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Generation of ovine induced pluripotent stem cellsSartori, Chiara January 2012 (has links)
Embryonic stem cells (ESCs) are pluripotent cells derived from the early embryo and are able to differentiate into cells belonging to the three germ layers. They are a valuable tool in research and for clinical use, but their applications are limited by ethical and technical issues. In 2006 a breakthrough report described the generation of induced pluripotent stem cells (iPSCs). IPSCs are ESC-like cells generated from somatic cells by forcing the ectopic expression of specific transcription factors. This circumvents the ethical issues about the use of embryos in research and provides multiple opportunities to understand the mechanisms behind pluripotency. The aim of this project was to generate sheep iPSCs and characterise them. In order to learn the technique I initially repeated the original iPSC methodology: the putative mouse iPSCs I have generated display a morphology typical of ESCs, characterised by a high nuclear to cytoplasmic ratio, and form colonies with neat edges and smooth domes. These cells are positive to Nanog, a marker of pluripotency, and can give rise to cells belonging to the mesodermal and the ectodermal lineages when differentiated in vitro. Since the main aim of the thesis was the derivation of sheep pluripotent cells, once established the protocol in mouse, I then moved to the generation of ovine iPSC colonies. The cells I have generated have a morphology similar to that of mouse ESCs, express markers of pluripotency such as alkaline phosphatase and Nanog and can differentiate in vitro and in vivo into cells belonging to the three germ layers. Additionally, these ovine iPSCs can contribute to live born chimeric lambs, although at low level.
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Investigation of the cell- and non-cell autonomous impact of the C9orf72 mutation on human induced pluripotent stem cell-derived astrocytesZhao, Chen January 2016 (has links)
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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Reprogramming a DNA methylation mutantHunter, Jennifer Margaret January 2016 (has links)
Chemical modification of the cytosine base via the addition of a methyl group to form 5-‐methylcytosine (5-‐mC) is a well-‐studied example of an epigenetic mark, which contributes to regulation of gene expression, chromatin organisation and other such cellular processes without affecting the underlying DNA sequence. In recent years it was shown that 5-‐mC is not the only DNA modification found within the vertebrate genome. 5-‐hydroxymethylcytosine (5-‐hmC) was first described in 1952 although it wasn’t until 2009 when it was rediscovered in mammalian tissues that it sparked intense interest in the field. Research has found that unlike the 5-‐mC base from which it is derived, 5-‐hmC displays variable levels and patterns across a multitude of tissue and cell types. As such the patterns of these DNA modifications can act as an identifier of cell state. This thesis aims to characterize the methyl and hydroxymethyl profiles of induced pluripotent stem cells (iPSCs), derived from control mouse embryonic fibroblast cell line (p53-‐/-‐) as well as and methylation hypomorphic (p53-‐/-‐, Dnmt1 -‐/-‐) mutant cell lines. As such both somatic cells were subject to reprogramming with Yamanaka factors (Oct4, cMyc, Klf4 and Sox2) via the piggyback transposition technique. Successful reprogramming was confirmed by a number of techniques and outcomes, including the de novo expression of a number of key pluripotency related factors (Nanog, Sall4 and Gdf3). Reprogrammed cells were then analysed for transcriptomic changes as well as alterations to their methyl and hydroxymethyl landscapes that accompany reprogramming. Through this work I have shown that the reprogramming of MEF derived cell lines results in a global increase in 5-‐hmC for both p53-‐/-‐ and (p53-‐/-‐, Dnmt1 -‐/-‐) hypomorphic mutant cell lines – possibly through the reactivation of an alternative form of DNMT1. I demonstrate by both antibody based dot blot assay and genome wide sequencing that the reprogramming of the (p53-‐/-‐, Dnmt1 -‐/-‐) somatic cells towards a pluripotent state brings about an increase in methylation levels within the cells. This latter observation may indicate that the reprogramming of the cells is driving them towards a more wild type phenotypic state. My studies suggest that lack of DNMT1 function is not a barrier to reprogramming of somatic cells.
