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Matz, Peggy. "Human induced pluripotent stem cell–based modeling of hepatogenesis." Doctoral thesis, Humboldt-Universität zu Berlin, Lebenswissenschaftliche Fakultät, 2016. http://dx.doi.org/10.18452/17530.
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In dieser Studie wurden nicht-integrative Vektorkonstrukte zur Reprogrammierung von zwei menschlichen Zelllinien (HFF1, HUVEC) verwendet, um integrations-freie, episomal generierte iPSC Zelllinien (E-iPSCs) zu generieren. Darüber hinaus wurden diese iPSCs zu sogenannten Leberzell-ähnlichen Zellen (HLCs) differenziert. Hierzu konnten die verschiedenen Stufen der Hepatogenese und die potentielle Reifung zu Leberzellen untersucht sowie mit fötalen und ausgereiften menschlichen Leberzellen verglichen werden. Diese Studie konnte Gen-regulierende Netzwerke aufdecken, welche eine pi-potentiale Vorläuferpopulation in den HLCs präsentieren. Zusätzlich deckte das Transkriptions-Profil auf, dass die iPSC-generierten HLCs unreif und ähnlicher den fötalen Leberzellen sind. Dennoch weisen die HLCs typische funktionelle Charakteristika von Leberzellen auf, z.B. Glykogen-Einlagerung, Aufnahme und Abgabe von Substanzen wie ICG und CDFDA, Sekretierung von Gallensäure und Harnstoff. Zusätzlich konnten typische Leber-Strukturen wie Gallenkanälchen mit Mikrovilli, Fettspeicherung und sogenannte tight junctions, Verbindungsgänge zwischen den Zellen nachgewiesen werden. Um die potentielle Reifung dieser HLCs voranzutreiben, wurde eine Langzeit-Kultivierung von HUVEC-iPSC-generierten HLCs durchgeführt. Dies sollte zugleich zeigen, ob die HLCs länger kultiviert und gleichzeitig reifen können. Ein zweiter Teil dieser Studie befasst sich mit der Generierung von endodermalen Vorläuferzellen (EPs). Es wurden HFF1-iPSCs zu EPs differenziert um die endodermale Entwicklung vor der Entstehung der Gallenwege und des Hepatoblasten zu untersuchen. Die EPs zeigen Merkmale dafür, dass sie sowohl in Hepatozyten, Cholangozyten und auch Pankreaszellen differenziert werden können. Mit Hilfe dieser multipotenten EPs könnte es möglich sein die endodermale Entwicklung des Darmes, der Lunge, Leber, Gallengänge und Gallenblase sowie der Bauchspeicheldrüse näher zu untersuchen.<br>This project generated and characterized integration-free, episomal-derived induced pluripotent stem cell lines (E-iPSCs) from human somatic cell lines of different origins. Two different somatic cell lines were used, the human fetal fibroblast cell line HFF1 and human umbilical vein endothelial cell line HUVEC. Both were reprogrammed into integration-free iPSCs and were comparable amongst themselves and to human embryonic stem cells, the gold standard of pluripotent stem cells. Furthermore, the iPSCs with different genetic background were differentiated to hepatocyte-like cells (HLCs). With the use of iPSC-derived hepatocytes different stages during hepatogenesis and the potential of maturation could be analyzed as well as compared to fetal liver and primary human hepatocytes (PHH). This study could uncover gene regulatory networks which presence bipotential progenitor populations in HLCs. Additionally, comparable transcriptome profile analyses revealed that the iPSC-derived HLCs are immature and more similar to fetal liver. However, the HLCs hold typical functionality characteristics of hepatocyte, e.g. glycogen storage, uptake and release of ICG and CDFDA, bile acid and urea secretion. Furthermore, typical structures of hepatocytes such as bile canaliculi with microvilli, lipid storage and tight junctions are visible. In order to analyze the maturation potential of HLCs a long-term culture experiment was performed using HUVEC-iPSC-derived HLCs which implies the possibility for long-term culture of HLCs while increasing maturation. Additionally, HFF1-derived iPSCs were differentiated to endodermal progenitors (EPs) to analyze the endodermal development before biliary tree and hepatoblast which can give rise to hepatocytes, cholangiocytes and pancreatic cells. The multipotent EPs hold a great potential to analyze the endodermal development of intestine, lung, liver, bile duct and gallbladder, as well as pancreas.
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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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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.<br>published_or_final_version<br>Medicine<br>Doctoral<br>Doctor of Philosophy
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Ratanasirintrawoot, Sutheera. "Defining markers and mechanisms of human somatic cell reprogramming." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:11236.
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Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by over expression of the transcription factors OCT4, SOX2, KLF4 and c-MYC. Using serial live cell immunofluorescence imaging of human fibroblasts undergoing reprogramming, we traced the emergence of nascent iPS cell colonies among heterogeneous cell populations and defined the kinetics of marker expression. We identified distinct colony types that morphologically resemble embryonic stem (ES) cells yet differ in molecular phenotype and differentiation potential. By analyzing expression of pluripotency markers, methylation at the OCT4 and NANOG promoters, and differentiation into teratomas, we determined that only one colony type represented bona fide iPS cells, whereas the others represented reprogramming intermediates. Proviral silencing and expression of TRA-1-60, DNMT3B, and REX1 distinguished the fully reprogrammed state, whereas Alkaline Phosphatase, SSEA-4, GDF3, hTERT and NANOG proved insufficient as markers. Reprogramming in chemically defined medium favored formation of bona fide iPS cell colonies relative to partially reprogrammed colonies. These data highlight the need for rigorous characterization and standardization of putative iPS cells.
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Zorzan, Irene. "Dissecting the role of TGF-beta pathway in human Pluripotent Stem Cells." Doctoral thesis, Università degli studi di Padova, 2019. http://hdl.handle.net/11577/3424722.
