to repair the damaged portions of the heart. In this study, whole porcine hearts were decellularized to create a 3D construct that was recellularized with cardiomyocytes (CM) differentiated from human induced pluripotent stem (IPS) cells. At day 4 of differentiation, IPS-derived CMs were implanted onto cardiac extracellular matrix (cECM) and ten days after recellularization, the cells started to beat spontaneously. After implantation, the progenitor CMs continued to proliferate and populate the cECM. A live/dead assay showed the potential of the cECM as a scaffold suitable for recellularization. Confocal microscopy images were taken to evaluate the organization of the cells within the matrix and the impact of the cECM on the growth and maturation of the CMs. Representative cardiac Troponin T (cTNT) and vimentin immunostaining images of CMs derived from iPSCs, on cECM and on standard cell culture plates showed that the cECM allowed the cells to organize and form fibrils with the fibroblasts, compared with CMs cultured in regular culture plates. The timeline of implantation of the cells was a key factor for the development of the heart tissue constructs. Progenitor CMs seeded onto cECM showed better organization and the ability to penetrate 96 µm deep within the collagen fibers and align to them. However, mature CMs seeded onto the matrix showed a disorganized network with very reduced interaction of CMs with fibroblasts, forming two different layers of cells; CMs on top of fibroblasts. In addition, the depth of penetration of the mature CMs within the matrix was only 20 µm. To evaluate the impact of the addition of support cells to the CM monolayer cultures, CMs were co-cultured with human umbilical vein endothelial cells (HUVEC) and it was demonstrated that at ratios of 2:1 HUVEC:CM the beating rate of the CMs was improved from 20 to 112 bpm, additionally, the CM monolayer cultures showed a more synchronized beating pace after the addition of HUVECs. Pharmacological stimulation was performed on CM monolayer cultures using norepinephrine as a stimulator and the results showed that the beating pace of the CMs was improved to 116 bpm after 5 minutes of drug exposure. For future studies, inosculation of the tissue constructs could be performed with the incorporation of membrane proteins to understand the mechanotransduction of the cells. As a preliminary study, the action of dual claudins was evaluated with HUVEC cultures and the results showed the potential of these membrane proteins in the healing of the damaged cell membrane.
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Zakariyah, Abeer. "The Characterization of a Human Disease-Associated Mutation Nkx2.5 R142C Using In vitro and In vivo Models." Thesis, Université d'Ottawa / University of Ottawa, 2017. http://hdl.handle.net/10393/35817.
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Nkx2.5 is a cardiac transcription factor that plays a critical role in heart development. In humans, heterozygous mutations in the NKX2.5 gene result in congenital heart defects (CHDs), but the molecular mechanisms by which these mutations cause the defects are still unknown. NKX2.5 R142C is a mutation that is found to be associated with atrial septal defect and atrioventricular block in 13 patients from one family. The R142C mutation is located within both the DNA-binding domain and the nuclear localization sequence of NKX2.5 protein. The pathogenesis of CHDs in humans with R142C point mutation is not well understood. Also, a previous study in our laboratory has identified Mypt1/PP1 as a novel interacting partner of Nkx2.5 in stem cells during cardiomyogenesis. Nkx2.5 has a PP1-binding consensus sequence RVxF located in the N-terminus of the homeodomain. Notably, the PP1-binding sequence, RVxF, is mutated from arginine to cysteine in patients with the R142C heterozygous mutation. However, the ability of the R142C mutation to bind to the Mypt1/PP1 complex has not been investigated yet. The following thesis addresses the functional deficit associated with R142C by utilizing a combination of in vitro, and in vivo models. It also addresses the interaction of Mypt1/PP1 with the R142C mutation. We have generated a heterozygous mouse embryonic stem cell (mESC) line, harboring the murine homologue (R141C) of the human mutation R142C in Nkx2.5 gene. We show reduced cardiomyogenesis and impaired subcellular localization of Nkx2.5 protein in Nkx2.5R141C/+ mESCs. Gene expression profiling of Nkx2.5R141C/+ mESCs revealed a global misregulation of genes important for heart development and identified putative direct target genes of Nkx2.5 that are affected by the R141C heterozygous mutation. We also generated a mouse model harboring the human mutation R142C. We show that the Nkx2.5R141C/R141C homozygous embryos are developmentally arrested around E10.5 with delayed heart morphogenesis. Moreover, Nkx2.5R141C/+ newborn mice are grossly normal but show variable cardiac defects and downregulation of ion channel genes that later cause AV block in adult mice. Finally, we show that the R141C mutant binds to the Mypt1/PP1 complex but is not inhibited or translocated to the perinuclear region in the presence of Mypt1/PP1 as the WT Nkx2.5 is.
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Silva, Daniela Nascimento. "Isolamento e caracterização de células-tronco obtidas de corações de camundongos adultos." reponame:Repositório Institucional da FIOCRUZ, 2014. https://www.arca.fiocruz.br/handle/icict/8001.
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Previous issue date: 2014<br>Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil<br>O uso de células-tronco representa uma alternativa para o tratamento das doenças que acometem o coração, devido à capacidade que essas células indiferenciadas têm de preservar sua própria população e de se diferenciar em células dos diversos tecidos, incluindo o cardíaco. Nesse trabalho comparamos as características de células-tronco isoladas a partir do tecido cardíaco e da medula óssea de camundongos transgênicos para a proteína fluorescente verde (GFP). As células-tronco cardíacas e da medula óssea apresentaram característica morfológica fibroblastóide e imunofenotípica de células-tronco mesenquimais, com alta expressão dos marcadores CD44, CD90, CD73, Sca-1 e baixa expressão dos marcadores de células hematopoiéticas. A análise citogenética revelou um cariótipo poliplóide a partir da terceira passagem das células-tronco isoladas do coração e da medula-óssea. A capacidade de diferenciação em vários tipos celulares, tais como adipócitos, osteócitos e condrócitos, também foi avaliada nas células-tronco de ambas as fontes. Tanto as células-tronco isoladas do coração como da medula óssea foram capazes de se diferenciar nessas três linhagens. Quando estimuladas com 5’azacitidina para testar o potencial cardiomiogênico das células isoladas do coração e da medula óssea, apenas as células-tronco cardíacas passaram a expressar alguns marcadores de cardiomiócitos, tais como troponina T cardíaca e GATA-4. As células-tronco cardíacas GFP+ foram injetadas na parede lateral do ventrículo esquerdo de camundongos C57BL/6. Nenhum animal morreu durante o procedimento, e os parâmetros funcionais cardíacos mantiveram-se inalterados. Após 48 horas e uma semana depois da injeção foi possível observar células GFP+ em secções do miocárdio. Os resultados indicam que células-tronco isoladas do coração e da medula óssea possuem características similares, porém o potencial cardiomiogênico das células-tronco cardíacas é maior. A injeção intramiocárdica mostrou-se segura podendo ser candidata à via de administração de células no miocárdio. Novos estudos no campo da medicina regenerativa que visam a utilização de células-tronco cardíacas poderão ser úteis para demonstrar sua aplicação clínica como opção de tratamento para as doenças cardíacas.<br>Stem cells are undifferentiated cells with the ability of self-renewal and differentiation into different cell types, with the potential to treat heart diseases. In the present study we compared the characteristics of stem cells isolated from the heart to bone marrow stem cells, both obtained from EGFP transgeneic mice. Cardiac and bone marrow stem cells presented fibroblastic morphology and an immunophenotype compatible with mesenchymal stem cells – high expression of CD44, CD90, CD73, Sca-1 and low expression of of hematopoietic lineage markers. Cytogenetic analysis demonstrated polyploid karyotypes after the third passage of the stem cells isolated from heart and bone marrow. Both bone marrow and heart stem cells were able to differentiate into adipocytes, osteocytes and chondrocytes. In order to test the potential of differentiation into cardiomyocytes, cells were stimulated with 5’azacytidine and only cardiac stem cells expressed heart-specific markers: cardiac T troponin and GATA-4. GFP+ cardiac stem cells were injected into the lateral wall of the left ventricule of C57bl/6 mice. The procedure did not alter the functional cardiac parameters or induced mortality. GFP+ cells were observed in the heart 48 hours and seven days after the intramyocardial injection. The results indicate that cardiac stem cells and bone marrow stem cells are similar cell populations, although cardiac stem cells appear to have an increased cardiomyogenic potential. Intramyocardial injection was a safe and useful procedure for the transplantation of cardiac stem cells. New studies in the field of regenerative medicine aimed at the use of cardiac stem cells may be useful to demonstrate its clinical application as a treatment option for heart disease.
