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Dissertations / Theses on the topic 'Induced-pluripotent Stem Cell-derived Hepatocytes'

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Published: 10 December 2022
Last updated: 28 January 2023

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1

Szkolnicka, Dagmara Maria. "MicroRNA regulation of drug metabolism in stem cell-derived hepatocytes." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/23421.

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The liver is a multi-functional and highly regenerative organ. While resilient, the liver is susceptible to organ damage and failure. In both the acute and chronic settings liver disease has dire consequences for health. A common cause of liver damage is adverse reactions to drugs which can lead to drug induced liver injury (DILI). This creates major problems for patients, clinicians, the pharmaceutical industry and regulatory authorities. In the context of drug overdose or serious adverse reactions, liver failure can be acute and life threatening, and in some cases require orthotopic liver transplantation. While transplantation is highly successful, such an approach has limitations and justifies basic science attempts to develop better human models to study liver injury and to develop scalable intervention strategies. With this in mind, we have studied the importance of microRNAs (miRs) in regulating human drug metabolism in pluripotent stem cell – derived hepatocytes and their potential to reduce liver toxicity in response to toxic levels of paracetamol. miRs are small non-coding RNAs that are approximately 20 - 24 nucleotides long and their major function is to fine tune gene expression of their target genes. Recently, it has been demonstrated that microRNAs play a role in regulating the first phase of drug metabolism however the second phase of drug metabolism, drug conjugation, has not been studied in detail. Drug conjugation is a crucial stage in human drug metabolism, and any alterations in this process can lead to changes in compound pharmacology, including therapeutic dose and clearance from the body. To test the importance of miRs in regulating phase II drug metabolism we opted to study the metabolism of a common used analgesic, paracetamol. When taken in the appropriate amounts paracetamol is modified by sulfotransferases (SULTs) and UDP - glucuronosyltransferases (UGTs) and removed from the body without organ damage. However, when paracetamol is taken above the recommended dose it is metabolised by phase I enzymes to generate a toxic intermediate N-acetyl-p-benzoquinone imine (NAPQI), which if untreated can lead to massive hepatocyte cell death and liver failure, placing the patient in a life threatening situation. In order to promote non-toxic drug metabolism, in the context of drug overdose, we employed candidate miRs to regulate different parts of the paracetamol metabolism pathway. In summary, we have focused on studying human drug metabolism in the major metabolic cell type of the liver, the hepatocyte. We have identified a novel microRNA (called miR-324-5p) which regulates phase II drug metabolism and reduces cell cytotoxicity. Additionally, a supportive role of anti-microRNA- 324 in response to fulminant plasma collected from paracetamol overdose patients is also observed. The findings of this project are novel, provide proof of concept and exemplify the power of stem cell based models to identify new approaches to treating human liver damage.
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2

Wills, Lauren Raquel. "Investigating Induced Pluripotent Stem Cells for Tissue Engineering and Hepatotoxicity Applications." Thesis, Virginia Tech, 2019. http://hdl.handle.net/10919/101006.

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Induced pluripotent stem cells (iPSCs) can be differentiated into multiple cell types in the body while maintaining proliferative capabilities. The generation of human iPSC-derived hepatocytes (iPSC-Heps) has resulted in a new source for hepatic cells. The current available options for human hepatocytes are primary human hepatocytes (PHHs) and cell lines. PHHs isolated from healthy human donors are difficult to obtain, while cell lines exhibit reduced hepatotoxic sensitivity. iPSC-Heps are being investigated as an alternative option as they are derived from a continuous, stable source and are able to maintain their original donor genotype, which opens the door for patient-specific studies. iPSC-Heps show promise for utilization in tissue engineering, hepatotoxicity studies as well as screening for patient-specific therapeutics. Various reports have concluded that iPSC-Heps exhibit reduced hepatocyte function in comparison to PHHs. Prior reports on iPSC-Heps have focused on improving their adult phenotype functions through variations in differentiation protocols or by altering their in vitro culturing environment. This thesis focuses on incorporating hepatic non-parenchymal cells to more closely mimic the tissue and cell architecture found in the liver tissue. We designed and assembled a 3D iPSC-Hep model that integrates liver sinusoidal endothelial cells, with the goal of achieving functional maturity. Hepatotoxicants were administered to our models and various hepatic markers were measured to analyze the toxic response. This work demonstrates the need for the inclusion of hepatic non-parenchymal cells in iPSC-derived liver tissues, specifically for hepatotoxicity applications.
Master of Science
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3

Chen, Xike. "Integration Capacity of Human Induced Pluripotent Stem Cell-Derived Cartilage." Kyoto University, 2019. http://hdl.handle.net/2433/242390.

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4

Fatica, Erica Marie. "Investigating Cardiac Metabolism in Barth Syndrome Using Induced Pluripotent Stem Cell-Derived Cardiomyocytes." Cleveland State University / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=csu1556630870935279.

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5

Nishimura, Koji. "Transplantation of murine induced pluripotent stem cell-derived neural progenitors into the cochlea." Kyoto University, 2012. http://hdl.handle.net/2433/157416.

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6

Sachamitr, Supatra. "Exploiting the use of induced pluripotent stem cell derived immune cells for immunotherapy." Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:89315b6b-a8cd-4a6f-8c43-3506d8dd1725.

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Immunotherapy traditionally made use of biological agents such as cytokines and monoclonal antibodies. Such first generation therapies lack antigen specificity and fail to induce immunological memory, suggesting that cell therapies may provide the next generation of treatments that are more discerning in their mode of action. Nevertheless, difficulties in obtaining sufficient immunologically-relevant cell types from patients has limited their success. Given that induced pluripotent stem cells (iPSC) may be generated from patients, we have investigated the feasibility of deriving two cell types whose availability is restricted in vivo: regulatory T cells (Tregs) and CD141+ cross-presenting dendritic cells (DCs). We describe the optimization of protocols for differentiation and purification of CD141+ DCs, focussing on their utility as a therapeutic vaccine for HIV-1. We investigate their phenotype, chemotactic capacity, phagocytic ability and propensity to harbour infectious virus. We also assess their immunostimulatory capacity and ability to cross-present exogenous antigen to MHC class I-restricted T cells. Our findings led us to speculate that iPSC-derived DCs (iPDCs) possess fetal phenotype, which is characterised by excessive secretion of IL-10 and failure to secrete IL-12, under all but the most stringent conditions. We hypothesised that constitutive secretion of IL-10 may be responsible for maintaining the fetal phenotype, a hypothesis we tested by developing an appropriate mouse model. iPSCs were derived from WT and IL-10-/- mice and were shown to differentiate into iPDCs which recapitulate the fetal phenotype observed among human cells. However, loss of the endogenous Il-10 gene failed to restore full immunogenicity and IL-12 secretion. Finally, we developed protocols for differentiation of FoxP3+ Tregs from iPSCs, a feat that has not previously been achieved. These findings pave the way for the differentiation of Tregs from iPSCs reprogrammed from antigen-specific pathogenic T cells, thereby creating a source of Tregs with matched specificity for therapeutic intervention.
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7

Zhang, Jiao, and 张姣. "Regulation of cell proliferation and modulation of differentiation in human induced pluripotent stem cell-derived mesenchumal stem cells." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hub.hku.hk/bib/B49617503.