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Human Embryonic Stem Cells (hESCs) and induced Pluripotent Stem cells (hiPSCs) are characterized by the ability to give rise all cell types found in the adult and to be expanded indefinitely in vitro. Understanding the molecular mechanisms controlling pluripotency is fundamental to differentiate human pluripotent cells into cells types useful for clinical applications. The signaling pathway of TGF-beta and FGF are known to maintain pluripotency in human cells. Only a handful of factors controlling pluripotency have previously been identified, such as the transcription factors OCT4, SOX2 and NANOG. Therefore, I used a systematic approach to identify novel components of the pluripotency network. Here I focused on the role of TGF-beta pathway, in order to find direct functional targets downstream of this pathway. Through comparative transcriptome analysis intersected with genome location data, I obtained a list of 21 putative transcription factors, out of which 8 were confirmed. Further functional assays led to the identification of four transcription factors that are able to maintain hESCs and hiPSCs undifferentiated in the absence of TGF-beta. Particularly, one of these four transcription factors has never been studied, so I focused on it. I then characterized the transcriptional program under the control of this factor in order to understand how it maintains the human pluripotency network. Interestingly, I found that this factor regulates both pluripotency and cell morphology. Finally, knockdown of this factor during the reprogramming strongly reduces the number of iPSCs obtained.<br>Le cellule staminali embrionali umane (hESCs) e le cellule staminali pluripotenti indotte (hiPSCs) sono caratterizzate dalla capacità di dare origine tutti i tipi cellulari presenti nell’adulto e di poterle espandere indefinitamente in vitro. Comprendere i meccanismi molecolari che controllano la pluripotenza è fondamentale per differenziare cellule pluripotenti umane in tutti i tipi cellulari utili per applicazioni cliniche. Le vie di segnalazione che mantengono la pluripotenza nelle cellule staminali pluripotenti umane sono TGF-beta e FGF. Ad oggi, sono stati identificati solo pochi fattori di trascrizione che controllano la pluripotenza, come i fattori di trascrizione OCT4, SOX2 e NANOG. Pertanto, ho utilizzato un approccio sistematico per identificare nuovi componenti del network di pluripotenza. Mi sono focalizzata sul ruolo di TGF-beta al fine di trovare target funzionali diretti che a valle di questa via di segnalazione siano in grado di mantenere lo stato di pluripotenza. Intersecando un’analisi comparativa del trascrittoma con dati relativi alla posizione nel genoma, ho ottenuto una lista di 21 fattori di trascrizione, di cui poi 8 sono stati confermati. Ulteriori test funzionali hanno portato all’identificazione di quattro fattori di trascrizione che sono in grado di mantenere hESCs e hiPSCs pluripotenti indifferenziate in assenza di TGF-beta. In particolare, uno di questi quattro fattori di trascrizione non è mai stato studiato, quindi mi sono focalizzata su di esso. Ho successivamente caratterizzato il programma trascrizionale controllato da questo fattore per capire come sia in grado di mantenere la pluripotenza. È interessante notare che questo nuovo fattore regola sia la pluripotenza che la morfologia cellulare, ossia l’identità epiteliale. Infine, il knockdown di questo fattore durante la riprogrammazione somatica riduce fortemente il numero di colonie di iPSCs ottenute.
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Yamashiro, Chika. "Generation of human oogonia from induced pluripotent stem cells in vitro." Kyoto University, 2019. http://hdl.handle.net/2433/242826.
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Rohani, Leili, Claire Fabian, Heidrun Holland, et al. "Generation of human induced pluripotent stem cells using non-synthetic mRNA." Universitätsbibliothek Leipzig, 2016. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-205889.
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Here we describe some of the crucial steps to generate induced pluripotent stemcells (iPSCs) usingmRNA transfection. Our approach uses a V. virus-derived capping enzyme instead of a cap-analog, ensuring 100% proper cap orientation for in vitro transcribedmRNA. V. virus\' 2′-O-Methyltransferase enzymecreates a cap1 structure found in higher eukaryotes and has higher translation efficiency compared to other methods. Use of the polymeric transfection reagent polyethylenimine proved superior to other transfection methods. The mRNA created via this method did not trigger an intracellular immune response via human IFN-gamma (hIFN-γ) or alpha (hIFN-α) release, thus circumventing the use of suppressors. Resulting mRNA and protein
were expressed at high levels for over 48 h, thus obviating daily transfections. Using this method, we demonstrated swift activation of pluripotency associated genes in human fibroblasts. Low oxygen conditions further facilitated colony formation. Differentiation into different germ layers was confirmed via teratoma assay. Reprogramming with non-synthetic mRNA holds great promise for safe generation of iPSCs of human origin. Using the protocols described herein we hope to make this method more accessible to other groups as a fast, inexpensive, and non-viral reprogramming approach.
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Lau, Kei-ling Kelly, and 劉己綾. "Human pluripotent stem cells as a source of dendritic cells to induce immune tolerance." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/197516.
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Dendritic Cells (DCs) are professional antigen presenting cells that play a crucial role in the induction of immune tolerance. Although DCs have been a potential target for immunotherapy, the amount of DCs in blood source is limited and ex vivo expansion has been inefficient. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) provide a great source in cell-based therapy because of their self-renewal ability and pluripotency. My project focuses on generating tolerogenic DCs (tDCs) from human pluripotent stem cells (i.e. hESCs and iPSCs) and their characterization.
Specifically, hESCs and hiPSCs were first differentiated to hematopoietic progenitor cells (HPCs) using three different methods (i.e. bone-marrow stromal cell co-culture and two previously reported defined medium methods). The hESC/iPSC-differentiated hematopoietic progenitor cells (HPCs) were characterized by their surface phenotype using flow cytometry. Then the hESC/iPSC-differentiated immature DCs were further expanded and differentiated from the hESC/iPSCdifferentiated CD34+ HPCs with the addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) and Interleukin 4 (IL-4). Tolerogenic properties were introduced by treating hESC-differentiated DCs with rapamycin. The treated DCs were characterized for their tolerogenicity by examining their expression of PDL1, PDL2, ICOS and CD40 etc., and their ability to promote regulatory T cells (Treg) differentiation. All these were compared with monocyte-derived tDCs.
In summary, this study has examined the potential of using pluripotent stem cells-derived DCs as a cell source for immune tolerance induction therapy.<br>published_or_final_version<br>Anatomy<br>Master<br>Master of Philosophy
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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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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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Awaya, Tomonari. "Selective Development of Myogenic Mesenchymal Cells from Human Embryonic and Induced Pluripotent Stem Cells." Kyoto University, 2013. http://hdl.handle.net/2433/180602.
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Li, Quan. "Synthetic Hydrogel-Based 3D Culture System for Maintenance of Human Induced Pluripotent Stem Cell." Thesis, Kansas State University, 2017. http://hdl.handle.net/2097/36189.