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Bondue, Antoine. "Mesp1 functions in multipotent cardiovascular progenitor specification." Doctoral thesis, Universite Libre de Bruxelles, 2009. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210319.
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During embryonic development, multipotent cardiovascular progenitor cells (MCPs) are specified from early mesoderm. Although the core cardiac transcriptional machinery acting during cardiac cell differentiation is relatively well known, the molecular mechanism acting upstream of these cardiac transcriptional factors, and promoting cardiac progenitor specification from early mesoderm remains poorly understood. We used embryonic stem cell (ESC) differentiation as a model to dissect the molecular mechanisms implicated in cardiovascular progenitor specification. Using ESCs, in which gene expression can be temporally regulated, we showed that transient expression of Mesp1 dramatically accelerates and enhances multipotent cardiovascular progenitor specification through an intrinsic and cellular autonomous mechanism. Using genome wide transcriptional analysis, we found that Mesp1 rapidly activates and represses a discrete set of genes. Using chromatin immunoprecipitation, we showed that Mesp1 directly binds to regulatory DNA sequences located in the promoter of many key genes belonging to the core cardiac transcriptional machinery, resulting in their rapid upregulation. Mesp1 also directly and strongly represses the expression of key genes regulating other early mesoderm and endoderm cell fates. Using engineered ESC expressing the green fluorescent protein under the control of the Mesp1 promoter, we isolated Mesp1 expressing cells in differentiating ESCs allowing characterization of the cellular and molecular mechanisms underlying cardiovascular specification. Our results demonstrate that Mesp1 acts as a key regulatory switch during cardiovascular specification, residing at the top of the hierarchy of the gene network responsible for cardiovascular cell fate determination. Moreover our results place Mesp1 upstream of the specification of both first and second heart fields and provide novel and important insights into the molecular mechanisms underlying the earliest step of cardiovascular specification. We identified cell surface markers expressed allowing the isolation of early cardiovascular progenitors and provide potentially novel methods for dramatically increasing the number of cardiovascular cells for cellular therapy in humans.<br>Doctorat en sciences médicales<br>info:eu-repo/semantics/nonPublished
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Paiva, Solenne. "Facteurs environnementaux et épigénétiques impliqués dans la différenciation cardiaque de cellules souches humaines pluripotentes induites MiRroring the Multiple Potentials of MicroRNAs in Acute Myocardial Infarction Acellular therapeutic approach for heart failure: in vitro production of extracellular vesicles from human cardiovascular progenitors." Thesis, Sorbonne université, 2019. http://www.theses.fr/2019SORUS457.
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L’objectif de cette thèse a été d’évaluer certains paramètres physiques et épigénétiques impliqués dans la différenciation cardiaque de cellules souches humaines pluripotentes induites. Un premier paramètre physique souvent sous-évalué a été étudié, celui de la rigidité. Classiquement, les cellules souches sont cultivées et adaptées à des rigidités in vitro allant de 1-10 GPa très éloignées des valeurs physiologiques, de l’ordre du kPa. L’impact de support de culture à 3, 12 et 25 kPa a été évalué sur les cellules souches initiales. Les résultats montrent que des rigidités inférieures à 25 kPa ne permettent pas le maintien de la pluripotence au bout de 6 passages. De plus, les colonies cellulaires se développent en 3D et créent leur propre microenvironnement. Un second paramètre étudié concerne les microRNAs appartenant à la famille let-7 dont la fonction exacte au niveau cardiaque reste à définir. Les résultats montrent qu’au cours de la différenciation son expression se caractérise par une augmentation transitoire précoce au moment de la formation du mésoderme, puis s’éteint pour ne ré-augmenter que plus tard lors de la maturation des cardiomyocytes. Des modulations via des mimics ou des inhibiteurs dans différents contextes cellulaires suggèrent qu’initialement let-7 contribue à une future spécification cardiaque, mais que plus tard cette famille devra être réprimée pour générer des progéniteurs cardiaques. À l’opposé, miR-1, spécifique au cœur, contribue toujours à la progression en cardiomyocytes. Ensemble, ces recherches contribuent à la recherche fondamentale sur le développement du cœur humain et à la recherche appliquée en ingénierie tissulaire cardiaque<br>The objective of this thesis was to evaluate some physical and epigenetic parameters involved during cardiac differentiation of human induced pluripotent stem cells. Environmentally, an often undervalued physical parameter remains, the stiffness defined by the Young’s modulus. Commonly stem cells are cultured and adapted to in vitro rigidities ranging between 1-10 GPa very far from physiological values, for instance 10-15 kPa for the heart. The impact of soft culture substrates with 3 kPa, 12 kPa and 25 kPa was studied on the initial stem cells. Globally, results indicated that rigidities lower than 25 kPa were not suited for total pluripotency maintenance after 6 passages. Also, cellular colonies started to grow in 3D suggesting that softness drove them to build their own microenvironment. Epigenetically, the exact role of one of the first discovered microRNAs, the let-7 family has not yet been fully elucidated. Throughout differentiation its expression was characterized by an early transient peak at the time of mesoderm formation, after which their expression extinguished to only gradually re-increase later in the course of cardiomyocytes maturation. Modulation experiments involving mimics or inhibitors of the let-7 family on different cellular contexts suggested that initially let-7 acted on future cardiac specification but later, this family had to be repressed in order for cardiac progenitors to emerge. Oppositely, the cardiac specific miR-1 always contributed to their progression into cardiomyocytes. Together these researches contribute to fundamental research on human heart development and to applied research on human engineered cardiac tissues
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Watson, Andrea. "Heat shock proteins in leukaemia cell differentiation and cell death." Thesis, Aston University, 1990. http://publications.aston.ac.uk/12533/.
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When HL60 cells were induced to differentiate to granulocyte-like cells with the agents N-methylformamide and tunicamycin an concentrations marginally below those which were cytotoxic, there was a decrease in the synthesis of the glucose- regulated proteins which preceded the expression of markers of a differentiated phenotype. There was a transient increase in the amount of hsp70 after 36 hours in NMF treated cells but in differentiated cells negligible amounts were detected. Inducers which were known to modulate hsp70 such as azetadine carboxylic acid did not induce differentiation suggesting early changes in the endoplasmic reticulum may be involved in the commitment to terminal differentiation of HL60 cells. These changes in group synthesis were not observed when K562 human chronic myelogenous leukemia cells were induced to differentiate to erythroid-like cells but there was a comparable increase in amounts of hsp70. When cells were treated with concentrations of drugs which brought about a loss in cell viability there was an early increase in the amount of hsp70 protein in the absence of any increase in synthesis. HL60 cells were treated with NMF (225mM), Adriamycin (1 jiM), or CB3717 (5iM) and there was an increase in the amounts of hsp70, in the absence of any new synthesis, which preceded any loss of membrane integrity and any significant changes in cell cycle but was concomitant with a later loss in viability of > 50% and a loss in proliferative potential. The amounts of hsp70 in the cell after treatment with any of the drugs was comprable to that obtained after a heat shock. Following a heat shock hsp70 was translocated from the cytoplasm to the nucleus, but treatment with toxic concentrations of drug caused hsp70 to remain localised in the cytoplasm. Changes in hsp70 turn-over was observed after a heat shock compared to NMF-treated cells. Morphological studies suggested that cells that had been treated with NMF and CB3717 were undergoing necrosis whereas the Adriamycin cells showed characteristics that were indicative of apoptosis. The data supports the hypothesis that an increase in amounts of hsp70 is an early marker of cell death.