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Functional mesenchymal stem cells (MSCs) derived from human induced pluripotent stem cells (iPSCs) may represent an unlimited cell source with superior therapeutic benefits for tissue regeneration to somatic tissue, such as bone marrow (BM)-derived MSC. In the first part of this project, I investigated whether the differential expression of ion channels in iPSC-MSCs was responsible for their higher proliferation capacity than that of BM-MSCs. The expression of ion channels for K+, Na+, Ca2+ and Cl- currents was assessed by reverse transcription-polymerase chain reaction (RT-PCR). The functional role of these ion channels were then verified by patch clamp experiments to compare the electrophysiological properties of iPSC-MSCs versus BM-MSCs. I detected significant mRNA expression of ion channel genes including KCa1.1, KCa3.1, KCNH1, Kir2.1, SCN9A, CACNA1C and Clcn3 in both human iPSC-MSCs and BM-MSCs; while Kir2.2 and Kir2.3 were only observed in human iPSC-MSCs. Furthermore, I identified five types of currents (BKCa, IKDR, IKir, IKCa and ICl) in iPSC-MSCs, while only four of them (BKCa, IKDR, IKir and IKCa) were observed in BM-MSCs. The rate of cell proliferation was 1.4 fold faster in iPSC-MSCs as compared to BM-MSCs. Interestingly, the proliferation rate of human iPSCMSCs was significantly reduced when inhibiting IKDR with shRNA and hEAG1 channel blockers, 4-AP and astemizole. Though to a lesser extent, the proliferation rate of human BM-MSCs also decreased by IKDR blockage. These results demonstrated that hEAG1 channel plays a crucial role in controlling the proliferation rate of human iPSC-MSCs but to a lesser extent in BM-MSCs. Next, I examined whether forced expression of a transcription factor- myocardin in iPSC-MSC using viral vectors (adenovirus or lentivirus) can further enhance their trans-differentiation to cardiomyocytes and improve their electrophysiological properties for cardiac regeneration. My results on RT-PCR and immunofluorescent staining revealed that myocardin induced the expression of several cardiac and smooth muscle cell markers, including α-MHC, cTnT, GATA4, α-actinin, and cardiac MHC, smooth muscle cell markers MYH11, calponin, and SM α-actin, but not the more mature cardiac markers such as β-MHC and MLC2v in iPSC-MSCs. These findings indicate that forced expression of myocardin in iPSC-MSC resulted in partial trans-differentiation into cardiomyocytes phenotype. Furthermore, I also discovered that myocardin altered the electrophysiological properties of iPSC-MSCs when examined by RT-PCR and patch clamp experiments. Forced expression of myocardin in iPSC-MSC enhanced the expression of Kv4.3, SCN9A and CACNA1C, but reduced that of KCa3.1 and Kir 2.2 in iPSC-MSCs. Moreover, BKCa, IKir, ICl, Ito and INa.TTX were detected in iPSC-MSC with ectopic expression of myocardin; while only BKCa, IKir, ICl, IKDR and IKCa were noted in iPSC-MSC transfected with green florescence protein. Furthermore, as measured by multi-electrode arrays recording plate, the conduction velocity of the neonatal rat ventricular cardiomyocytes cocultured iPSC-MSC monolayer was significantly increased after ectopic expression of myocardin. Taken together, I have demonstrated that hEAG1 channel is important in the regulation of iPSC-MSC proliferation and forced expression of myocardin in iPSC-MSC resulted in their partial transdifferentiation into cardiomyocytes phenotype and improved the electrical conduction during integration with mature cardiomyocytes.
published_or_final_version
Medicine
Doctoral
Doctor of Philosophy
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8

Jambi, Majed. "Differentiation of Human Atrial Myocytes from Endothelial Progenitor Cell-Derived Induced Pluripotent Stem Cells." Thèse, Université d'Ottawa / University of Ottawa, 2014. http://hdl.handle.net/10393/31158.

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Recent advances in cellular reprogramming have enabled the generation of embryoniclike cells from virtually any cell of the body. These inducible pluripotent stem cells (iPSCs) are capable of indefinite self-renewal while maintaining the ability to differentiate into all cell types. Nowhere will this technology have a greater impact than in the ability to generate disease and patient-specific cell lines. Here we explore the capacity of human iPSCs reprogrammed from peripheral blood endothelial progenitor cells lines to differentiate into atrial myocytes for the study of patient specific atrial physiology. Methods and Results: Late outgrowth endothelial progenitor cells (EPCs) cultured from clinical blood samples provided a robust cell source for genetic reprogramming. Transcriptome analysis hinted that EPCs would be comparatively more amenable to pluripotent reprogramming than the traditional dermal fibroblast. After 6 passages, EPCs were transduced with a doxycycline inducible lentivirus system encoding human transcription factors OCT4, SOX2, KLF4 and Nanog to permit differentiation after removal of doxycycline. The high endogenous expression of key pluripotency transcripts enhanced the ease of iPSC generation as demonstrated by the rapid emergence of typical iPSC colonies. Following removal of doxycycline, genetically reprogrammed EPC-iPSC colonies displayed phenotypic characteristics identical to human embryonic stem cells and expressed high levels of the pluripotent markers SSEA-4, TRA1-60 and TRA1-81. After exposure to conditions known to favor atrial identity, EPC- iPSC differentiating into sheets of beating cardiomyocytes that expressed high levels of several atrial-specific expressed genes (CACNA1H, KCNA5, and MYL4). Conclusions: EPCs provide a stable platform for genetic reprogramming into a pluripotent state using a doxycycline conditional expression system that avoids reexpression of oncogenic/pluripotent factors. Human EPC-derived iPSC can be differentiated into functional cardiomyocytes that express characteristic markers of atrial identity.
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9

Ruiz-Torres, Sonya Jomara. "Modeling Fanconi Anemia in Squamous Epithelium using Human Induced Pluripotent Stem Cell-Derived Organoids." University of Cincinnati / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1573573103136768.

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10

Cleary, Elaine Marie. "Effect of C9orf72 hexanucleotide repeat expansions on human induced pluripotent stem cell derived oligodendrocytes." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/28816.

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A hexanucleotide repeat expansion in the C9orf72 gene is the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia. Genetic testing for this pathogenic mutation is challenging due to its GC rich, repetitive nature. I developed PCR based assays to detect the presence of the pathogenic variant, which were used in screening an archival cohort of Scottish ALS patients, and have also been implemented within a diagnostic setting. These PCR assays allow amplification of larger repeat expansions than have previously been reported, and can determine whether a C9orf72 expansion of greater than 100 repeats is present or not. It is not well understood how the repeat expansion leads to disease, but several potential mechanisms have been hypothesised, including reduced expression, RNA toxicity and protein toxicity via dipeptide repeat proteins produced through repeat associated non-AUG translation. Motor neurons are an understandably well studied target in amyotrophic lateral sclerosis, however the role of glia, particularly oligodendrocytes, in the pathogenesis of the disease has recently been highlighted from studies on rodent models and post mortem tissue. To investigate the effect of the C9orf72 repeat expansion on oligodendrocytes, we have applied a differentiation protocol to hiPSCs with the expansion and controls, including an isogenic control which has been generated in the lab. There was no difference in the production of neuronal and glial cell types between these cell lines. I went on to look for evidence of the main proposed pathological mechanisms of C9orf72 repeat expansions: loss of function or gain of function through either RNA or protein toxicity. hiPSC derived oligodendrocytes from both carrier and control showed low expression of C9orf72 mRNA, and there was no difference due to the presence of a repeat expansion. Carrier hiPSC derived oligodendrocytes displayed sense RNA foci, which did not appear to have an effect on cellular morphology. The detection of dipeptide repeat proteins proved challenging, and the results were inconclusive as to their presence in hiPSC derived oligodendrocytes. I went on to show there was no evidence of mislocalisation of TDP-43 in C9orf72 carrier oligodendrocytes. Finally, the study showed similar levels of cell death in basal conditions in carrier and control cells, and no clear difference in the response to endoplasmic reticulum stress. Further research will be required to elucidate the role of oligodendrocytes in C9orf72 related amyotrophic lateral sclerosis.
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11

Songstad, Allison Elaine. "Generating patient-specific induced pluripotent stem cell-derived choroirdal endothelium to study and treat macular degeneration." Diss., University of Iowa, 2016. https://ir.uiowa.edu/etd/2278.