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Master of Science<br>Department of Grain Science and Industry<br>X. Susan Sun<br>Human induced pluripotent stem cells (hiPSCs) are generated from human somatic cells using defined transcription factors. These cells possess characteristics very similar to that of human embryonic stem cells including the ability to differentiate into cell types of all three germ layers. HiPSCs show great potential in clinical researches like drug screening and regenerative medicine, that all require large amount of cells cultured under well-defined conditions. The most common culture methods used for hiPSCs are 2D culture methods using Matrigel or vitronectin coated culture plates or flasks. 2D culture methods require large surface area to produce the same amount of cells compared to 3D methods. In addition, cells cultured in 2D culture environment are far from that in vivo. In this study, we developed a robust 3D culture condition based on hiPSC-qualified PGmatrix (PGmatrix-hiPSC) hydrogel. This 3D culture system provide hiPSCs with well-defined, more in vivo-like environment that encapsulate cells in liquid rich hydrogel with appropriate oxygen supply that resembles the hypoxia condition in vivo. Two hiPSC lines grown continuously in PGmatrix-hiPSC showed higher total population expansion and higher viability, with more consistency compared to the same cell lines grown in 2D on Matrigel or Vitronectin-XF. After grown in 3D PGmatrix-hiPSC for over 25 passages, major pluripotency markers, such as Oct4, Sox2, Nanog, and SSEA4 are expressed in most hiPSCs examined by flow cytometry. RT-qPCR also confirmed adequate expression levels of major pluripotency related genes. In addition, karyotype analysis of hiPSC after 37 passages in 3D PGmatrix-hiPSC was found normal. The same hiPSC lines cultured continuously in parallel in 2D and 3D showed differences in gene expression and surface marker TRA-1-81 expression. These results indicated the 3D PGmatrix-hiPSC system is likely superior in maintaining hiPSC growth as well as pluripotency. The findings also suggest that it is very important to study cells in 3D culture environment to better understand the mechanism of pluripotency maintenance.
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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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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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Nakamura, Sou. "Expandable Megakaryocyte Cell Lines Enable Clinically Applicable Generation of Platelets from Human Induced Pluripotent Stem Cells." Kyoto University, 2015. http://hdl.handle.net/2433/202779.
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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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Palandri, Chiara. "DIFFERENT METHODS TO MODEL CARDIAC ARRHYTHMOGENIC DISEASES: FROM TRANSFECTED CELLS TO CARDIOMYOCYTES DERIVED FROM HUMAN INDUCED PLURIPOTENT STEM CELLS." Doctoral thesis, Università di Siena, 2021. http://hdl.handle.net/11365/1143558.
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Cardiomyopathies are a heterogeneous group of diseases of the heart muscle, associated to alterations of cardiac currents and ions handling that cause the impairment of the cardiac function leading to the insurgence of arrhythmias, heart failure and sudden death. In this project, we focus our attention on ion channel diseases. I will use different models, from expression system, to human adult cardiomyocytes isolated from septal samples, until human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CM) to design cardiomyopathies and used it to test the efficacy and safeness of “old” drugs in restore cardiac function. I analysed the electrophysiological feature of those models, by calcium and sodium fluorescence and by patch clamp technique to better investigate the cardiac impairment.
In the cell-based model of a mixed phenotype between BrS and LQT3s obtained in HEK293 cells transfected with the wt-SCN5A gene or the 1795insD-SCN5A, together with the β-subunits, we tested the potential efficacy of Mexiletine in increase the altered peak sodium current (INa) and in reducing the late sodium current (INaL) (that were respectively reduce and increase by the mutation on the cardiac sodium channel). The double efficacy make it a possible effective treatment for the overlap syndrome. Our results confirmed this hypothesis, Mexiletine (500µM) in chronic treatment (48h incubation, washed before recordings) is able to increase INa.
In cardiomyocytes isolated from human septal samples of obstructive HCM (OHCM) patients undergoing surgical myectomy, we analysed the efficacy and safeness of Disopyramide. Less is known about the complexity of its action, beside the sodium channel block, so we investigated its effects in reducing arrhythmic event by measuring action potential (AP) and cardiac ion currents by patch clamp technique and Calcium transient features, using calcium sensing fluorescent dye. Our results show that it is able to reduced AP duration, the upstroke velocity and the incidence of EAD and DAD reducing the intensity of INa-L and ICa-L and inhibiting Ik and calcium sparks from RYR. It’s action on multiple ion channel make Disopyramide a safe drug for OHCM, more than the actual treatment suggested by the guidelines.
hiPSCs are a novel tool to obtain cells with preserved genetic background of healthy individuals or patients. The limitations of this model consist in the maturity of the cardiomyocytes, that are consistently different from an adult ventricular cardiomyocytes (for both morphological and electrophysiological features). To improve the maturation level of our cells we used long term culture (until 90 days post differentiation) associated to patterned surfaces. By measuring calcium handling and action potential profile at different time points (30, 60 and 90 days), we tried to understand how the electrophysiological features of these cardiomyocytes evolve in the control and DMD line. hiPSC-CM could be the future of disease modelling but the limits in the maturation protocols need to be overpass, our methods shows how is possible do that using a long term culture associated with patterned surfaces and how it could give us the chance to understand the disease progression during cardiomyocytes development and which pathway target with drugs to inhibits disease progression.
Pay attention in choosing the right model for our purpose is essential, this could let our work to successfully and reliable result with high translational value. The troubles in modelling cardiomyopathy associated with elevated arrhythmic risk make the decision even harder and probably the combination of different model will be a valid option. Our results also highlight the importance of a personalized therapy for those patients, in which every subject has a different background and different phenotype manifestation; studying, not the disease, but the patient’s phenotype will help us to identify specific drug treatment suitable for that subject.
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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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Martewicz, Sebastian. "Human pluripotent stem cell-based microtechnologies for in vitro modeling of cardiac diseases." Doctoral thesis, Università degli studi di Padova, 2015. http://hdl.handle.net/11577/3424118.
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Human pluripotent stem cells are quickly emerging as a fundamental tool for in vitro studies. In particular, the advent of “induced pluripotency” opened completely new horizons for in vitro disease modeling and patient-specific disease-on-a-dish therapeutic approach screening. The easy access to cell types of human origin hardly available otherwise, with virtually infinite amounts in a donor-unrestricted manner, unlocked in vitro studies for human tissues such as brain, pancreas and the heart. In the latter case, the need for new models of human cardiac physiology and physiopathology is highlighted by the severe fallouts of heart conditions on worldwide health and economy.