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Rao, Srinivas Venkateswarlu. "Examination of the role of porcine heat shock protein during adipocyte differentiation /." The Ohio State University, 1991. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487757723995836.
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Doroodian, Paymon. "Overexpression of Differentiation and Greening-Like Alters Stress Response of Arabidopsis thaliana." Ohio University / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1596227767908937.
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Stiening, Chad Michael. "GENOMIC REGULATION OF BOVINE MAMMARY EPITHELIAL CELL GROWTH AND DIFFERENTIATION." Diss., Tucson, Arizona : University of Arizona, 2005. http://etd.library.arizona.edu/etd/GetFileServlet?file=file:///data1/pdf/etd/azu%5Fetd%5F1252%5F1%5Fm.pdf&type=application/pdf.
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Guyette, Jacques Paul. "Conditioning of Mesenchymal Stem Cells Initiates Cardiogenic Differentiation and Increases Function in Infarcted Hearts." Digital WPI, 2012. https://digitalcommons.wpi.edu/etd-dissertations/32.
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Current treatment options are limited for patients with myocardial infarction or heart failure. Cellular cardiomyoplasty is a promising therapeutic strategy being investigated as a potential treatment, which aims to deliver exogenous cells to the infarcted heart, for the purpose of restoring healthy myocardial mass and mechanical cardiac function. While several cell types have been studied for this application, only bone marrow cells and human mesenchymal stem cells (hMSCs) have been shown to be safe and effective for improving cardiac function in clinical trials. In both human and animal studies, the delivery of hMSCs to infarcted myocardium decreased inflammatory response, promoted cardiomyocyte survival, and improved cardiac functional indices. While the benefits of using hMSCs as a cell therapy for cardiac repair are encouraging, the desired expectation of cardiomyoplasty is to increase cardiomyocyte content that will contribute to active cardiac mechanical function. Delivered cells may increase myocyte content by several different mechanisms such as differentiating to a cardiomyocyte lineage, secreting paracrine factors that increase native stem cell differentiation, or secreting factors that increase native myocyte proliferation. Considerable work suggests that hMSCs can differentiate towards a cardiomyocyte lineage based on measured milestones such as cardiac-specific marker expression, sarcomere formation, ion current propagation, and gap junction formation. However, current methods for cardiac differentiation of hMSCs have significant limitations. Current differentiation techniques are complicated and tedious, signaling pathways and mechanisms are largely unknown, and only a small percentage of hMSCs appear to exhibit cardiogenic traits. In this body of work, we developed a simple strategy to initiate cardiac differentiation of hMSCs in vitro. Incorporating environmental cues typically found in a myocardial infarct (e.g. decreased oxygen tension and increased concentrations of cell-signaling factors), our novel in vitro conditioning regimen combines reduced-O2 culture and hepatocyte growth factor (HGF) treatment. Reduced-O2 culturing of hMSCs has shown to enhance differentiation, tissue formation, and the release of cardioprotective signaling factors. HGF is a pleiotropic cytokine involved in several biological processes including developmental cardiomyogenesis, through its interaction with the tyrosine kinase receptor c-Met. We hypothesize that applying a combined conditioning treatment of reduced-O2 and HGF to hMSCs in vitro will enhance cardiac-specific gene and protein expression. Additionally, the transplantation of conditioned hMSCs into an in vivo infarct model will result in differentiation of delivered hMSCs and improved cardiac mechanical function. In testing our hypothesis, we show that reduced-O2 culturing can enhance hMSC growth kinetics and total c-Met expression. Combining reduced-O2 culturing with HGF treatment, hMSCs can be conditioned to express cardiac-specific genes and proteins in vitro. Using small-molecule inhibitors to target specific effector proteins in a proposed HGF/c-Met signaling pathway, treated reduced-O2/HGF hMSCs show a decrease in cardiac gene expression. When implanted into rat infarcts in vivo, reduced-O2/HGF conditioned hMSCs increase regional cardiac mechanics within the infarct region at 1 week and 1 month. Further analysis from the in vivo study showed a significant increase in the retention of reduced-O2/HGF conditioned hMSCs. Immunohistochemistry showed that some of the reduced-O2/HGF conditioned hMSCs express cardiac-specific proteins in vivo. These results suggest that a combined regimen of reduced-O2 and HGF conditioning increases cardiac-specific marker expression in hMSCs in vitro. In addition, the implantation of reduced-O2/HGF conditioned hMSCs into an infarct significantly improves cardiac function, with contributing factors of improved cell retention and possible increases in myocyte content. Overall, we developed a simple in vitro conditioning regimen to improve cardiac differentiation capabilities in hMSCs, in order to enhance the outcomes of using hMSCs as a cell therapy for the diseased heart.
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Hosie, Andrew. "Differentiating thermal from non-thermal eccrine sweating during exercise and heat stress." Access electronically, 2002. http://www.library.uow.edu.au/adt-NWU/public/adt-NWU20041105.114628/index.html.
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Haddad, Nisrine. "The effects of heat-shock and differentiation on nuclear structure/functions in mammalian cellular systems." Thesis, University of Ottawa (Canada), 2002. http://hdl.handle.net/10393/6292.
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The work delineated in this research thesis aims to achieve advances in understanding nuclear structure-function interrelationships. Two natural physiological processes---heat-shock and differentiation---have been used in order to provide a clearer understanding of the "big picture" that links structure and functions together. I have performed experiments using mild or severe heat-shock on HeLa S3 cells and I differentiated L6E9 myoblasts into skeletal muscle fibers. Microscopic and biochemical analysis were used in order to assess the effects of heat-shock and differentiation on nuclear morphology. A series of antibodies specific to the nuclear periphery such as: lam in-associated-polypeptides, emerin, lamins A/C and lamin B were used to analyze structural changes that occurs at the nuclear periphery. Similarly, nuclear matrix antigens such as the proliferating cell nuclear antigen or PCNA, the nucleocytoplasmic shuttling protein 2A7, 2H12 and fibrillarin, two nucleolar proteins, were used as tools to assess structural changes that occur in the nuclear morphology. (Abstract shortened by UMI.)
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Rusnak, Lauren Elizabeth. "Investigating Heat Shock Protein 70 as a Binding Partner of MK-STYX, and the Role of MK-STYX in Neuronal Differentiation." W&M ScholarWorks, 2013. https://scholarworks.wm.edu/etd/1539626944.
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Huber, Adrian Thomas. "Multi-organ non-invasive tissue characterization of fibrosis, adipose tissue, edema and inflammation with magnetic resonance (MR) imaging : applications to myocardium, skeletal muscle and liver interactions Cardiac MR strain: a noninvasive biomarker of fibro-fatty remodeling of the left atrial myocardium Comparison of MR T1 and T2 mapping parameters to characterize myocardial and skeletal muscle involvement in systemic Idiopathic Inflammatory Myopathy (IIM) Non-invasive differentiation of acute viral myocarditis and idiopathic inflammatory myopathy with cardiac involvement using magnetic resonance imaging T1 and T2 mapping CT predicts liver fibrosis: Prospective evaluation of morphology- and attenuationbased quantitative scores in routine portal venous abdominal scans." Thesis, Sorbonne université, 2019. http://www.theses.fr/2019SORUS135.