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Age-related macular degeneration (AMD) is a leading cause of irreversible blindness in the Western world. Although, the majority of stem cell research to date has focused on production of RPE and photoreceptor cells for the purpose of evaluating disease pathophysiology and cell replacement, there is strong evidence that the choroidal endothelial cells (CECs) that form the choriocapillaris vessels are the first to be affected in this disease. As such, to accurately evaluate disease pathophysiology and develop an effective treatment, production of patient-specific stem cell-derived CECs will be required. During the first stage of my Ph.D work, represented in Chapter 1 of this dissertation, I developed a co-culture system to differentiate mouse stem cells into CECs. I reprogrammed dermal fibroblasts from the Tie2-GFP mouse into two independent iPSC lines. TheTie2-GFP iPSCs were differentiated into CECs using a co-culture method with either the monkey RF/6A CEC line or primary mouse CECs. IPSC-derived CECs were characterized via rt-PCR and immunocytochemistry (ICC) for EC- and CEC-specific markers. The mouse iPSC-derived CECs described in Chapter 1 expressed the CEC-specific marker carbonic anhydrase IV (CA4), eNOS, FOXA2, PLVAP, CD31, CD34, ICAM-1, Tie2, TTR, VE-cadherin, and vWF. These Tie2-GFP iPSC-derived CECs paved the way for the rest of my Ph.D, in which I transitioned into using human iPSCs to generate patient-specific CECs. During the second phase of my graduate work, presented in Chapter 3, I developed a novel stepwise differentiation protocol suitable for generating human iPSC-derived CECs. I used previously published RNA-seq data of the monkey CEC line, RF/6A and two statistical screens to develop media comprised of various protein combinations. In both screens, I identified connective tissue growth factor (CTGF) as the key component required for driving CEC development. I also found that a second factor, called TWEAKR, promoted iPSC to CEC differentiation by inducing endogenous CTGF secretion. CTGF-driven iPSC-derived CECs formed capillary tube-like vascular networks, and expressed the EC-specific markers CD31, ICAM1, PLVAP, vWF, and the CEC-restricted marker CA4. These patient-specific iPSC-derived CECs made it possible for me to proceed into the next phase of my Ph.D work, in which I started working with AMD patient-specific iPSC-derived CECs to evaluate AMD pathophysiology. In the final stage of my Ph.D, represented in Chapter 4, I used the novel CEC differentiation method I developed to generate AMD iPSC-derived CECs and use these cells for AMD disease modeling. In line with previous studies that the membrane attack complex (MAC) forms in the AMD choriocapillaris, I showed that the AMD iPSC-derived CECs were much more susceptible to MAC formation and cell death when the cells were antagonized with complement components. I also demonstrated that, unlike the control CECs, the AMD CECs lost their capillary tube-like structures when the cells were cultured for over ten days, indicating that the AMD CECs may also exhibit other disease phenotypes other than susceptibility to MAC and cytolysis. Overall, the work I present in this dissertation will help push the AMD research field forward by providing a way to directly study AMD patient-specific iPSC-derived CECs and how they differ from healthy iPSC-derived CECs. In combination with RPE and photoreceptor cells, these patient-specific iPSC-CECs will make it possible to study AMD patient-specific CECs in vitro to better understand AMD pathogenesis and to develop autologous cell replacement therapies to replenish patients’ damaged choroids with healthy CECs.
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12

Kamakura, Tsukasa. "Ultrastructural Maturation of Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes in a Long-Term Culture." Kyoto University, 2015. http://hdl.handle.net/2433/199202.

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13

Kitahata, Shohei. "Critical Functionality Effects from Storage Temperature on Human Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium Cell Suspensions." Kyoto University, 2019. http://hdl.handle.net/2433/242417.

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14

Kimura, Azuma. "Small molecule AT7867 proliferates PDX1-expressing pancreatic progenitor cells derived from human pluripotent stem cells." Kyoto University, 2019. http://hdl.handle.net/2433/242422.

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15

Nakane, Takeichiro. "Impact of Cell Composition and Geometry on Human Induced Pluripotent Stem Cells-Derived Engineered Cardiac Tissue." Kyoto University, 2018. http://hdl.handle.net/2433/232090.

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16

Shiba, Takeshi. "Functional evaluation of the pathological significance of MEFV variants using induced pluripotent stem cell-derived macrophages." Kyoto University, 2020. http://hdl.handle.net/2433/253163.

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17

Juthaporn, Assawachananont. "Transplantation of embryonic and induced pluripotent stem cell-derived 3D retinal sheets into retinal degenerative mice." Kyoto University, 2015. http://hdl.handle.net/2433/199159.

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18

Tangruksa, Benyapa. "Cardiac hypertrophy in human stem cells-derived cardiomyocytes : Biomarker identification and pathway analysis of endotheline-1 induced cardiac hypertrophy in human induced pluripotent stem cells-derived cardiomyocytes." Thesis, Högskolan i Skövde, Institutionen för biovetenskap, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-18902.

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Cardiac hypertrophy is when heart muscles thicken as an adaptive response to several stimuli. Prolonged pathological cardiac hypertrophy can lead to heart failure and severe cardiovascular diseases. Scientists have faced challenges in studying cardiac hypertrophy due to the lack of human cardiomyocytes available. Recently, hypertrophic model using human induced pluripotent stem cell-derived cardiomyocytes was introduced. In this study, expression profiles of in vitroendothelin-1 induced cardiac hypertrophy model were investigated at different time points. The study aimed to examine molecular pathways associated with cardiac hypertrophy, identify biomarker candidates for cardiac hypertrophy, and investigate if there were known pharmaceuticals that putatively are targeting the suggested candidate biomarkers. Using the Ingenuity pathway analysis (IPA) software, GRM1, NPPA, and STC1 gene were identified as biomarker candidates for cardiac hypertrophy model across all time points. More biomarker candidates unique to the cardiac hypertrophy-stages were also identified using IPA. In vivomicroarray data of hypertrophied heart profiles were also used to compare to the in vitro data and preliminarily validate the gene candidates identified by IPA. Four genes were identified by IPA and were presented in the in vivo data. IPA also revealed the in activation of specific pathways of the early-stage cardiac hypertrophy model. The result suggested that the molecular mechanisms of the in vitro cardiac hypertrophy model did not fully represent the actual hypertrophic condition of the heart. More research and validation are required to understand the underlying mechanism fully and potentially, in the future, utilize the identified genes as cardiac hypertrophy biomarkers.
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Robertson, Abigail. "Targeting the Hippo signalling pathway to enhance the protective effect of iPS cell derived cardiomyocytes." Thesis, University of Manchester, 2017. https://www.research.manchester.ac.uk/portal/en/theses/targeting-the-hippo-signalling-pathway-to-enhance-the-protective-effect-of-ips-cell-derived-cardiomyocytes(f80b12c7-5289-46f9-8829-28224f0c6270).html.