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Human Cerebral Organoids in Pillar/Perfusion Plates for Modeling Neurodevelopmental DisordersAcharya, Prabha 05 1900 (has links)
Human induced pluripotent stem cell (iPSCs)-derived brain organoids have potential to recapitulate the earliest stages of brain development, serving as an effective in vitro model for studying both normal brain development and disorders. In this study, we demonstrate a straightforward approach of generating multiple cerebral organoids from iPSCs on a pillar plate platform, eliminating the need for labor-intensive, multiple transfer and encapsulation steps to ensure the reproducible generation of cerebral organoids. We formed embryoid bodies (EBs) in an ultra-low attachment (ULA) 384-well plate and subsequently transferred them to the pillar plate containing Matrigel, using a straightforward sandwiching and inverting method. Each pillar on the pillar plate contains a single spheroid, and the success rate of spheroid transfer was in a range of 95 - 100%. Using this approach, we robustly generated cerebral organoids on the pillar plate and demonstrated an intra-batch coefficient of variation (CV) below 9 – 19% based on ATP-based cell viability and compound treatment. Notably, our spheroid transfer method in combination with the pillar plate allows miniaturized culture of cerebral organoids, alleviates the issue of organoid variability, and has potential to significantly enhance assay throughput by allowing in situ organoid assessment as compared to conventional organoid culture in 6-/24-well plates, petri dishes, and spinner flasks.
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Molecular and cellular basis of hematopoietic stem cells maintenance and differentiationDuong, Khanh Linh 01 December 2014 (has links)
The blood system consists of two main lineages: myeloid and lymphoid. The myeloid system consists of cells that are part of the innate immune response while the lymphoid system consist of cells that are part of humoral response. These responses protect our bodies from foreign pathogens. Thus, malignancies in these systems often cause complications and mortality. Scientists world wide have been researching alternatives to treat hematologic disorders and have explored induced pluripotent stem cells (iPSCs) and the conversion of one cell type to another.
First, iPS cells were generated by overexpression of four transcription factors: Oct4, Sox2, Klf4 an cMyc. These cells closely resemble embryonic stem cells (ESCs) at the molecular and cellular level. However, the efficiency of cell conversion is less than 0.1%. In addition, many iPS colonies can arise from the same culture, but each has a different molecular signature and potential. Identifying the appropriate iPS cell lines to use for patient specific therapy is crucial. Here we demonstrate that our system is highly efficient in generating iPS cell lines, and cell lines with silent transgenes are most efficient in differentiating to different cell types .
Second, we are interested in generating hematopoietic stem cells (HSCs) from fibroblasts directly, without going through the pluripotent state, to increase efficiency and to avoid complications associated with a stem cell intermediate. However, a robust hematopoietic reporter system remains elusive. There are multiple hematopoietic reporter candidates, but we demonstrate that the CD45 gene was the most promising. CD45 is expressed early during hematopoiesis on the surface of HSCs; and as HSCs differentiate CD45 levels increase. Furthermore, the CD45 reporter is only active in hematopoietic cells. We were able to confirm the utility of the CD45 reporter using an in vitro and an in vivo murine model.
In conclusion, The goal of this research was to expand the knowledge of stem cell reprogramming, specifically the reprogramming of iPS cells. Furthermore, it is our desire that the CD45 reporter system will undergo further validation and find utility in clinical and cell therapy environments.
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Transplantation of embryonic and induced pluripotent stem cell-derived 3D retinal sheets into retinal degenerative mice. / 網膜変性モデルマウスへのES/iPS細胞由来立体網膜シート移植Juthaporn, Assawachananont 23 March 2015 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18850号 / 医博第3961号 / 新制||医||1007(附属図書館) / 31801 / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 吉村 長久, 教授 中畑 龍俊 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM
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Protective Effects of Human iPS-Derived Retinal Pigmented Epithelial Cells in Comparison with Human Mesenchymal Stromal Cells and Human Neural Stem Cells on the Degenerating Retina in rd1 Mice. / 変性網膜におけるiPS由来網膜色素上皮細胞移植による保護効果―間葉系幹細胞及び神経幹細胞との比較Sun, Jianan 23 March 2016 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19561号 / 医博第4068号 / 新制||医||1013(附属図書館) / 32597 / 京都大学大学院医学研究科医学専攻 / (主査)教授 吉村 長久, 教授 戸口田 淳也, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM
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Humanized mouse models with endogenously developed human natural killer cells for in vivo immunogenicity testing of HLA class I-edited iPSC-derived cells / HLAクラスI編集iPS細胞由来細胞のインビボ免疫原性検証を可能とする内在発生ヒトNK細胞を有するヒト化マウスモデルFlahou, Charlotte Astrid Denise 25 September 2023 (has links)