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Cette thèse réalise une preuve de concept pour quantifier la déformation de l’oreillette gauche (OG) en IRM, ainsi que la relaxométrie IRM dans le myocarde, dans les muscles squelettiques et dans le foie. Grâce à l’interaction entre radiologues et ingénieurs, deux logiciels différents ont été développés, appliqués et validés pour l'analyse de la déformation myocardique multi-chambre et pour la cartographie quantitative du T1 multi-organes. La première publication a montré une forte corrélation de la déformation de l’OG, avec le degré de remplacement fibro-graisseux en histologie. Ce biomarqueur d'imagerie fonctionnelle est prometteur, puisque le remodelage structurel du myocarde est un substrat morphologique connu du dysfonctionnement électro-physiologique et de la fibrillation atriale. La deuxième publication a démontré l'influence de la composition et de la vascularisation de différents tissus sur les paramètres cartographiques T1. ΔT1 (prise de contraste musculaire relative) et EHF (prise de contraste musculaire normalisée par la prise de contraste dans le sang) ont été introduits comme alternatives simples au volume extracellulaire (ECV). Dans la troisième publication, les paramètres de relaxométrie appliqués aux muscles squelettiques ont permis une discrimination entre patients avec myocardite aiguë et patients avec des myosites systémiques. La quatrième publication a introduit le T1 du foie pour quantifier l’insuffisance cardiaque chez des patients avec des cardiomyopathies idiopathiques dilatées, montrant de meilleures performances que les paramètres fonctionnels établis tels que les volumes, la fraction d'éjection ou la déformation myocardique<br>This thesis provides a proof of concept for MR atrial strain, as well as MR relaxometry in the myocardium, in skeletal muscles and in the liver. Thanks to a close interaction between radiologist and software engineers, two different softwares were developed, applied and validated: one for multiorgan T1 mapping in the myocardium, skeletal muscle and liver, another one for cardiac four-chamber strain analysis and volumetry. The first publication showed a strong correlation of LA strain with the degree of fibro-fatty replacement in histology. Such functional imaging biomarker in combination with LA volumetry could help to guide clinical decisions, since myocardial structural remodeling is a known morphologic substrate of LA dysfunction, atrial fibrillation and adverse outcome. In the second publication, MR relaxometry parameters applied to the myocardium and skeletal muscles in IIM patients and healthy volunteers were used as a model to demonstrate influences of different tissue composition and vascularization on T1 mapping parameters. ΔT1 and EHF were introduced as simple alternatives to ECV in highly vascularized tissues such as the myocardium. In the third publication, MR relaxometry parameters applied to the skeletal muscls allowed for an accurate discrimination of AVM and IIM with cardiac involvement. However, when applied to the myocardium, parametric mapping did not separate between the two groups. The fourth publication introduced native T1 of the liver an easily accessible and accurate non-invasive imaging associate of congestive HF in IDCM patients with better performance than established functional parameters such as LV volumes, ejection fraction or strain
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Mohammed, Sara Basher Taha [Verfasser]. "The Role of Heat Shock Protein 90 (HSP90) in the Transformation of Theileria annulata- infected Cells and the Parasite Stage Differentiation / Sara Basher Taha Mohammed." Berlin : Freie Universität Berlin, 2015. http://d-nb.info/1077478321/34.
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Raad, Farah. "Characterization and Application of Bioengineered Heart Muscle as a New Tool to Study Human Heart Development and Disease." Doctoral thesis, 2016. http://hdl.handle.net/11858/00-1735-0000-002B-7C05-A.
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Lopes, Floro Kylie Biotechnology & Biomolecular Sciences Faculty of Science UNSW. "Dissecting the requirement for Cited2 during heart development and left-right patterning of the mouse embryo." 2007. http://handle.unsw.edu.au/1959.4/40531.
Full textAbstract:
Cited2 is a member of the Cited gene family, which has no homology to any other genes. It encodes a transcriptional co-factor that is expressed during early heart formation (cardiogenesis). Embryos lacking Cited2 display a range of cardiac defects including bilaterally identical atria, aortic arch abnormalities, rotation of the aorta and pulmonary artery, and malseptation of the cardiac chambers. The latter results in communication between the aorta and pulmonary artery, the aorta and both ventricles, and the atria and ventricles (with themselves and each other). Cardiogenesis is complex, and requires many different cell types and processes to occur correctly. Some of these cells and processes are external to the primary heart. For example, once the initial muscle cells of the heart form a tube, cells from other regions such as the secondary heart field (adjacent mesoderm) and cardiac neural crest (ectoderm) migrate into this tube, and are required for the formation of additional muscle cells and septa. Furthermore, cardiogenesis also requires correct left-right patterning of the embryo to be established prior to heart formation. To understand the developmental origins of the cardiac defects observed in Cited2-null embryos, the expression pattern of Cited2 and the anatomy of Cited2-null embryo hearts were studied. Subsequently, the expression of genes required for left-right patterning were studied in both Cited2-null and Cited2 conditionally-deleted embryos. This demonstrated that Cited2 may be required in many, possibly all, of the processes required for cardiogenesis. Next this study focused on the role of Cited2 in patterning the left-right axis of the embryo. Firstly, Cited2 was found to regulate the expression of the master regulator of left-right patterning (Nodal). Secondly, Cited2 was shown to regulate the expression of the left-specific transcription factor Pitx2 independently of Nodal. Thirdly, gene expression and conditional deletions of Cited2 suggested that Cited2 might regulate left-right patterning in the paraxial mesoderm, a tissue which has not previously been shown to regulate the left-right axis in the mouse. Lastly, an argument is made suggesting the possibility that all the cardiac defects found in Cited2-null embryos may directly or indirectly stem from a failure of correct left-right patterning.
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"Gene expression profiles in neonatal heart development and functional roles of calcyclin binding protein/Siah-interacting protein in terminal differentiation of cardiomyocytes." 2004. http://library.cuhk.edu.hk/record=b6073675.
Full textAbstract:
by Au Ka Wing.<br>"June 2004."<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2004.<br>Includes bibliographical references (p. 153-162).<br>Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Mode of access: World Wide Web.<br>Abstracts in English and Chinese.
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"Purification of cardiomyocytes derived from differentiated embryonic stem cells and study of the cytokines' effect on embryonic stem cell differentiation." 2008. http://library.cuhk.edu.hk/record=b5893596.