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Cell based therapy using stem cell derived cardiomyocytes, has emerged as a potential therapeutic approach for cardiac diseases such as myocardial infarction and heart failure. Induced pluripotent stem cells (iPS cells) could be an ideal source of cardiomyocytes (iPS-CM). Challenges facing cell therapy include the high number of viable cells needed to survive in pathological conditions. The Hippo signalling pathway has been described as a key pathway involved in regulating cardiomyocyte proliferation and survival in both embryonic and adult hearts. We hypothesise that modification of the Hippo pathway will enhance the efficiency of iPS-CM generation and will increase iPS-CM survival and viability in pathological conditions. Skin fibroblasts were reprogrammed to iPS cells and then differentiated to cardiomyocytes. The Hippo signalling pathway was modified by genetic ablation of MST1, a major upstream regulator of the Hippo pathway, or by overexpressing YAP, the main downstream effector of the pathway. Cell proliferation was analysed using an EdU incorporation assay and staining for cytokinesis markers Ki67 and phospho-histone H3. Cell death and viability were analysed by measuring caspase 3/7 and MTT activity and by trypan blue staining in both normal and hypoxic conditions (CoCl2 treatment). Analysis of cell proliferation shows that genetic ablation of Mst1 leads to significantly increased proliferation (+12±1.5% P < 0.001), survival and viability (+20±4.3% P < 0.001) of iPS cells in both normal and hypoxic (CoCl2 treatment) conditions compared to controls. In addition, overexpression of YAP, which is normally inhibited by upstream Hippo pathway components, and overexpression of mutated constitutively active form of YAP (S127A) increases cell proliferation in iPS-CM compared to control iPS-CM as shown with EdU assay (46±2.60% P < 0.01) and Ki67 staining (4.9±0.9% P < 0.001). Overexpression of YAP leads to up regulation of genes associated with inhibition of apoptosis and promotion of cell proliferation. Preliminary studies show mouse iPS-CM are retained in the myocardium following intra-cardiac injection and do not cause any adverse effects confirmed with histological, echocardiography and electrocardiogram analysis. In conclusion targeting the Hippo pathway in iPS cells and iPS-CM significantly increases proliferation and survival in both normal and hypoxic conditions. Therefore, modulation of the Hippo pathway could become a new strategy to enhance the therapeutic potential of iPS-CM.
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Maifoshie, Evie. "Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for studying the role of MAP4K4 kinase in cell death." Thesis, Imperial College London, 2014. http://hdl.handle.net/10044/1/44977.

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Kinases comprise universal signalling cascades potentially pivotal to cardiac cell death pathways. Cell death is divided into several modes, chiefly apoptosis and necrosis. Both contribute to the pathophysiology of heart disease, with overlapping signalling pathways of caspase activation and apoptogen release from the mitochondria. This project focuses on the genetic validation of the stress-activated protein kinase, MAP4K4 / HGK, which functions as a control point integrating diverse signals for cardiomyocyte cell death. MAP4K4 and its reported target MAP3K7 / TAK1 appear to operate upstream of JNK in driving cardiac muscle cell death. MAP4K4 is activated in diseased mouse and human myocardium and in cultured rat cardiomyocytes subjected to death signals. Its overexpression in the rodent cardiomyocytes is sufficient to induce cardiomyocyte apoptosis, whereas knockdown of endogenous MAP4K4 using shRNA suppresses it. However, there is no direct or concrete evidence that MAP4K4 has functional importance in a human cardiac muscle cell. Given this unmet need, we deployed shRNAmir-based technology in cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) to knockdown MAP4K4 and investigate inhibition of cardiac muscle cell death, in response to death-inducing agents (i.e. H2O2 and c2-ceramide). First, we characterized the purity of the proposed human system by cardiac-specific antibodies and confirmed the cells’ cardiac identity, with a purity of 95% for the most prevalent marker tested. Then, via a series of optimization efforts using assays for caspase-3 and caspase-8 activities, diminished mitochondrial membrane potential and loss of surface membrane integrity, we developed a short-term maintenance protocol for hiPSC-CMs entailing 2 days in defined (transition) medium prior to death induction. Using this method, the cells were refractory to serum-induced proliferation, thus providing us with conditions under which the cell death assays could be carried out without confounding effects on purity. Prior to phenotypic assessment of MAP4K4 knockdown in hiPSC-CMs, we validated the efficacy of multiple shRNAmirs in a heterologous system. Three potent shRNAmirs were selected for virus production and proven to inhibit MAP4K4 (approximately 60-70%) in hiPSC-CMs. We then defined the conditions for provoking cell death with H2O2 and c2-ceramide, and identified the biochemical responses to each of these complementary death signals. Cell death with various H2O2 concentrations at different times was characterized by diminished mitochondrial potential, decreased caspase-8 activity and lack of caspase-3 activation. Cell death with 20μM c2-ceramide was evident at 16h associated with increased caspase-3 activity. Suppression of MAP4K4 in hiPSC-CMs with the most potent shRNAmir resulted in a 2-fold reduction of death (p < 0.001) measured by DRAQ7 and 2-fold reduction (p < 0.05) of cells with diminished mitochondrial potential, compared to the non-silencing control (NSC) upon H2O2 treatment. Interestingly, the reduction of cell death was associated with increased caspase-8 activity. Thus, we prove that MAP4K4 is pro-apoptotic in human cardiac muscle cells, as its knockdown leads to salvage from cell death. This proof-of-concept study will serve as a technical and scientific springboard for later, “higher bandwidth” studies of the entire human kinome or other essential mediators of cardiomyocyte demise.
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Li, Xiang. "Mitochondrial transfer from induced pluripotent stem cell-derived mesenchymal stem cells to airway epithelial and smooth muscle cells attenuates oxidative stress-induced injury." Thesis, Imperial College London, 2016. http://hdl.handle.net/10044/1/58260.