The main focus of this thesis are human cardiomyocytes derived through differentiation of pluripotent stem cells, and their application as an in vitro model of the human cardiac tissue. In particular, the stress point of the work is their early and immature phenotype, that often limits their application and frustrates the potential of a human heart model in a Petri dish.
After introducing the current scenario of study models for heart diseases and describing the main features of human pluripotent stem cells (hPSCs) and their cardiac derivatives (hPSC-CMs), this thesis will separately focus on the two main aspects of the cardiomyocyte physiology: structural and functional features and metabolic profile. From these perspectives, human cardiomyocytes derived from hPSCs display in vitro an early and immature phenotype, closely resembling cardiomyocytes at early stage of the development, such as fetal cardiomyocytes.
Cell ultrastructural organization and functional performance are two strictly related features that find in adult cardiomyocytes perfect synthesis, with a very specialized function performed through a finely orchestrated sequence of events hugely relying on the right spatial distribution of key molecular components. In Chapter 2, biomaterials and microengineered substrates are employed to address the molecular mechanisms triggering cardiac maturation in vitro, in order to provide insight in the process and drive hPSC-CMs towards more adult-like phenotypes, better suiting disease modeling and drug screening.
Cardiac metabolism is likewise a characterizing feature of the tissue supporting in a unique fashion the impressive workload of the heart. In Chapter 3, hPSC-CM metabolism is described and a novel microfluidic technology is developed for metabolic maturation screening of cardiac cultures. With this approach, hPSC-CMs are shown to positively respond to an optimized metabolic maturation protocol, similar to the very rapid fetal-to-adult metabolism switch in hCMs after birth in response to changing metabolite availability.
Finally, in perspective of the maturation approaches previously described and their feasible application to human cardiac cultures, in Chapter 4 are discussed two human genetic diseases affecting the heart muscle. For both Duchenne’s muscular dystrophy and arrhythmogenic right ventricular cardiomyopathy/dysplasia, cardiac cellular models are set up and proven to display in vitro the molecular hallmarks of the disease, thus providing the biological substrate for further studies on human cardiomyocyte cultures.<br>Le cellule pluripotenti umane stanno velocemente emergendo come strumenti fondamentali nella ricerca in vitro. In particolare, l’avvento delle cellule pluripotenti indotte ha aperto nuovi orizzonti sullo studio e la modellazione delle malattie umane e lo screening di approcci terapeutici. La possibilità di avere in coltura cellule di origine umana provenienti da tessuti da cui è difficile ottenere campioni bioptici, ha permesso di delineare nuove prospettive di studi in vitro per tessuti come il cervello, il pancreas o il cuore. Specialmente in quest’ultimo caso, il bisogno di nuovi modelli di studio è esaltato dall’impatto che le malattie cardiache hanno sulla sanità e sull’economia mondiali, a cui i modelli di studio tradizionali non riescono a far fronte in modo efficace.
L’oggetto di studio di questa tesi di dottorato sono i cardiomiociti umani derivati per differenziamento da cellule staminali pluripotenti e la loro applicazione come modello di studio del tessuto cardiaco. In particolar modo, ci si è focalizzati sul loro fenotipo, ritenuto precoce ed immaturo rispetto al cardiomiocita adulto, che limita enormemente il loro impiego in campo medico e scientifico, impedendo il pieno sviluppo del loro potenziale.
Dopo aver introdotto i modelli sperimentali attualmente impiegati nello studio di patologie cardiache, verranno descritte le principali caratteristiche delle cellule pluripotenti umane (hPSC) e dei loro derivati cardiaci (hPSC-CM). Successivamente, l’attenzione verrà focalizzata su due caratteristiche fondamentali che descrivono la fisiologia di un cardiomiocita: l’organizzazione strutturale della cellula legata alla sua funzionalità ed il suo profilo metabolico. In entrambe queste categorie, i hPSC-CM vengono spesso paragonati a cellule cardiache fetali, lontane in termini di sviluppo dal fenotipo del cardiomiocita adulto.
L’organizzazione ultrastrutturale di una cellula cardiaca è strettamente correlata con la sua capacità funzionale: nei cardiomiociti adulti si assiste ad una perfetta concertazione spazio-temporale di diverse componenti molecolari la cui azione coordinata permette alle cellule del cuore di svolgere l’attività contrattile. Nel Capitolo 2 di questa tesi, vengono impiegati biomateriali e substrati micro- ingegnerizzati per studiare i meccanismi molecolari che promuovono la maturazione strutturale e funzionale dei cardiomiociti umani in vitro, rendendoli così più vicini fenotipicamente ad una cellula adulta su cui svolgere studi farmacologici e di modellazione di patologie.
Il metabolismo cardiaco è una caratteristica altrettanto unica e caratterizzante per un cardiomiocita, essendosi adattato ad sostenere un’attività costante ed energicamente dispendiosa com’è la generazione di forza meccanica. Nel Capitolo 3 viene descritto il metabolismo di un cardiomiocita e viene proposta una nuova piattaforma microfluidica da utilizzare per la validazione di protocolli di maturazione metabolica in vitro. Con il saggio funzionale messo a punto viene inoltre dimostrato come l’induzione di un metabolismo maturo è possibile attraverso la variazione dei substrati energetici presenti nel mezzo di coltura.
Infine, in prospettiva dell’applicazione dei protocolli di maturazione descritti nei capitoli precedenti a colture cardiache umane, nel Capitolo 4 viene presentato l’allestimento di due modelli cellulari di patologie genetiche: la distrofia muscolare di Duchenne e la cardiomiopatia aritmogena del ventricolo destro. Vengono presentati e caratterizzati cardiomiociti umani derivati per differenziamento da cellule hiPS di pazienti affetti, mostrando come i cardiomiociti in coltura presentino il fenotipo molecolare aberrante caratterizzante le malattie prese in esame. Si fornisce, così, un modello cellulare cardiaco umano che può trovare impiego nella modellazione in vitro delle due patologie.
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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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Nakajima, Taiki. "Modeling human somite development and fibrodysplasia ossificans progressiva with induced pluripotent stem cells." Kyoto University, 2019. http://hdl.handle.net/2433/242429.