Full textAbstract:
Leung, Sze Lee Cecilia.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2008.<br>Includes bibliographical references (leaves 144-153).<br>Abstracts in English and Chinese.<br>Abstract --- p.i<br>Abstract in Chinese (摘要） --- p.iii<br>Acknowledgements --- p.v<br>Table of Content --- p.vi<br>Abbreviations --- p.xv<br>Chapter CHAPTER 1 --- INTRODUCTION<br>Chapter 1.1 --- Stem cells --- p.1<br>Chapter 1.1.1 --- Adult stem cells --- p.2<br>Chapter 1.1.2 --- Embryonic stem cells --- p.2<br>Chapter 1.1.3 --- Pros and cons of embryonic and adult stem cells --- p.5<br>Chapter 1.1.4 --- Human embryonic stem cells (hESCs) --- p.6<br>Chapter 1.1.5 --- Mouse embryonic stem cells (mESCs) --- p.7<br>Chapter 1.1.6 --- Characteristics of ESC-derived cardiomyocytes --- p.7<br>Chapter 1.2 --- Cardiovascular Diseases (CVD) --- p.9<br>Chapter 1.2.1 --- Causes and statistics of CVD --- p.9<br>Chapter 1.2.2 --- Current treatment for CVD --- p.10<br>Chapter 1.2.3 --- Current hurdles of putting hESC-CMs into clinical use --- p.11<br>Chapter 1.3 --- Myosin light chain2v --- p.13<br>Chapter 1.4 --- Genetic-engineering of hESCs & their cardiac derivatives by lentiviral-mediate gene transfer --- p.14<br>Chapter 1.5 --- Cytokines secretion during myocardial infarction --- p.15<br>Chapter 1.6 --- Aims of the Project --- p.19<br>Chapter 1.7 --- Significance of the Project --- p.19<br>Chapter CHAPTER 2 --- MATERIALS AND METHODS<br>Chapter 2.1 --- Subcloning --- p.20<br>Chapter 2.1.1 --- Amplification of MLC-2v --- p.20<br>Chapter 2.1.2 --- Purification of DNA product --- p.21<br>Chapter 2.1.3 --- Restriction enzyme digestion --- p.21<br>Chapter 2.1.4 --- Ligation of MLC-2v promoter with DuetO 11 vector --- p.22<br>Chapter 2.1.5 --- Transformation of ligation product into competent cells --- p.22<br>Chapter 2.1.6 --- PCR confirmation of successful ligation --- p.23<br>Chapter 2.1.7 --- Small-scale preparation of bacterial plasmid DNA --- p.23<br>Chapter 2.1.8 --- Restriction enzyme digestions to reconfirm positive clones --- p.24<br>Chapter 2.1.9 --- DNA sequencing of the cloned plasmid DNA --- p.25<br>Chapter 2.1.10 --- Large-scale preparation of target recombinant expression vector --- p.25<br>Chapter 2.2 --- Mouse Embryonic Fibroblast (MEF) Culture --- p.26<br>Chapter 2.2.1 --- Derivation of MEF --- p.26<br>Chapter 2.2.2 --- Mouse embryonic fibroblast cells culture --- p.27<br>Chapter 2.2.3 --- Irradiation of mouse embryonic fibroblast --- p.28<br>Chapter 2.3 --- HESC culture --- p.29<br>Chapter 2.3.1 --- Thawing and Plating hESCs --- p.29<br>Chapter 2.3.2 --- Splitting hESCs --- p.30<br>Chapter 2.3.3 --- "Culture maintainence, selection and colony removal" --- p.31<br>Chapter a) --- Distinguish differentiated and undifferentiated cells and colonies<br>Chapter b) --- "Remove differentiated cells by ""Picking to Remove"""<br>Chapter c) --- "Remove undifferentiated cells by ""Picking to Keep"""<br>Chapter 2.3.4 --- Freezing hESCs --- p.31<br>Chapter 2.3.5 --- Differentiation of hESCs --- p.32<br>Chapter 2.3.6 --- "HESC culture on feeder free system, mTeSR TM1" --- p.34<br>Chapter a) --- Preparation of mTeSRTMl<br>Chapter b) --- Preparation of BD MatrigelTM hESC-qualified Matrix aliquots<br>Chapter c) --- Coating plates with BD MatrigelTM hESC-qualified Matrix<br>Chapter d) --- Human Embryonic stem cells culture in mTeSRTMl<br>Chapter 2.4 --- ES Cell Characterization (Chemicon Cat# SCR001) --- p.36<br>Chapter 2.4.1 --- Alkaline Phosphatase Staining --- p.36<br>Chapter 2.4.2 --- Immunofluorescence staining --- p.37<br>Chapter 2.5 --- MESC culture --- p.38<br>Chapter 2.5.1 --- Thawing and Plating mESCs --- p.38<br>Chapter 2.5.2 --- Splitting mESCs --- p.38<br>Chapter 2.5.3 --- Differentiation of mESCs --- p.39<br>Chapter 2.5.4 --- To study the effects of cytokines on mESC differentiation --- p.40<br>Chapter 2.6 --- Lentivirus (LV) Packaging --- p.41<br>Chapter 2.6.1 --- Transfection of lentiviral vectors into HEK293FT cells --- p.41<br>Chapter 2.6.2 --- LV titering --- p.42<br>Chapter 2.7 --- MultipleTransduction --- p.43<br>Chapter 2.8 --- Selection of transduced cells by hygromycin --- p.43<br>Chapter 2.8.1 --- Determination of hygromycin selection dosage --- p.43<br>Chapter 2.8.2 --- Selection of stable clones --- p.44<br>Chapter 2.9 --- Isolation of green fluorescent cardiomyocytes derived from differentiated hESCs --- p.45<br>Chapter 2.9.1 --- Collagenase digestion of embryoid bodies into single cells --- p.45<br>Chapter 2.9.2 --- FACS --- p.46<br>Chapter 2.10 --- Gene expression study<br>Chapter 2.10.1 --- Primer design --- p.46<br>Chapter 2.10.2 --- RNA extraction --- p.46<br>Chapter 2.10.3 --- DNase Treatment --- p.47<br>Chapter 2.10.4 --- Synthesis of Double-stranded cDNA from Total RNA --- p.47<br>Chapter 2.10.5 --- Quantitative real-time PCR --- p.48<br>Chapter 2.10.6 --- Quantification of mRNA expression --- p.49<br>Chapter 2.11 --- Protein Expression study --- p.49<br>Chapter 2.11.1 --- Crude protein extraction --- p.49<br>Chapter 2.11.2 --- Quantitation of protein samples --- p.50<br>Chapter 2.11.3 --- SDS-PAGE --- p.50<br>Chapter 2.11.4 --- Western Blot --- p.51<br>Chapter 2.11.5 --- Western blot luminal detection --- p.52<br>Chapter 2.11.6 --- Quantification of protein expression --- p.52<br>Chapter CHAPTER 3 --- PURIFICATION OF CARDIOMYOCYTES DERIVED FROM DIFFERENTIATED HESCs<br>Chapter 3.1 --- Subcloning --- p.57<br>Chapter 3.1.1 --- Linearization of DuetO11 and excision of UBC promoter --- p.58<br>Chapter 3.1.2 --- PCR cloning of MLC-2V --- p.59<br>Chapter 3.1.3 --- Ligation of MLC-2v promoter to linearized DuetO11 --- p.60<br>Chapter 3.1.3.1 --- Colony PCR to screen for positive clones --- p.61<br>Chapter 3.1.3.2 --- Restriction digestion to confirm the success of ligation --- p.61<br>Chapter 3.2 --- Lentivirus (LV) packaging --- p.62<br>Chapter 3.2.1 --- Transfection --- p.63<br>Chapter 3.2.2 --- LV titering --- p.64<br>Chapter 3.3 --- HESC culture --- p.66<br>Chapter 3.4 --- Multi-transduction of hESCs with LVs --- p.67<br>Chapter 3.5 --- Differentiation after transduction --- p.69<br>Chapter 3.6 --- Antibiotic selection --- p.71<br>Chapter 3.6.1 --- Characterization of hESCs on feeder free system --- p.72<br>Chapter 3.6.1.1 --- Alkaline Phosphatase (AP) staining --- p.72<br>Chapter 3.6.1.2 --- Immunostaining with pluripotency marker --- p.73<br>Chapter 3.6.2 --- Determination of hygromycin dosage by MTT assay --- p.74<br>Chapter 3.6.3 --- HESCs after selection in feeder free system --- p.75<br>Chapter 3.7 --- Differentiation of hESCs after selection --- p.76<br>Chapter 3.8 --- FACS --- p.77<br>Chapter 3.9 --- QPCR of cells after FACS --- p.80<br>Chapter 3.9.1 --- Gene expression of Nkx2.5 --- p.81<br>Chapter 3.9.2 --- Gene expression of c-Tnl --- p.82<br>Chapter 3.9.3 --- Gene expression of c-TnT --- p.83<br>Chapter 3.9.3 --- Gene expression of MLC-2v --- p.84<br>Chapter CHAPTER 4 --- THE STUDY OF CYTOKINES' EFFECT ON MESC DIFFERENTIATION<br>Chapter 4.1 --- mESC culture --- p.85<br>Chapter 4.2 --- The effect of cytokines on the differentiation of mESCs --- p.86<br>Chapter 4.2.1 --- Beating curves of mESCs treated with different concentrations of cytokines