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Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease characterized by persistent airflow limitation that is not fully reversible and is usually caused by cigarette smoke (CS). The disease is predicted to be the fourth leading cause of death by 2030, but none of the currently available treatments can alleviate the progressive decline in lung function. Mesenchymal stem cells (MSCs) are fibroblast-like multipotent stem cells that can be isolated from various tissues such as bone marrow (BM-MSCs). Despite numerous reports of their efficacy in COPD-related pre-clinical models, BM-MSCs have not demonstrated efficacy in a clinical trial of COPD, highlighting the need for improved MSC-based therapy. The in vitro derivation of MSCs from induced pluripotent stem cells (iPSCs) has provided a new source of MSCs. Compared to BM-MSCs, iPSC-derived MSCs (iPSC-MSCs) are a more abundant source, have a higher expanding capacity and are possibly not subject to the ageing-associated dysfunction seen in BM-MSCs. In this study I determined the effects of human iPSC-MSCs in a rat COPD model using BM-MSCs as comparison. Rats were exposed to CS for 1 hr/day for 56 days. iPSC-MSCs or BM-MSCs were administrated at days 29 and 43. iPSC-MSCs demonstrated superior effects over BM-MSCs in attenuating CS-induced lung airspace enlargement, fibrosis, inflammation and apoptosis. In a mouse model of ozone-induced lung damage, intravenous administration of iPSC-MSCs 24 hours before ozone exposure for 3 hours alleviated airway hyper-responsiveness, inflammation and apoptosis in the lung. There is increasing evidence demonstrating that mitochondrial dysfunction may play an important role in COPD pathogenesis, indicating mitochondria as a potential therapeutic target. Meanwhile, mitochondrial transfer from MSCs to injured airway cells has been reported as a novel mechanism of action for MSCs. In this study mitochondrial transfer from iPSC-MSCs to the airway epithelium of CS-exposed rats was detected. iPSC-MSCs also transferred mitochondria to bronchial epithelial BEAS-2B cells and primary airway smooth muscle cell (ASMCs) in vitro in a direct co-culture system, an effect that was enhanced by CS medium (CSM). Direct co-culture with iPSC-MSCs alleviated CSM-induced ATP deprivation in BEAS-2B cells, as well as CSM-induced mitochondrial reactive oxygen species (ROS), apoptosis and reduction of mitochondrial membrane potential (ΔΨm) in ASMCs. Administration of iPSC-MSCs also prevented ozone-induced mitochondrial ROS and ΔΨm reduction in mouse lungs. The paracrine effects of iPSC-MSCs were also investigated. iPSC-MSC-derived conditioned medium (iPSC-MSCs-CdM) protected BEAS2-B cells from CSM-induced apoptosis. The effect was reduced by depleting stem cell factor (SCF) from iPSC-MSCs-CdM. However, both iPSC-MSCs-CdM and trans-well inserts containing iPSC-MSCs were only able to alleviate CSM-induced mitochondrial ROS, but not ΔΨm reduction and apoptosis, in ASMCs. I demonstrated the capacity of iPSC-MSCs to alleviate oxidative stress-induced COPD phenotype in vivo. Mitochondrial transfer from iPSC-MSCs was able to alleviate oxidative stress-induced mitochondrial dysfunction and apoptosis in target cells. The full capacity of iPSC-MSCs to achieve these effects may rely on a combination of cell-cell contact and release of paracrine factors. These findings define iPSC-MSCs as a promising candidate for the development of MSCs-based therapy of COPD.
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Shoji, Emi. "Early pathogenesis of Duchenne muscular dystrophy modelled in patient-derived human induced pluripotent stem cells." Kyoto University, 2015. http://hdl.handle.net/2433/200495.

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Kocharyan, Avetik. "Derivation and Characterization of Pax7 Positive Skeletal Muscle Precursor Cells from Control and HGPS-derived induced Pluripotent Stem Cells." Thesis, Université d'Ottawa / University of Ottawa, 2018. http://hdl.handle.net/10393/37517.

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Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic disorder associated with premature aging in various tissues and organs of the afflicted individuals, including accelerated skeletal muscle atrophy. Classical HGPS manifests due to single-base substitution in the LAMNA gene which encodes Lamin A/C proteins. As a result of the mutation, a truncated form of Lamin (known as Progerin) is produced which undergoes persistent farnesylation during post-translational modification. Accumulation of Progerin in the nucleus has been linked to various cellular abnormalities including abnormal nuclear morphologies and altered chromatin organization, among others. However, the exact molecular mechanisms leading to skeletal muscle atrophy have not yet been elucidated. In this study, the iPSC approach was implemented in order to study the skeletal muscle phenotype of HGPS by generating and characterizing a population of Pax7 positive skeletal muscle precursor cells (SMPs). During the course of this project, we have demonstrated the need for excessive optimization of the previously developed directed differentiation protocol for successful application on induced Pluripotent Stem Cells. Furthermore, we have successfully modified the protocol to allow for a more rapid expansion of the SMPs through regular passaging of the myogenic cells starting on day 20 of differentiation. Additionally, this new method produced more uniform distribution of the myogenic cells and allowed for successful freezing/thawing of the myogenic cells. When compared to the controls, the HGPS-derived SMPs did not appear to be defective in formation, proliferation or differentiation. Abnormal nuclear morphology and DNA damage, documented in HGPS fibroblasts and vascular smooth muscle cells, were not detected the in myogenic cells. Furthermore, we were not able to detect Progerin protein accumulation in the generated myogenic cultures, offering an explanation for the absence of these phenotypes in the skeletal muscle system.
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Zhao, Chen. "Investigation of the cell- and non-cell autonomous impact of the C9orf72 mutation on human induced pluripotent stem cell-derived astrocytes." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/25903.

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Amyotrophic lateral sclerosis (ALS) is a late onset neurodegenerative disorder characterised by selective loss of upper and lower motor neurons (MNs). Recently, the GGGGCC (G4C2) hexanucleotide repeat expansion in chromosome 9 open reading frame 72 (C9orf72) has been identified as the most common genetic cause of ALS, highlighting the importance of studying the pathogenic mechanisms underlying this mutation. Accumulating evidence implicates that ALS is a multisystem and multifactor disease. Specifically, non-neuronal cells, astrocytes in particular, are also affected by toxicity mediated by ALS-related mutations, and they can contribute to neurodegeneration, suggesting astrocytes as a key player in ALS pathogenesis. Here, a human induced pluripotent stem cells (iPSCs)-based in vitro model of ALS was established to investigate the impact of the C9orf72 mutation on astrocyte behaviour—both cell- and non-cell autonomous. Work in this study shows that patient iPSC-derived astrocytes recapitulate key pathological features associated with C9orf72-mediated ALS, such as formation of G4C2 repeat RNA foci, production of dipeptide repeat (DPR) proteins and reduced viability under basal conditions compared to controls. Moreover, C9orf72 mutant astrocytes in co-culture result in reduced viability and structural defects of human MNs. Importantly, correction of the G4C2 repeat expansion in mutant astrocytes through targeted gene editing reverses these phenotypes, strongly confirming that the C9orf72 mutation is responsible for the observed findings. Altogether, this iPSC-based in vitro model provides a valuable platform to gain better understandings of ALS pathophysiology and can be used for future exploration of potential therapeutic drugs.
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Achberger, Kevin [Verfasser]. "Human retinal organoids - Exploration of a human induced pluripotent stem cell-derived in vitro model / Kevin Achberger." Tübingen : Universitätsbibliothek Tübingen, 2021. http://d-nb.info/1231790687/34.

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Yulius, Hermanto. "Transplantation of feeder-free human induced pluripotent stem cell-derived cortical neuron progenitors in adult male Wistar rats with focal brain ischemia." Kyoto University, 2019. http://hdl.handle.net/2433/242389.

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O'Brien, Susanne. "Induced human pluripotent stem cell-derived NK cells as an alternative source of lymphocytes for anti-cancer immunotherapy." Thesis, University College London (University of London), 2018. http://discovery.ucl.ac.uk/10041819/.