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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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Maus, Andreas [Verfasser]. "Human induced pluripotent stem cell models used in the study of doxorubicin-induced cardiomyopathy / Andreas Maus." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2020. http://d-nb.info/1228364427/34.
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Ikuno, Takeshi. "Efficient and robust differentiation of endothelial cells from human induced pluripotent stem cells via lineage control with VEGF and cyclic AMP." Kyoto University, 2017. http://hdl.handle.net/2433/227586.
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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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Feldman, Danielle A. (Danielle Anagela). "Human induced pluripotent stem cell models of Rett Syndrome reveal deficits in early cortical development." Thesis, Massachusetts Institute of Technology, 2016. http://hdl.handle.net/1721.1/107869.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Brain and Cognitive Sciences, 2016.<br>Cataloged from PDF version of thesis.<br>Includes bibliographical references.<br>Rett Syndrome (RTT) is a pervasive, X-linked neurodevelopmental disorder that predominantly affects girls. The clinical patient features of RTT are most commonly reported to emerge between the ages of 6-18 months and as such, RTT has largely been considered to be a postnatal disorder. The vast majority of cases are caused by sporadic mutations in the gene encoding methyl CpG-binding protein 2 (MeCP2), which is expressed in the brain during prenatal neurogenesis and continuously throughout adulthood. MeCP2 is a pleiotropic gene that functions as a complex, high-level transcriptional modulator. It both regulates and is regulated by coding genes and non-coding RNAs including microRNAs (miRNAs). The effects of MeCP2 are mediated by diverse signaling, transcriptional, and epigenetic mechanisms. Whereas the postnatal effects of MeCP2 have been widely studied, pre-symptomatic stages of RTT have yet to be thoroughly investigated. Recent evidence from our lab among others suggests a role for MeCP2 during prenatal neurogenesis that may contribute to the neuropathology observed later in life. We sought to characterize the course of neurogenesis in MeCP2-deficient human neurons with the use of induced pluripotent stem cells (iPSCs) derived from RTT patient skin samples. We generated a variety of monolayer and 3D neuronal models and found that RTT phenotypes are present at the earliest stages of brain development including neuroepithelial expansion, neural progenitor migration and differentiation, and later stages of membrane and synaptic physiological development. We established a link between MeCP2 and key microRNAs that are misregulated in RTT and lie upstream of signalling pathways that contribute to aberrant neuronal maturation in the absence of MeCP2. We have uncovered novel roles of MeCP2 in human neurogenesis. Whereas the processes that comprise early neural development were previously considered irrelevant to RTT pathology, the deficits we observed in neuronal differentiation, migration, and maturation are a crucial component to the larger picture of RTT pathogenesis and provide additional insight into the emergence of RTT patient symptoms.<br>by Danielle A. Feldman.<br>Ph. D.
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Nishizawa, Masatoshi. "Epigenetic variation between human induced pluripotent stem cell lines is an indicator of differentiation capacity." Kyoto University, 2017. http://hdl.handle.net/2433/218003.
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Rashid, Sheikh Tamir. "Human induced pluripotent stem cells for in vitro modeling and cell based therapy of α-1 antitrypsin deficiency". Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610175.
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Requena, Osete Jordi. "Advancing induced pluripotent stem cell (iPSC) technology by assessing genetic instability and immune response." Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/457970.
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Induced pluripotent stem cells (iPSC) can be made from adult somatic cells by reprogramming them with Oct4, Sox2, Klf4 and c-Myc. IPSC have given rise to a new technology to study and treat human disease (Takahashi et al., 2007). However, before iPSC clinical application, we need to step back and address two main challenges:
(i) Genetic stability of iPSC.
(ii) Immune response of iPSC-derived cells.
To address these key issues, the overall mission of this PhD thesis is to advance iPSC technology by addressing two objectives. First, is to replace c-Myc with Cyclin D1 in the reprogramming cocktail (Oct4, Sox2, Klf4 and c-Myc or Cyclin D1) and second, to study the immune response of iPSC-derived cells.
The quality of the starting iPSC determines the quality of the differentiated cells to be transplanted for clinical applications. In terms of genetic stability, aberrant cell reprogramming leads to genetic and epigenetic modifications that are the most significant barriers to clinical applications of patient iPSC derivatives (Gore et al., 2011). Such aberrations can result from the cellular stress that accompanies reprogramming or from the reprogramming factors themselves (Lee et al., 2012a). IPSC made with c-Myc are neoplastic in mouse models and have a higher tumorigenic potential than embryonic stem cells, prompting a search for new pluripotency factors that can replace the oncogenic factors Klf4 and c-Myc (Huangfu et al., 2008; Miura et al., 2009; Okita et al., 2007). We chose Cyclin D1 to replace c-Myc because of previous observation it can be used to reprogram cells to iPSC (Edel et al., 2010) and because of its DNA repair function (Chalermrujinanant et al., 2016). In this thesis we adopt a synthetic mRNA method to demonstrate that Cyclin D1 and c-Myc made iPSC have equal pluripotency using standard methods of characterisation. Moreover, no significant changes in copy number variation were found between starting skin cells and iPSC highlighting it is the method of choice for generating high quality iPSC. Further in- depth analysis revealed that Cyclin D1 made iPSC have reduced genetic instability assessed by: (i) reduced DNA double strand breaks (DSB), (ii) higher nuclear amount of the homologous recombination key protein Rad51, (iii) reduced multitelomeric signals (MTS) and (iv) reduced teratoma growth kinetics in vivo, compared to c-Myc made iPSC. Moreover, we demonstrate that Cyclin D1 iPSC derived neural stem cells engraft successfully, survive long term and differentiate into mature neuron cell types with high efficiency, with no evidence of pathology in a spinal cord injury rat model.