at differentiation day 2 to 6 before attachment --- p.88<br>Chapter 4.2.2 --- qPCR to determine the cytokines' effect on the differentiation of mESCs --- p.94<br>Chapter 4.2.2.1 --- The effect of IL-1α on the expression of cardiac specific genes --- p.95<br>Chapter 4.2.2.2 --- The effect of IL-1β on the expression of cardiac specific genes --- p.98<br>Chapter 4.2.2.3 --- The effect of IL-6 on the expression of cardiac specific genes --- p.101<br>Chapter 4.2.2.4 --- The effect of IL-10 on the expression of cardiac specific genes --- p.104<br>Chapter 4.2.2.5 --- The effect of IL-18 on the expression of cardiac specific genes --- p.107<br>Chapter 4.2.2.6 --- The effect of TNF-α on the expression of cardiac specific genes --- p.110<br>Chapter 4.2.3 --- Western blot analysis of the cytokines' effect on the differentiation of mESCs --- p.113<br>Chapter 4.2.3.1 --- The effect of IL-lα on the abundance of cardiac specific proteins --- p.114<br>Chapter 4.2.3.2 --- The effect of IL-1β on the abundance of cardiac specific proteins --- p.116<br>Chapter 4.2.3.3 --- The effect of IL-6 on the abundance of cardiac specific proteins --- p.118<br>Chapter 4.2.3.4 --- The effect of IL-10 on the abundance of cardiac specific proteins --- p.120<br>Chapter 4.2.3.5 --- The effect of IL-18 on the abundance of cardiac specific proteins --- p.122<br>Chapter 4.2.3.6 --- The effect of TNF-α on the abundance of cardiac specific proteins --- p.124<br>Chapter CHAPTER 5 --- DISCUSSION<br>Chapter 5.1 --- Purification of cardiomyocytes derived from differentiated hESCs --- p.127<br>Chapter 5.2 --- Study on the effect of cytokines on mESC differentiation --- p.135<br>Chapter 5.3 --- Conclusion --- p.142<br>REFERENCES --- p.144
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"Role of reactive oxygen species (ROS) in cardiomyocyte differentiation of mouse embryonic stem cells." 2009. http://library.cuhk.edu.hk/record=b5894101.
Full textAbstract:
Law, Sau Kwan.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2009.<br>Includes bibliographical references (leaves 111-117).<br>Abstract also in Chinese.<br>Thesis Committee --- p.i<br>Acknowledgements --- p.ii<br>Contents --- p.iii<br>Abstract --- p.vii<br>論文摘要 --- p.x<br>Abbreviations --- p.xi<br>List of Figures --- p.xiii<br>List of Tables --- p.xxiii<br>Chapter CHAPTER ONE --- INTRODUCTION<br>Chapter 1.1 --- Embryonic Stem (ES) Cells<br>Chapter 1.1.1 --- Characteristics of ES Cells l<br>Chapter 1.1.2 --- Therapeutic Potential of ES Cells --- p.3<br>Chapter 1.1.3 --- Myocardial Infarction and ES cells-derived Cardiomyocytes --- p.4<br>Chapter 1.1.4 --- Current Hurdles of Using ES cells-derived Cardiomyocytes for Research and Therapeutic Purposes --- p.6<br>Chapter 1.2 --- Transcription Factors for Cardiac Development<br>Chapter 1.2.1 --- GATA-binding Protein 4 (GATA-4) --- p.8<br>Chapter 1.2.2 --- Myocyte Enhancer Factor 2C (MEF2C) --- p.10<br>Chapter 1.2.3 --- "NK2 Transcription Factor Related, Locus 5 (Nkx2.5)" --- p.11<br>Chapter 1.2.4 --- Heart and Neural Crest Derivatives Expressed 1 /2 (HANDI/2) --- p.11<br>Chapter 1.2.5 --- T-box Protein 5 (Tbx5) --- p.13<br>Chapter 1.2.6 --- Serum Response Factor (SRF) --- p.14<br>Chapter 1.2.7 --- Specificity Protein 1 (Spl) --- p.15<br>Chapter 1.2.8 --- Activator Protein 1 (AP-1) --- p.16<br>Chapter 1.3 --- Reactive Oxygen Species (ROS)<br>Chapter 1.3.1 --- Cellular Production of ROS --- p.18<br>Chapter 1.3.2 --- Maintenance of Redox balance --- p.18<br>Chapter 1.3.3 --- Redox Signaling --- p.19<br>Chapter 1.4 --- Nitric Oxide (NO) and NO Signaling --- p.20<br>Chapter 1.5 --- Aims of the Study --- p.22<br>Chapter CHAPTER TWO --- MATERIALS AND METHODS<br>Chapter 2.1 --- Mouse Embryonic Fibroblast (MEF) Culture<br>Chapter 2.1.1 --- Derivation of MEF --- p.23<br>Chapter 2.1.2 --- Maintenance of MEF Culture --- p.24<br>Chapter 2.1.3 --- Irradiation of MEF --- p.25<br>Chapter 2.2 --- Mouse ES Cell Culture<br>Chapter 2.2.1 --- Maintenance of Undifferentiated Mouse ES Cell Culture --- p.26<br>Chapter 2.2.2 --- Differentiation of Mouse ES Cells --- p.26<br>Chapter 2.2.3 --- Exogenous addition of hydrogen peroxide (H2O2) and NO --- p.27<br>Chapter 2.3 --- ROS Localization Study<br>Chapter 2.3.1 --- Frozen Sectioning --- p.28<br>Chapter 2.3.2 --- Confocal microscopy for ROS detection --- p.28<br>Chapter 2.4 --- Intracellular ROS Measurement<br>Chapter 2.4.1 --- "Chemistry of 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA)" --- p.29<br>Chapter 2.4.2 --- Flow Cytometry for ROS Measurement --- p.29<br>Chapter 2.5 --- Gene Expression Study<br>Chapter 2.5.1 --- Primer Design --- p.30<br>Chapter 2.5.2 --- RNA Extraction --- p.31<br>Chapter 2.5.3 --- DNase Treatment --- p.32<br>Chapter 2.5.4 --- Reverse Transcription --- p.32<br>Chapter 2.5.5 --- Quantitative Real Time PCR --- p.33<br>Chapter 2.5.6 --- Quantification of mRNA Expression --- p.34<br>Chapter 2.6 --- Protein Expression Study<br>Chapter 2.6.1 --- Total Protein Extraction --- p.34<br>Chapter 2.6.2 --- Nuclear and Cytosolic Protein Extraction --- p.35<br>Chapter 2.6.3 --- Measurement of Protein Concentration --- p.36<br>Chapter 2.6.4 --- De-sumoylation Assay --- p.36<br>Chapter 2.6.5 --- De-phosphorylation Assay --- p.37<br>Chapter 2.6.6 --- De-glycosylation Assay --- p.38<br>Chapter 2.6.7 --- Western Blot --- p.39<br>Chapter 2.7 --- Statistical Analysis --- p.41<br>Chapter CHAPTER THREE --- RESULTS<br>Chapter 3.1 --- Study of Endogenous ROS<br>Chapter 3.1.1 --- Level and Distribution of Endogenous ROS --- p.47<br>Chapter 3.1.2 --- Quantification of intracellular ROS --- p.48<br>Chapter 3.2 --- Effect of Exogenous Addition of Nitric Oxide (NO) on Cardiac Differentiation<br>Chapter 3.2.1 --- Beating Profile of NO-treated Embryoid Bodies (EBs) --- p.50<br>Chapter 3.3 --- Effect of Exogenous Addition of H2O2 on Cardiac Differentiation<br>Chapter 3.3.1 --- Beating Profile of H2O2-treated EBs --- p.51<br>Chapter 3.3.2 --- mRNA Expression of Cardiac Structural Genes --- p.52<br>Chapter 3.3.3 --- Protein Expression of Cardiac Structural Genes --- p.54<br>Chapter 3.3.4 --- mRNA Expression of Cardiac Transcription Factors --- p.58<br>Chapter 3.3.5 --- Protein Expression of Cardiac Transcription Factors --- p.67<br>Chapter 3.3.6 --- Post-translational Modifications of Cardiac Transcription Factors --- p.74<br>Chapter 3.3.7 --- Translocation of Cardiac Transcription Factors --- p.89<br>Chapter CHAPTER FOUR --- DISCUSSION<br>Chapter 4.1 --- Changes in the Level of Endogenous ROS During Cardiac Differentiation of Mouse ES Cells --- p.96<br>Chapter 4.2 --- H2O2 and NO Have Opposite Effects Towards Cardiac Differentiation --- p.97<br>Chapter 4.3 --- Exogenous Addition of H2O2 Advances Differentiation of Mouse ES Cells into Cardiac Lineage --- p.99<br>Chapter 4.4 --- Possible Role of H2O2 in Mediating Cardiac Differentiation of Mouse ES Cells --- p.103<br>Chapter 4.5 --- Future Directions --- p.108<br>Conclusions --- p.110<br>References --- p.111
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"Mechanisms underlying the self-renewal characteristic and cardiac differentiation of mouse embryonic stem cells." 2009. http://library.cuhk.edu.hk/record=b5896594.