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Human induced pluripotent stem cells (hiPSCs) hold great promise in regenerative medicine as they have the potential to differentiate into any specialized cell type and retain unlimited self-renewal capacity in vitro. The use of hiPSC-derived lymphocytes for adoptive cell transfer therapies is appealing as it provides a potentially indefinite cell source and a platform for genetically modifying cells. Natural killer (NK) cell-based therapy has shown promising results in the treatment of haematological malignancies and the unlimited derivation of NK cells from hiPSCs could overcome the current obstacle of insufficient NK cell numbers for cellular anti-cancer immunotherapy. In this study, we initially used a two-stage culture system to differentiate human pluripotent stem cells (hPSCs) into NK cells. First, haematopoietic stem cells (HSCs) were derived by co-culture with murine OP9 stromal cells. The CD34-expressing fraction was enriched with magnetic activated cell sorting (MACS) and phenotypically characterised by cell surface marker expression. Colony forming unit (CFU) assay confirmed that hPSC-derived CD34+ cells had multi-lineage differentiation potential. Subsequently, HSCs were co-cultured with murine EL08.1D2 feeder cells and under feeder-free conditions with GBGM medium to generate NK like cells. Interestingly, we observed a 100-fold lower cell expansion of hiPSC-derived CD34+ cells in NK cell differentiation conditions compared to umbilical cord blood (UCB)-derived CD34+ cells. Despite this observation the phenotype of hiPSC-derived NK like cells was comparable to UCB CD34+-derived NK cells and freshly isolated peripheral blood natural killer cells (PBNKs). As an alternative to the two-stage culture system, we aimed to drive NK cell differentiation by overexpressing the NK cell-specific transcription factor E4bp4 during in vitro derivation. Our data suggest that the overexpression of E4bp4 does not improve the efficiency of hiPSC-derived NK like cell differentiation, nor can E4bp4 alone induce the NK cell-specific transcriptional network in fibroblasts.
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Miyawaki, Yoshifumi. "Zonisamide promotes survival of human induced pluripotent stem cell-derived dopaminergic neurons in the striatum of female rats." Kyoto University, 2020. http://hdl.handle.net/2433/259730.

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Garg, Pretty [Verfasser], Kurt [Gutachter] Gottmann, and Christine R. [Gutachter] Rose. "Differentiation and characterization of induced pluripotent stem cell derived astrocytes / Pretty Garg ; Gutachter: Kurt Gottmann, Christine R. Rose." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2017. http://d-nb.info/1131799488/34.

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Borooah, Shyamanga. "Developing a patient-derived induced pluripotent stem cell model to understand the clinical and pathological changes in macular degeneration." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/25471.

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Late-onset retinal macular degeneration (L-ORMD) is a fully penetrant autosomal dominant macular degeneration resulting from a Ser163Arg substitution in the gene encoding the protein C1QTNF5. Clinically L-ORMD results in dark adaptation delay in the fifth decade, central visual loss in the sixth decade and further progressive visual field loss in successive decades of life. Pathologically the disease results in thick sub-retinal deposits, which have a similar composition to drusen seen in AMD, retinal pigment epithelial (RPE) loss, and neuro-retinal atrophy. The function of C1QTNF5 is incompletely understood however within the eye it is expressed most strongly by the RPE cells. An in vitro model for L-ORMD was developed using human induced pluripotent stem cells (hiPSCs) derived from patients and with stem cells from patient’s unaffected siblings used as controls. The hiPSCs were differentiated to RPE (hiPSC-RPE). L-ORMD hiPSC-RPE shared baseline characteristics with sibling control hiPSC-RPE. In order to model in vivo conditions hiPSC-RPE were grown on permeable supports in human serum enriched media. Case hiPSC-RPE cell lines were found to activate the complement pathway resulting in increased deposition of the terminal complement complex (TCC) C5b-9 when compared to control hiPSC-RPE. Using depleted serum, deposition was not affected by depletion of classical and lectin pathway components but was reduced by depletion of alternative complement pathway components. Depletion of complement components C3 and C5 abolished TCC deposition. The addition of a monoclonal antibody against C5 also reduced TCC deposition. The role of complement dysregulation in L-ORMD pathogenesis was confirmed by immunostaining of L-ORMD and age-matched control human donor retinal sections. L-ORMD retinal sections displayed increased C3d and C5b-9 deposition. Using mutant and wild type-protein generated from a bacterial expression system it was found that the mutant protein was less stable than the wild-type. In addition the wild type protein formed multimers whilst the mutant was mainly monomeric. A surface plasmon resonance (SPR) study showed an increased affinity of wild-type C1QTNF5, especially in multimeric form for complement factor H (CFH), a key regulator of the alternative complement pathway when compared to mutant protein. Taken together these studies implicate dysfunction of the alternative complement pathway in L-ORMD disease mechanism and have suggested a role for C1Q TNF5 in the extracellular matrix. The studies also show that L-ORMD and AMD share a pathogenic and clinical similarities.
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Korogi, Yohei. "In Vitro Disease Modeling of Hermansky-Pudlak Syndrome Type 2 Using Human Induced Pluripotent Stem Cell-Derived Alveolar Organoids." Kyoto University, 2019. http://hdl.handle.net/2433/243303.

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Hatani, Takeshi. "Nano-structural Analysis of Engrafted Human Induced Pluripotent Stem Cell-derived Cardiomyocytes in Mouse Hearts Using a Genetic-probe APEX2." Kyoto University, 2019. http://hdl.handle.net/2433/236616.

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Guessoum, Celina Isabelle [Verfasser]. "Analysis of catecholamine-induced beta-adrenergic signaling in TTS by patient-specific pluripotent stem cell-derived cardiomyocytes / Celina Isabelle Guessoum." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2020. http://d-nb.info/121670368X/34.

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Zhong, Rujia [Verfasser], and Martin [Akademischer Betreuer] Borggrefe. "Effects of Gastrointestinal Cancer Cell Secretion on Ion Channel Functions of Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes / Rujia Zhong ; Betreuer: Martin Borggrefe." Heidelberg : Universitätsbibliothek Heidelberg, 2020. http://d-nb.info/1228539901/34.

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Hayano, Mamoru. "The Development of a Patient-Derived Induced Pluripotent Stem Cell Model for the Investigation of SCN5A-D1275N- Related Cardiac Sodium Channelopathy." Kyoto University, 2018. http://hdl.handle.net/2433/230995.

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36

Moad, Mohammad. "Influence of cell type of origin to the differentiation potential of induced pluripotent stem cells derived from human urinary tract cells." Thesis, University of Newcastle upon Tyne, 2015. http://hdl.handle.net/10443/2803.

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Background: Direct reprogramming of human somatic cells to pluripotent embryonic stem (ES) cell -like cells, termed induced pluripotent stem (iPS) cells, can be achieved by expression of defined transcription factors. The potential use of iPS cells derived from the urinary tract provides a substantial opportunity in developing new disease models, drug screening and tissue engineering. We aimed to generate, for the first time, human induced pluripotent stem cells derived from the urinary tract (UT-iPS) cells and to assess capacity for directed differentiation into bladder lineages. Methods: Human primary culture cells derived from benign bladder and ureters were transduced with OCT4, SOX2, KLF4 and C-MYC genes to generate human UT-iPS cells. Generated cells were characterised using RT-PCR and immunofluorescence. Differentiation capacity was evaluated by embryoid body formation in vitro and teratoma assay in vivo. Established co-culture based directed differentiation into bladder cells was assessed in comparison with classical skin-derived iPS cells. Results: We demonstrated successful re-programming of adult urinary tract cells from both bladder and ureter into human UT-iPS cells. Most of the clones showed efficient transgene silencing and maintained a normal diploid karyotype. Specifically, we showed expression of ES cell markers and functional pluripotency by the generation of endodermal, ectodermal and mesodermal lineages. Differentiation into bladder lineages was demonstrated by expression of urothelial-specific markers, uroplakins (UPIb, UPII, UPIIIa, and UPIIIb), claudins (CLD1 and CLD5) and cytokeratin (CK7); and stromal smooth muscle markers a-SMA, calponin, and desmin. Human UT-iPS cells were shown to be more efficient than skin-derived iPS cells in undergoing bladder differentiation, underlining the importance of the origin of the parent cell for re-programming. Conclusions: We demonstrated that the induction of human urinary tract cells into iPS cells is possible, offering a new exciting opportunity for tissue engineering and for the study of bladder disease.
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Kikuchi, Tetsuhiro. "Survival of human induced pluripotent stem cell-derived midbrain dopaminergic neurons in the brain of a primate model of Parkinson's disease." Kyoto University, 2012. http://hdl.handle.net/2433/159389.