As we move towards the clinic with iPSC-derived cells for cell transplantation, the immunogenic response is thought to be one of the main advantages of iPSC technology for clinical application, because of its perceived lack of immune rejection of autologous cell therapy. We hypothesize that iPSC derived cells are unlikely to provoke an immune response. Here we have performed an analysis of the innate and adaptive immune response of human skin cells (termed F1) reprogramed to iPSC and then compared to iPSC-derived cells (termed F2) using proteomic and methylome arrays. We found little differences between MHCI expression and function; however, we discovered a short isoform of the Toll-like receptor 3 (TLR3), essential for viral dsRNA innate immune
recognition, which is predominantly upregulated in all iPSC derived cells analysed and not seen in normal endogenous cells. High levels of the TLR3 isoform is associated with unresponsiveness to viral stimulation measured by lack of IL6 secretion in iPSC derived neural stem cells. We propose a new model that TLR3 short isoform competes with the full length wild type isoform destabilizing the essentially required TLR3 dimerization process. These differences could result in supressed inflammatory effects for transplanted human iPSC-derived cells in response to viral or bacterial insult. Further work to determine the in vivo effects is warranted and calls for screening of iPSC lines for TLR3 isoform expression levels before clinical use. In conclusion, this thesis has advanced iPSC technology by defining a new method that is a significant advance with novel insights that has immediate impact on current methods to generate iPSC for clinical application and more accurate disease modelling.<br>Les cèl·lules mare pluripotents induïdes (iPSC) es poden derivar de cèl·lules somàtiques adultes mitjançant la reprogramació amb Oct4, Sox2, Klf4 i c-Myc. Les iPSC han donat lloc a una nova tecnologia per estudiar i tractar malalties humanes (Takahashi et al., 2007). No obstant, abans de la aplicació clínica de les iPSC, dos problemes principals han de ser adreçats:
(i) Estabilitat genètica de les iPSC.
(ii) Resposta immune de les cèl·lules derivades de iPSC.
Per adreçar aquests dos qüestions cabdals, la missió principal d’aquest doctorat és avançar la tecnologia de les iPSC adreçant dos objectius. El primer, és la substitució de c-Myc per Ciclina D1 al còctel de reprogramació (Oct4, Sox2, Klf4 and c-Myc o Ciclina D1) i segon, estudiar la resposta immune de les cèl·lules derivades de iPSC.
Hem escollit Ciclina D1 per substituir c-Myc atès a observacions prèvies que pot ser emprat per reprogramar (Edel et al., 2010) i donada la seva funció en reparació de l’ADN (Chalermrujinanant et al., 2016). Les iPSC reprogramades amb Ciclina D1 presenten una pluripotència similar a les reprogramades amb c-Myc, l’anàlisi en profunditat mostra però, que les iPSC reprogramades amb Cyclin D1 tenen una reduïda inestabilitat genètica adreçada per: (i) reducció en ruptures de doble cadena de DNA,
(ii) major quantitat nuclear de la proteïna clau en la recombinació homòloga Rad51, (iii) reducció en senyals multitelomèriques (MTS) i (iv) reducció en la cinètica de creixement de teratomes in vivo, en comparació amb iPSC reprogramades amb c-Myc. A més a més, demostrem que les cèl·lules mare neuronals derivades d’aquestes iPSC son capaces de implantar-se exitosament, sobreviure a llarg termini i diferenciar a neurones madures sense evidències de patologia en un model de dany medul·lar.
També hem realitzat un anàlisi del sistema immune innat i adaptatiu de cèl·lules humanes de la pell (nomenades F1) reprogramades a iPSC i comparades amb cèl·lules derivades de iPSC (nomenades F2). Hem descobert una isoforma curta del Toll-Like Receptor 3 (TLR3), essencial en el reconeixement de RNA de doble cadena d’origen víric, que està predominantment sobreexpresada en totes les cèl·lules derivades de iPSC analitzades i no trobat en cèl·lules endògenes. Nosaltres proposem un nou model per el qual la isoforma curta del TLR3 competeix amb la isoforma llarga wild type desestabilitzant el procés essencial de dimerització del TLR3.
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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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Fujiwara, Masataka. "Induction and enhancement of cardiac cell differentiation from mouse and human induced pluripotent stem cells with cyclosporin-A." Kyoto University, 2011. http://hdl.handle.net/2433/142089.
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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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Sasaki, Ben. "Transient FOXO1 inhibition in pancreatic endoderm promotes the generation of NGN3+ endocrine precursors from human iPSCs." Kyoto University, 2020. http://hdl.handle.net/2433/259709.
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Kalra, Spandan Kaur. "Towards the development of human induced pluripotent stem cell models for Duchenne muscular dystrophy-associated cardiomyopathy." Thesis, University of Nottingham, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.715123.
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Duchenne Muscular Dystrophy (DMD) is a fatal X-linked condition that affects 1 in 4710 boys. Causative mutations in the DMD gene result in a loss of functional expression of the dystrophin protein. Patients usually die in their second or third decade due to cardiac or respiratory failure. Incidence of cardiomyopathy increases with age, such that clinical symptoms are observed in 25% of boys below 6 years of age, but by the teenage years prevalence is 100%. The current treatments are palliative and there are no cures. Numerous cell-based and animal models for DMD exist, but each has its limitations. This includes inter-species differences, incomplete phenocopying of DMD pathophysiology and limited access to diseased human cardiac cells. Therefore, to complement these models, cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) from DMD patients were used to investigate therapeutic strategies and to evaluate the phenotype to better understand the disease. Towards these goals, protocols were optimised for differentiation, characterisation and enrichment of CMs from DMD hiPSC lines. These lines had been derived in our laboratory by four factor lentiviral reprogramming (LIN28, SOX2, NANOG and OCT4) of skin biopsies from boys clinically and genetically diagnosed as having DMD (Dick et al., 2011). Specifically, lines DMD19 and DMD16 carried premature stop codon mutations in exons 35 and 70 respectively, while DMD4 harboured frameshift deletion in exons 48-50 and were used in the study. In the first instance, no CM differentiation was observed. However, manipulation of cell seeding density and BMP, TGFP and WNT signalling pathways enabled efficiencies to be improved to up to 100%. In addition, culture of the hiPSC-CMs in low glucose / high lactate medium lead to preferential survival of the CMs to improve purity to >90%. Improved efficiency of hiPSC-CM production and enrichment provided material to evaluate methods of gene therapy. This included delivery of micro-dystrophin and gene correction of the genomic mutation with RNA guided nucleases (RGNs) in both DMD16 and 19 lines. In each case, restoration of dystrophin protein was observed. In parallel to the gene therapy studies, methods to perform phenotypic analysis were developed. Thus, hiPSC-CMs were analysed for: a) sarcolemma permeability by measuring response of stress on LDH release from cells; b) mitochondrial function in response to different pharmacological challenges by a Seahorse XF assay, and c) Ca2+ profiles, which were quantified using a method termed SALVO (synchronization, amplitude, length and variability of oscillations). Even under severe stress, by treatment with doxorubicin or hypo-osmolarity, no difference was observed in sarcolemma permeability between CMs derived from DMD or healthy hiPSCs. The alterations in mitochondrial function were observed in CMs from DMD hiPSCs relative to healthy CMs. Particularly in DMD16 hiPSCCMs, significant reduction in the ATP production, maximum respiration, spare reserve capacity and non-mitochondrial respiration was observed, while the proton leak was increased. While the data on calcium profile was derived, the number of replicates was not enough to overcome variability in recordings and draw any conclusions. Additional experiments are currently ongoing to confirm whether these phenotypic observations can be rescued by the gene therapy approaches employed. In summary, the work in this thesis has developed optimised protocols for CM differentiation from hiPSCs carrying mutation in the DMD gene, which has been used for pilot investigation into suitability as a supplementary model for investigating phenotype and evaluating genetic therapies. Further development of this in vitro model should lead to increased understanding and progress toward the development of therapies for DMD.