Full textAbstract:
Ng, Sze Ying.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2009.<br>Includes bibliographical references (leaves 110-124).<br>Abstract also in Chinese.<br>Thesis Committee --- p.i<br>Acknowledgements --- p.ii<br>Contents --- p.iii<br>Abstract --- p.vii<br>論文摘要 --- p.x<br>Abbreviations --- p.xi<br>List of Figures --- p.xiii<br>List of Tables --- p.xvii<br>Chapter CHAPTER ONE --- INTRODUCTION --- p.1<br>Chapter 1.1 --- Embryonic Stem Cells (ESCs) --- p.1<br>Chapter 1.1.1 --- What are ESCs and the characteristics of ESCs --- p.1<br>Chapter 1.1.1.1 --- Pluripotent markers --- p.2<br>Chapter 1.1.1.2 --- Germ layers' markers --- p.3<br>Chapter 1.1.2 --- Mouse ESCs (mESCs) --- p.4<br>Chapter 1.1.2.1 --- mESCs co-culture with mitotically inactivated mouse embryonic fibroblast (MEF) feeder layers --- p.4<br>Chapter 1.1.2.2 --- Feeder free mESCs --- p.4<br>Chapter 1.1.3 --- Promising uses of ESCs and their shortcomings --- p.5<br>Chapter 1.1.4 --- Characteristics of ESC-derived cardiomyocytes (ESC-CMs) --- p.6<br>Chapter 1.2 --- Cardiovascular diseases (CVD) --- p.7<br>Chapter 1.2.1 --- Background --- p.7<br>Chapter 1.2.2 --- Current treatments --- p.8<br>Chapter 1.2.3 --- Potential uses of ESC-CMs for basic science research and therapeutic purposes --- p.9<br>Chapter 1.2.4 --- Current hurdles in application of ESC-CMs for clinical uses --- p.10<br>Chapter 1.3 --- Cardiac gene markers --- p.13<br>Chapter 1.3.1 --- Atrial-specific --- p.13<br>Chapter 1.3.2 --- Ventricular-specific --- p.19<br>Chapter 1.4 --- Lentiviral vector-mediated gene transfer --- p.27<br>Chapter 1.5 --- Cell cycle in ESCs --- p.29<br>Chapter 1.5.1 --- Cell cycle --- p.29<br>Chapter 1.5.2 --- Characteristics of cell cycle in ESCs --- p.30<br>Chapter 1.6 --- Potassium (K+) channels --- p.31<br>Chapter 1.6.1 --- Voltage gated potassium (Kv) channels --- p.32<br>Chapter 1.6.2 --- Role of Kv channels in maintenance of membrane potential --- p.32<br>Chapter 1.7 --- Objectives and significances --- p.33<br>Chapter CHAPTER TWO --- MATERIALS AND METHODS --- p.35<br>Chapter 2.1 --- Mouse embryonic fibroblast (MEF) culture --- p.35<br>Chapter 2.1.1 --- Derivation of MEF --- p.3 5<br>Chapter 2.1.2 --- MEF culture --- p.37<br>Chapter 2.1.3 --- Irradiation of MEF --- p.37<br>Chapter 2.2 --- mESC culture and their differentiation --- p.38<br>Chapter 2.2.1 --- mESC culture --- p.38<br>Chapter 2.2.2 --- Differentiation of mESCs --- p.39<br>Chapter 2.3 --- Subcloning --- p.40<br>Chapter 2.3.1 --- Amplification of Irx4 --- p.40<br>Chapter 2.3.2 --- Purification of DNA products --- p.41<br>Chapter 2.3.3 --- Restriction enzyme digestion --- p.42<br>Chapter 2.3.4 --- Ligation of Irx4 with iDuet101A vector --- p.43<br>Chapter 2.3.5 --- Transformation of ligation product into competent cells --- p.43<br>Chapter 2.3.6 --- Small scale preparation of bacterial plasmid DNA --- p.44<br>Chapter 2.3.7 --- Confirmation of positive clones by restriction enzyme digestion --- p.45<br>Chapter 2.3.8 --- DNA sequencing of the cloned plasmid DNA --- p.45<br>Chapter 2.3.9 --- Large scale preparation of target recombinant expression vector --- p.45<br>Chapter 2.4 --- Lentiviral vector-mediated gene transfer to mESCs --- p.47<br>Chapter 2.4.1 --- Lentivirus packaging --- p.47<br>Chapter 2.4.2 --- Lentivirus titering --- p.48<br>Chapter 2.4.3 --- Multiple transduction to mESCs --- p.48<br>Chapter 2.4.4 --- Hygromycin selection on mESCs --- p.49<br>Chapter 2.5 --- Selection of stable clone --- p.49<br>Chapter 2.5.1 --- Monoclonal establishment and clone selection --- p.49<br>Chapter 2.6 --- Differentiation of cell lines after selection --- p.50<br>Chapter 2.7 --- Gene expression study on control and Irx4-overexpressed mESC lines --- p.50<br>Chapter 2.8 --- Analysis of mESCs at different phases of the cell cycle --- p.55<br>Chapter 2.8.1 --- Go/Gi and S phase synchronization --- p.55<br>Chapter 2.8.2 --- Cell cycle analysis by propidium iodide (PI) staining followed by flow cytometric analysis --- p.55<br>Chapter 2.8.3 --- Gene expression study by qPCR of Kv channel isoforms --- p.56<br>Chapter 2.8.4 --- Membrane potential measurement by membrane potential-sensitive dye followed by flow cytometry --- p.57<br>Chapter 2.9 --- Apoptotic study --- p.58<br>Chapter 2.10 --- Determination of pluripotent characteristic of mESCs --- p.59<br>Chapter 2.10.1 --- Expression of germ layers' markers by qPCR --- p.59<br>Chapter 2.10.2 --- Differentiation by hanging drop method and suspension method --- p.61<br>Chapter CHAPTER THREE --- RESULTS --- p.62<br>Chapter 3.1 --- mESC culture --- p.62<br>Chapter 3.1.1 --- Cell colony morphology of feeder free mESCs --- p.62<br>Chapter 3.2 --- Subcloning --- p.63<br>Chapter 3.2.1 --- PCR cloning of Irx4 --- p.63<br>Chapter 3.2.2 --- Restriction digestion on iDuet101A --- p.64<br>Chapter 3.2.3 --- Ligation of Irx4 to iDuet101A backbone --- p.66<br>Chapter 3.2.4 --- Confirmation of successful ligation --- p.67<br>Chapter 3.3 --- Lentivirus packaging --- p.68<br>Chapter 3.3.1 --- Transfection --- p.68<br>Chapter 3.4 --- Multiple transduction of mESCs and hygromycin selection of positively-transduced cells --- p.69<br>Chapter 3.5 --- FACS --- p.70<br>Chapter 3.6 --- Irx4 and iduet clone selection --- p.71<br>Chapter 3.7 --- Characte rization of mESCs after clone selection --- p.74<br>Chapter 3.7.1 --- Immunostaining of pluripotent and differentiation markers --- p.74<br>Chapter 3.8 --- Differentiation of cell lines after selection --- p.77<br>Chapter 3.8.1 --- Size of EBs of the cell lines during differentiation --- p.77<br>Chapter 3.9 --- Gene expression study by qPCR --- p.79<br>Chapter 3.10 --- Kv channel expression and membrane potential of mESCs at Go/Gi phase and S phases --- p.84<br>Chapter 3.10.1 --- Expression of Kv channels subunits at G0/Gi phase and S phase --- p.86<br>Chapter 3.10.2 --- Membrane potential at Go/Gi phase and S phase --- p.87<br>Chapter 3.11 --- Effects of TEA+ on feeder free mESCs --- p.89<br>Chapter 3.11.1 --- Apoptotic study --- p.89<br>Chapter 3.11.2 --- Expression of germ layers´ة markers --- p.91<br>Chapter 3.11.3 --- Embryo id bodies (EBs) measurement after differentiation --- p.92<br>Chapter CHAPTER FOUR --- DISCUSSION --- p.95<br>Chapter 4.1 --- Effect of overexpression of Irx4 on the cardiogenic potential of mESCs --- p.95<br>Chapter 4.2 --- Role of Kv channels in maintaining the chacteristics of mESCs --- p.99<br>Chapter 4.2.1 --- Inhibition of Kv channels led to a redistribution of the proportion of cells in different phases of the cell cycle: importance of Kv channels in cell cycle progression in native ESCs --- p.99<br>Chapter 4.2.2 --- Inhibition of Kv channels led to a loss of pluripotency at molecular and functional levels: importance of Kv channels in the fate determination of mESCs --- p.102<br>Chapter 4.3 --- Insights from the present investigation on the future uses of ESCs --- p.105<br>Conclusions --- p.108<br>References --- p.110
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Lajiness, Jacquelyn D. "Shp2 deletion in post-migratory neural crest cells results in impaired cardiac sympathetic innervation." Thesis, 2014. http://hdl.handle.net/1805/5495.
Full textAbstract:
Indiana University-Purdue University Indianapolis (IUPUI)<br>Autonomic innervation of the heart begins in utero and continues during the neonatal phase of life. A balance between the sympathetic and parasympathetic arms of the autonomic nervous system is required to regulate heart rate as well as the force of each contraction. Our lab studies the development of sympathetic innervation of the early postnatal heart in a conditional knockout (cKO) of Src homology protein tyrosine phosphatase 2 (Shp2). Shp2 is a ubiquitously expressed non-receptor phosphatase involved in a variety of cellular functions including survival, proliferation, and differentiation. We targeted Shp2 in post-migratory neural crest (NC) lineages using our novel Periostin-Cre. This resulted in a fully penetrant mouse model of diminished cardiac sympathetic innervation and concomitant bradycardia that progressively worsen.
Shp2 is thought to mediate its basic cellular functions through a plethora of signaling cascades including extracellular signal-regulated kinases (ERK) 1 and 2. We hypothesize that abrogation of downstream ERK1/2 signaling in NC lineages is primarily responsible for the failed sympathetic innervation phenotype observed in our mouse model. Shp2 cKOs are indistinguishable from control littermates at birth and exhibit no gross structural cardiac anomalies; however, in vivo electrocardiogram (ECG) characterization revealed sinus bradycardia that develops as the Shp2 cKO ages. Significantly, 100% of Shp2 cKOs die within 3 weeks after birth. Characterization of the expression pattern of the sympathetic nerve marker tyrosine hydroxylase (TH) revealed a loss of functional sympathetic ganglionic neurons and reduction of cardiac sympathetic axon density in Shp2 cKOs. Shp2 cKOs exhibit lineage-specific suppression of activated pERK1/2 signaling, but not of other downstream targets of Shp2 such as pAKT (phosphorylated-Protein kinase B). Interestingly, restoration of pERK signaling via lineage-specific expression of constitutively active MEK1 (Mitogen-activated protein kinase kinase1) rescued TH-positive cardiac innervation as well as heart rate. These data suggest that the diminished sympathetic cardiac innervation and the resulting ECG abnormalities are a result of decreased pERK signaling in post-migratory NC lineages.
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Lankeit, Mareike Katharina. "Neue Biomarker und Multimarkerstrategien für eine optimierte Risikostratifizierung von Patienten mit Lungenembolie." Doctoral thesis, 2010. http://hdl.handle.net/11858/00-1735-0000-0006-AF7C-A.
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Kang, Susey H. "Characterization of gene expression profiles during differentiation, lipopolysaccharide stimulation, and heat shock repression in human U937 monocyte cells." 2004. http://link.library.utoronto.ca/eir/EIRdetail.cfm?Resources__ID=95285&T=F.
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Kaiser, Diana. "Der Einfluss mechanischer Last auf das Potential multipotenter adulter Keimbahnstammzellen zur kardialen Regeneration." Doctoral thesis, 2011. http://hdl.handle.net/11858/00-1735-0000-0006-ADEB-D.
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Montgomery, Amy. "Combining induced pluripotent stem cells and fibrin matrices for spinal cord injury repair." Thesis, 2014. http://hdl.handle.net/1828/5272.
Full textAbstract:
Spinal cord injuries result in permanent loss of motor function, leaving those affected with long term physical and financial burdens. Strategies for spinal cord injury repair must overcome unique challenges due to scar tissue that seals off the injury site, preventing regeneration. Tissue engineering can address these challenges with scaffolds that serve as cell- and drug-delivery tools, replacing damaged tissue while simultaneously addressing the inhibitory environment on a biochemical level. To advance this approach, the choice of cells, biomaterial matrix, and drug delivery system must be investigated and evaluated. This research seeks to evaluate (1) the behaviour of murine induced pluripotent stem cells in previously characterized 3D fibrin matrices; (2) the 3D fibrin matrix as a platform to support the differentiation of human induced pluripotent stem cells; and (3) the ability of an affinity-based drug delivery system to control the release of emerging spinal cord injury therapeutic, heat shock protein 70 from fibrin scaffolds.<br>Graduate<br>0541<br>amy.lynn.montgomery@gmail.com