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Ryosaka, Makoto. "Expansion of human iPSC-derived ureteric bud organoids with repeated branching potential." Kyoto University, 2021. http://hdl.handle.net/2433/261606.

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Okuyama, Hideaki. "Transplantation of multiciliated airway cells derived from human iPS cells using an artificial tracheal patch into rat trachea." Kyoto University, 2020. http://hdl.handle.net/2433/253142.

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Komatsu, Kenichi. "Overexpressed wild-type superoxide dismutase 1 exhibits amyotrophic lateral sclerosis-related misfolded conformation in induced pluripotent stem cell-derived spinal motor neurons." Kyoto University, 2018. http://hdl.handle.net/2433/232077.

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Goldszer, Isaac M. "Biochemical fractionation of induced pluripotent stem cell derived motor neurons from an ALS patient with the Glycine-298-Serine TDP-43 mutation." Thesis, Boston University, 2013. https://hdl.handle.net/2144/12112.

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Thesis (M.A.)--Boston University
Transactive response DNA-binding protein (TDP-43), and fused in sarcoma/translocated in liposarcoma (FUS/TLS) form protein aggregates in amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration (FTLD). The sequencing of the TDP-43 gene TARDBP in a large patient population has shown more than 40 missense mutations that are now known to cause disease. The effect of genetic mutation on protein aggregation, and the pathogenesis of ALS is the focus of this study. In order to determine the effect of the TARDBP Glycine-298- Serine (G298S) missense mutation on protein aggregation in disease, induced pluripotent stem cells (iPSCs) were reprogrammed from control and G298S mutant fibroblasts, and differentiated into motor neurons using defined factors, and fractio- nated to determine the soluble and insoluble TDP-43 burden. There was an increase in insoluble TDP-43 in the ALS-patient-derived motor neuron lysates over a normal control, but the significance could not be assessed because of the small sample size. A toxicity assay using fluorescence activated cell sorting showed an unexpected trend towards healthier control neurons. Future studies should include quantified immunohistochemical analysis of motor neurons and use novel pharmaceuticals to attempt to correct aberrant TDP-43-mediated RNA processing.
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Lambertz, Pascal [Verfasser], and Dobromir [Akademischer Betreuer] Dobrev. "Target validation of adenylyl cyclase 5 and adenylyl cyclase 6 in human induced pluripotent stem cell-derived cardiomyocytes / Pascal Lambertz ; Betreuer: Dobromir Dobrev." Duisburg, 2021. http://d-nb.info/1241963231/34.

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Hildebrandt, Susanne [Verfasser]. "Generation of induced Pluripotent Stem Cell derived Endothelial Cells from Fibrodysplasia Ossificans Progressiva patients - a disease model of ActivinA and BMP-induced ALK2 receptor activation / Susanne Hildebrandt." Berlin : Freie Universität Berlin, 2021. http://d-nb.info/1233286528/34.

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Gomes, Sara Ferreira Martins [Verfasser], and Alexandra [Gutachter] Schubert-Unkmeir. "Induced Pluripotent Stem Cell-derived Brain Endothelial Cells as a Cellular Model to Study Neisseria meningitidis Infection / Sara Ferreira Martins Gomes ; Gutachter: Alexandra Schubert-Unkmeir." Würzburg : Universität Würzburg, 2019. http://d-nb.info/1196972168/34.

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Takaki, Tadashi. "Optical recording of action potentials in human induced pluripotent stem cell-derived cardiac single cells and monolayers generated from long QT syndrome type 1 patients." Kyoto University, 2019. http://hdl.handle.net/2433/242346.

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Tsurumi, Fumitoshi. "The intracellular Ca²⁺ concentration is elevated in cardiomyocytes differentiated from hiPSCs derived from a Duchenne muscular dystrophy patient." Kyoto University, 2020. http://hdl.handle.net/2433/253462.

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Ahmad, Faizzan Syed. "A novel human stem cell platform for probing adrenoceptor signaling in iPSC derived cardiomyocytes including those with an adult atrial phenotype." Thesis, University of Oxford, 2017. http://ora.ox.ac.uk/objects/uuid:17972018-6750-4e5c-8cc9-42e9c381f531.

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Scientific research is propelled by two objectives: Understanding and recognizing the essential biology of life, and deciphering this to uncover possible therapeutics in order to improve quality of life as well as relieve pain from disease. The aim of the work described in this thesis was to dissect the fundamental requirements of induced pluripotent stem cells both in pluripotency and differentiation with a particular focus on atrial specificity. Drug targeting of atrial-specific ion channels has been difficult because of lack of availability of appropriate cardiac cells, and preclinical testing studies have been carried out in non-cardiac cell lines, heterogeneous cardiac populations or animal models that have been unable to accurately represent human cardiomyocyte physiology. Therefore, we sought out to develop a preparation of cardiomyocytes showing an atrial phenotype with adult characteristics from human induced-pluripotent stem cells. A culture programme involving the use of Gremlin 2 allowed differentiation of cardiomyocytes with an atrial phenotype from human induced-pluripotent stem cells. When these differentiated cultures were dissociated into single myocytes a substantial fraction of cells showed a rod-shaped morphology with a single central nucleus that was broadly similar to that observed in cells isolated from atrial chambers of the heart. Immunolabelling of these myocytes for cardiac proteins (including RyR2 receptors, actinin-2, F-actin) showed striations with a sarcomere spacing of slightly less than 2um. The isolated rod-shaped cells were electrically quiescent unless stimulated to fire action potentials with an amplitude of 100 mV from a resting potential of approximately -70 mV. Proteins expressed included those for IK1, IKur channels. Ca2+ Transients recorded from spontaneously beating cultures showed increases in amplitude in response to stimulation of adrenoceptors (both alpha and beta). With the aim of identifying key signaling mechanisms in directing cell fate, our new protocol allowed differentiation of human myocytes with an atrial phenotype and adult characteristics that show functional adrenoceptor signaling pathways and are suitable for investigation of drug effects.
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Forsgren, Elin. "Using patient-derived cell models to investigate the role of misfolded SOD1 in ALS." Doctoral thesis, Umeå universitet, Institutionen för farmakologi och klinisk neurovetenskap, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-138948.