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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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Chang, Chia-Wei. "Polycistronic lentiviral vector for hit and run reprogramming of mouse and human somatic cells to induced pluripotent stem cell." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2009. https://www.mhsl.uab.edu/dt/2009p/changc.pdf.
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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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Suzuki, Naoya. "Pluripotent cell models of Fanconi anemia identify the early pathological defect in human hemoangiogenic progenitors." Kyoto University, 2015. http://hdl.handle.net/2433/199215.
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Crutchley, James E. B. "Automation and scale-up of human induced pluripotent stem cell models of cardiovascular disease for drug screening." Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/32207/.
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The global cost of heart failure is USD$45 billion and set to double in the next 15 years. The only method of treatment is heart transplant but demand far exceeds supply and is projected to increase. Meanwhile, global pharmaceutical development has been hindered by poor drug development success rates. Of the drugs that make it to phase I clinical trials, only 8 % pass phase III and existing drug screens do not always accurately predict or detect adverse cardiac events. Cardiotoxicity is the underlying reason for 26 % of safety related drug withdrawals between 1990-2006. Therefore, a source of human cardiomyocytes (CMs) is required to fill the need for regenerative medicine and drug screening applications. Differentiation of human pluripotent stem cells (hPSCs) to CMs is a viable solution to this bottleneck but the number of cells required is staggering; up to 5 million novel compounds are registered annually by pharmaceutical and academic institutions, while cell replacement studies in primates suggest that 10 billion CMs will be required per patient to repair the damaged myocardium post infarction. The objective of this thesis was to evaluate whether automated high throughput manufacture of hPSCs and CMs was possible, and to demonstrate that hPSC-CMs could be used in automated high throughput drug screening by carrying out assays in 384-well plates. This thesis started by carrying out three manual differentiation methods; an embryoid body (EB) based method and two monolayer methods. Batch variability in mouse embryonic fibroblast conditioned medium (MEF-CM) led to erratic and variable differentiation outcomes (as high as 94+/-0.3 % to as low as 25.6+/-39.7 % beating EBs per 96 well plate). Two monolayer methods, using defined media (mTeSR and E8) increased cell yields by up to 12-fold and 65-fold respectively and simplified the process technically. When these methods were automated, EB differentiation failed to generate spontaneously beating EBs, whereas both monolayer methods succeeded in generating spontaneously beating cardiomyocytes of purities >90 %. Finally, cryopreserved stocks of hiPSC-CMs produced by automation were used to evaluate whether cardiotoxicity from the anticancer drug doxorubicin could be decreased by co-treating with dexrazoxane (an existing doxorubicin cardio-protectant), carvedilol (a β-blocker), sildenafil (a vasoactive agent) and isoprenaline (a β-adrenoreceptor agonist). This was carried out in a real-time, fully automated assay setup to monitor induction of apoptosis by the marker propidium iodide using the Operetta confocal plate reader. The concentration of doxorubicin that led to 50 % hiPSC-CM death (TD50) was significantly reduced by co-treatment with dexrazoxane, carvedilol and sildenafil. Carvedilol showed the highest level of cardioprotection by increasing TD50 of doxorubicin by 7.5-fold. In contrast, isoprenaline reduced TD50 of doxorubicin, suggesting that isoprenaline would be contraindicated in patients undergoing doxorubicin treatment. Thus, this thesis demonstrated that automated differentiation of cardiomyocytes was technically feasible with capability of generating high yields (up to 39 million cells per flask) and high purity (>90 %) cardiomyocytes. Furthermore, this system was compatible with high content assays in 384-well plates for evaluating drug toxicity.
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Matsubara, Masaki. "Analysis of mitochondrial function in human induced pluripotent stem cells from patients with mitochondrial diabetes due to the A3243G mutation." Kyoto University, 2018. http://hdl.handle.net/2433/232452.
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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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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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Yoshinaga, Daisuke. "Phenotype-Based High-Throughput Classification of Long QT Syndrome Subtypes Using Human Induced Pluripotent Stem Cells." Kyoto University, 2020. http://hdl.handle.net/2433/253171.
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Hirose, Sayako. "Propranolol Attenuates Late Sodium Current in a Long QT Syndrome Type 3-Human Induced Pluripotent Stem Cell Model." Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/265195.
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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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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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Maurissen, Thomas Luc. "Synergistic gene editing in human iPS cells via cell cycle and DNA repair modulation." Kyoto University, 2020. http://hdl.handle.net/2433/254520.
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GIANI, ALICE MARIA. "GENERATION OF AUTHENTIC HUMAN NEOCORTICAL NEURONS FROM INDUCED PLURIPOTENT STEM CELLS TO INVESTIGATE 7Q11.23 GENE DOSAGE IMBALANCES." Doctoral thesis, Università degli Studi di Milano, 2018. http://hdl.handle.net/2434/561835.
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Questo lavoro di tesi ha avuto lo scopo di studiare lo sviluppo della neocorteccia umana ed i meccanisimi alla base della sua compromissione che risultano nell’insorgenza di patologie del neurosviluppo mediante un’analisi dei profili trascrizionali e della morfologia di neuroni neocorticali umani generati a partire da cellule staminali pluripotenti indotte (iPSCs).
Data l’importanza di basarsi su un paradigma di neurogenesi in vitro riproducibile e affidabile nel generare neuroni neocoritcali umani autentici, prima di adottare questo sistema modello per lo studio di patologie del neurosviluppo, nella prima fase di questa ricerca abbiamo eseguito un’ampia caratterizzazione trascrizionale, molecolare e funzionale del protocollo di differenziamento. Le dinamiche trascrizionali che regolano il neurosviluppo in vitro sono state studiate effettuando esperimenti di RNA-sequencing sia a livello di popolazione che di singola cellula. In combinazione con diverse analisi bioinformatiche tra cui l’analisi delle componeti principali (PCA), l’analisi dei geni differenzialemtne espressi e l’analisi WGCNA. L’analisi dei profili trascrizionali è stata accompagnata da un’ampia analisi di d’immunocitochimica che ha permesso di confermare l’identità e lo stadio di sviluppo delle cellule in coltura. Inoltre, la maturità funzionale dei neuroni derivati da iPSCs è stata ulteriormente confermata dalla loro capacità di generare potenziali d’azione, sostenere pattern di scarica complessi e sviluppare attività sinaptica spontanea eccitatoria ed inibitoria. Complessivamente, i risultati ottenuti da questo ampio e diversificato pannello di analisi hanno permesso di stabilitre la riproducibilità del protocollo di differenziamento e la sua competenza nel generare con elevata efficienza principalmente neuroni neocorticali autentici.
Successivamente abbiamo applicato questo protocollo di differenziamento neocorticale come sistema modello per studiare due patologie del neurosviluppo dovute alla delezione e duplicazione di una regione comprendente circa 1.5 - 1.8 Mb (megabasi) collacata sul braccio lungo (q) del cormosoma 7 nella banda 11.23. Duplicazioni e delezioni di questa regione sono di particolare interesse in quanto le due sindromi che ne risultano, rispettivamente la sindrome di Willams (WS) e la sindrome da duplicazione 7q11.23 (7q11DUP), presentano fenotipi cognitivi e comportamentali caratterizzati da profili simili e tratti simmetricamente opposti. La frequente comorbidità della sindrome da duplicazione 7q11.23 con altre patologie del neurosviluppo come l’autismo e la schizofrenia in contrasto con la sindrome di Williams che è una sindrome ben caraterizzata non associata ad altre patologie del neurosviluppo, rende lo studio dell’ alterato dosaggio genico del locus 7q11.23 estremamente interessante per identificare con precisione i meccanismi molecolari caratteristici di ciascuna condizione clinica, condivisi da entrabme le sindromi e comuni anche ad altre patologie del neurosviluppo.
A questo scopo, abbiamo generato diverse linee di iPS a partire da un ampio gruppo di individui, comprendente individui sani e pazienti affetti dalla sindrome di Williams (WS) e dalla sindrome di duplizazione 7q11.23, che sono poi state differenziate in neuroni neocorticali applicando il protocollo precedentemetne caraterizzato. Confermata l’identità e l’autenticità dei neuroni neocorticali generati da iPSCs, stiamo attualmente identificando i geni ed i meccanismi molecolari disregolati in specifici sottotipi di neuroni che abbiano la maggior rilvenza clinica. Inoltre, l’analisi morfologica dei neuroni neocorticali umani ottenuti da pazienti WS e soggetti sani ha permesso di confermare nell’uomo molte alterazioni morfologiche dei neuroni neocorticali osservate in un modello murino knockout per Dnajc30, un gene ancora funzionalmente non caraterizzato compreso nel locus 7q11.23.<br>This research project has been aimed to investigate human neocortical development in healthy and diseased subjects by analyzing and comparing the transcriptional profiles and cellular morphologies of human neocortical cells derived from induced pluripotent stem cells (iPSCs).
Given the importance to rely on a solid and highly reproducible iPSCs-based differentiation protocol that generates authentic neocortical neurons in vitro with high efficiency before applying it as a model system of human neurodevelopmental disorders, in the first phase of this study we performed a comprehensive transcriptional, cellular and physiological characterization of the in vitro neurodevelopmental paradigm. The transcriptional dynamics regulating in vitro neocortical development have been investigated by performing RNA-sequencing (RNA-seq) at both population and single- cell level in combination with several bioinformatics analyses including principal component analysis (PCA), differential gene expression analysis and weighted gene co-expression network analysis (WGCNA). The transcriptional results were corroborated by the widespread positivity for a selected panel of informative cell-fate and cell-stage specific markers detected through immunocytochemistry and the physiological maturity of our iPSCs-derived neocortical neurons was further confirmed by their ability to generate action potentials, develop complex firing patterns and sustain excitatory and inhibitory spontaneous synaptic activity. Overall, these results fully validated the reproducibility of the differentiation protocol and its efficiency and reliability in generating physiologically mature authentic neocortical neurons.
Subsequently, we applied this extensively characterized neocortical differentiation paradigm to model in vitro two human neurodevelopmental disorders caused by symmetrical copy number variations (CNVs) of the Williams-Beuren syndrome chromosome region (WBSCR) located on the long arm (q) of chromosome 7 at position 11.23 (7q.11.23 locus). 7q11.23 CNVs are of special interest as the two disorders resulting from the deletion (Williams syndrome, WS) and duplication (7q.11.23 duplication syndrome, 7q11DUP) of this region exhibit cognitive and behavioral phenotypes marked by both similar features and symmetrically opposite traits. The association of 7q11DUP to complex neurodevelopmental disorders such as autism spectrum disorder and schizophrenia, while WS is a well-characterized syndrome without clear overlap to complex neurodevelopmental disorders make the study of this locus extremely interesting to identify the molecular mechanisms unique to each clinical condition, common to both syndromes and shared with other complex neurodevelopmental disorders. To this aim, we generated several iPSCs lines from a large cohort comprising WS individuals, 7q11DUP patients and healthy subjects and differentiated them into neocortical neurons by applying the previously in-depth characterized protocol. Having assessed the quality of our iPSCs-derived neocortical neurons, we are currently identifying neuronal subtypes specific genes and gene networks having the most statistically significant relationship to these disorders through single cell RNA-sequencing analysis. Furthermore, morphometric analysis of WS and control iPSCs-derived neocortical neurons has confirmed in humans many neuronal morphological abnormalities observed in a mouse knockout for Dnajc30, a previously uncharacterized gene contained in the 7q11.23 locus.
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BOTTI, SOFIA. "Mathematical modeling of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs): from ionic currents to 3D ventricle models." Doctoral thesis, Università degli studi di Pavia, 2022. https://hdl.handle.net/11571/1467309.
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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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