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Protein misfolding and aggregation underlie several neurodegenerative proteinopathies including amyotrophic lateral sclerosis (ALS). Superoxide dismutase 1 (SOD1) was the first gene found to be associated with familial ALS. Overexpression of human mutant or wild type SOD1 in transgenic mouse models induces motor neuron (MN) degeneration and an ALS-like phenotype. SOD1 mutations, leading to the destabilization of the SOD1 protein is associated with ALS pathogenesis. However, how misfolded SOD1 toxicity specifically affects human MNs is not clear. The aim of this thesis was to develop patient-derived, cellular models of ALS to help understand the pathogenic mechanisms underlying SOD1. To understand which cellular pathways impact on the level of misfolded SOD1 in human cells, we established a model using patient-derived fibroblasts and quantified misfolded SOD1 in relation to disturbances in several ALS-related cellular pathways. Misfolded SOD1 levels did not change following reduction in autophagy, inhibition of the mitochondrial respiratory chain, or induction of endoplasmic reticulum (ER)-stress. However, inhibition of the ubiquitin-proteasome system (UPS) lead to a dramatic increase in misfolded SOD1 levels. Hence, an age-related decline in proteasome activity might underlie the late-life onset that is typically seen in SOD1 ALS. To address whether or not SOD1 misfolding is enhanced in human MNs, we used mixed MN/astrocyte cultures (MNCs) generated in vitro from patient-specific induced pluripotent stem cells (iPSCs). Levels of soluble misfolded SOD1 were increased in MNCs as well as in pure iPSC-derived astrocytes compared to other cell types, including sensory neuron cultures. Interestingly, this was the case for both mutant and wild type human SOD1, although the increase was enhanced in SOD1 FALS MNCs. Misfolded SOD1 was also found to exist in the same form as in mouse SOD1 overexpression models and was identified as a substrate for 20S proteasome degradation. Hence, the vulnerability of motor areas to ALS could be explained by increased SOD1 misfolding, specifically in MNs and astrocytes. To investigate factors that might promote SOD1 misfolding, we focussed on the stability of SOD1 mediated by a crucial, stabilizing C57-C146 disulphide bond and its redox status. Formation of disulphide bond is dependent on oxidation by O2 and catalysed by CCS. To investigate whether low O2 tension affects the stability of SOD1 in vitro we cultured fibroblasts and iPSC-derived MNCs under different oxygen tensions. Low oxygen tension promoted disulphide-reduction, SOD1 misfolding and aggregation. This response was much greater in MNCs compared to fibroblasts, suggesting that MNs may be especially sensitive to low oxygen tension and areas with low oxygen supply could serve as foci for ALS initiation. SOD1 truncation mutations often lack C146, and cannot adopt a native fold and are rapidly degraded. We characterized soluble misfolded and aggregated SOD1 in patient-derived cells carrying a novel SOD1 D96Mfs*8 mutation as well as in cells fom an unaffected mutation carrier. The truncated protein has a C-terminal fusion of seven non-native amino acids and was found to be extremely prone to aggregation in vitro. Since not all mutation carriers develop ALS, our results suggested this novel mutation is associated with reduced penetrance. In summary, patient derived cells are useful models to study factors affecting SOD1 misfolded and aggregation. We show for the first time that misfolding of a disordered and disease associated protein is enhanced in disease-related cell types. Showing that misfolded SOD1 exists in human cells in the same form as in transgenic mouse models strengthens the translatability of results obtained in the two species. Our results demonstrate disulphide-reduction and misfolding/aggregation of SOD1 and suggest that 20S proteasome could be an important therapeutic target for early stages of disease. This model provides a great opportunity to study pathogenic mechanisms of both familial and sporadic ALS in patient-derived models of ALS.
Varje år insjuknar omkring 5300 personer i världen i motorneuronsjukdomen Amyotrofisk lateralskleros (ALS). Sjukdomen kännetecknas av degeneration av motorneuron i hjärnan och ryggmärgen, de nervceller som styr kroppens muskler, vilket leder till musklerförtvining och gradvis förlamning. ALS-patienter avlider oftast till följd av andningssvikt när sjukdomen når andningsmuskulaturen. I de allra flesta fall uppkommer ALS sporadiskt (SALS), det vill säga utan känd genetisk orsak, medan ärftliga fall (FALS) drabbar omkring 10 % och beror på mutationer i ett antal kända gener. Upp till 6 % av alla ALS fall kan härledas till mutationer i genen superoxid dismutas 1 (SOD1). SOD1 är ett enzym som ansvarar för att omvandla och oskadliggöra fria syreradikaler som bildas vid normal ämnesomsättning. 206 olika SOD1 mutationer har identifierats, alla orsakar inte ALS men många leder till att den tredimensionella proteinstrukturen förändras, vilket ökar proteinets benägenhet att felveckas. Initialt trodde man att SOD1 mutationer förhindrade proteinets normalfunktion och följaktligen orsakade ALS. Studier har emellertid visat att den enzymatiska funktionen ofta bevaras, även hos muterade proteiner. Däremot kan små mängder felveckat SOD1 störa andra viktiga cellulära funktioner. Felveckat SOD1 har en benägenhet att klumpa ihop sig och bilda aggregat i det centrala nervsystemet (CNS). Dessa aggregat återfinns hos patienter med såväl FALS som SALS vilket tyder på att även vildtyps-SOD1 kan felveckas och vara involverat i sjukdomsutvecklingen. De flesta studier är baserade på transgena musmodeller som uttrycker extremt stora mängder av muterat humant SOD1. Det är dock oklart hur väl studier i möss överensstämmer med sjukdomsutvecklingen hos ALS-patienter, där mängden SOD1 är betydligt lägre. En central fråga som fortfarande står obesvarad är varför just motorneuron degenererar i ALS, trots att SOD1 uttrycks i alla kroppens celler. Det övergripande syftet med den här avhandlingen har varit att karakterisera felveckat SOD1 i patientceller för att studera dess roll i ALSrelaterade sjukdomsmekanismer med fysiologiskt relevanta nivåer av SOD1. Samtliga studier är gjorda in vitro med celler från friska donatorer med vildtyps-SOD1, celler från patienter med SOD1-FALS, FALS som bär andra ALS-associerade gener, samt SALS. I de allra flesta fallen har vi analyserat både lösligt felveckat SOD1 samt aggregerade former av SOD1 proteinet.
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Lepperhof, Vera [Verfasser], Jürgen [Akademischer Betreuer] Hescheler, and Stephan [Akademischer Betreuer] Rosenkranz. "Retention and therapeutic effect of co-injected murine mesenchymal stem cells and induced pluripotent stem cell derived cardiomyocytes in cryoinjured hearts of syngeneic mice / Vera Lepperhof ; Akademische Betreuer: Jürgen Hescheler, Stephan Rosenkranz." Köln : Deutsche Zentralbibliothek für Medizin, 2019. http://d-nb.info/1196549877/34.

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Li, Wener [Verfasser], Kaomei [Akademischer Betreuer] Guan-Schmidt, Swen [Gutachter] Hülsmann, and Viacheslav [Gutachter] Nikolaev. "Functional analysis of ryanodine receptor 2 mutations in induced pluripotent stem cell-derived cardiomyocytes from CPVT patients / Wener Li ; Gutachter: Swen Hülsmann, Viacheslav Nikolaev ; Betreuer: Kaomei Guan-Schmidt." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2017. http://d-nb.info/1128902737/34.

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You might also be interested in the bibliographies on the topic 'Induced-pluripotent Stem Cell-derived Hepatocytes' for other source types:
Journal articles
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