京都大学 / 新制・課程博士 / 博士(医科学) / 甲第24885号 / 医科博第152号 / 新制||医科||10(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 河本 宏, 教授 濵﨑 洋子, 教授 上野 英樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM
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Indução da pluripotência celular e diferenciação in vitro no modelo suíno como modelo translacional / Induction of cell pluripotency and in vitro differentiation in swine as a translational modelMachado, Lucas Simões 20 December 2018 (has links)
Em 2006, Takahashi e colaboradores demonstraram ser possível a obtenção de células-tronco pluripotentes por indução gênica (induced pluripotent stem cells ou iPSCs). Desde o surgimento desta tecnologia diversos modelos animais foram gerados, ampliando as possibilidades de seu uso na pesquisa, como por exemplo, na criação de modelos para doenças genéticas humanas como esclerose lateral amiotrófica, autismo, esquizofrenia, doença de Parkinson e Alzheimer, além do aprimoramento de características relevantes para produção animal. O modelo suíno é considerado vantajoso sobre os outros modelos animais principalmente pela criação já bem estabelecida e similaridades fisiológicas com os humanos. O intuito deste projeto foi reprogramar fibroblastos embrionários suínos através do sistema integrativo à iPSCs, para então diferenciá-las em células progenitoras neurais (neural progenitor cells, NPCs). Para isso, os fibroblastos foram transduzidos com vetores contendo sequencias humanas ou murinas dos genes OCT4, SOX2, c-Myc e KLF4 (hOSKM ou mOSKM) para formação das iPSCs. Estas foram caracterizadas quanto a morfologia, presença de fosfatase alcalina, a expressão dos genes exógenos e endógenos (OSKM, HS OCT4, OCT4, NANOG) através de imunofluorescência e RT-qPCR e formação de corpos embrióides. Então foram submetidas durante 14 dias ao meio de indução neural sob matriz extracelular comercial, gerando células potencialmente similares às NPCs. Estas foram caracterizadas morfologicamente, por imunofluorescência das proteínas NESTINA, BETA TUBULINA III e VIMENTINA, além da expressão de NESTINA e GFAP por RT-qPCR. Foram produzidas com sucesso 3 linhagens de iPSC em diferentes estágios de reprogramação e células positivas para todos os marcadores neurais testados. Os resultados apresentados deverão contribuir para a utilização do modelo suíno em futuros estudos voltados à medicina regenerativa e translacional. / In 2006, Takahashi and collaborators reported the induction into pluripotency of somatic cells (induced pluripotent stem cells, iPSCs). Since then, this technique has much been developed; many animal models have been created opening a new series of opportunities in research. They enable the creation of models for human genetic diseases, for example, amyotrophic lateral sclerosis, autism, schizophrenia, Parkinson´s disease, Alzheimer´s disease and the enhancement of relevant characteristics in agriculture. The swine model is considered to present many advantages over others, including the well-known production and maintenance and physiological similarities to humans. The aim of this project was to reprogram porcine embryonic fibroblasts (pEF) into iPSCs using the lentiviral integrative system, followed by its differentiation into neural progenitor cells (NPCs). The cells were reprogrammed using vector containing either the human sequences (hOSKM) or the mouse sequences (mOSKM) for the OCT4, SOX2, c-Myc and KLF4 genes to form the iPSCs. They were characterized regarding the presence of the Alkaline Phosphatase enzyme, expression of exogenous and endogenous genes (OSKM, HS OCT4, OCT4, NANOG) through immunofluorescence and RT-qPCR, and embryoid body formation. Then, the cells were cultured with neural induction media for 14 days in commercial extracellular matrix, generating cells potentially like NPCs. Those were characterized regarding their morphology, immunofluorescence for NESTINA, BETA TUBULIN III and VIMENTINA and gene expression of NESTINA and GFAP. iPSCs were successfully reprogramed, generating 3 cell lines at different stages of reprograming and cells positive for all the neural markers tested were produced. The results shown will contribute to the use of the porcine model in future regenerative and translational medicine research.
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Systems biology approaches to somatic cell reprogramming reveal new insights into the order of events, transcriptional and epigenetic control of the processScharp, Till 03 November 2014 (has links)
Die Reprogrammierung somatischer Zellen hat sich kürlich als leistungsfähige Technik für die Herstellung von induzierten pluripotenten Stammzellen (iPS Zellen) aus terminal differenzierten Zellen bewährt. Trotz der großen Hoffnung, die sie speziell im Bezug auf patientenspezifische Stammzelltherapie darstellt, gibt es viele Hindernisse auf dem Weg zur Anwendung in der Humanmedizin, die sich von niedrigen Effizienzen bei der technischen Umsetzung bis hin zur unerwünschten Integration von Onkogenen in das menschliche Genom erstrecken. Aus diesem Grund ist es unabdingbar, unser Verständnis der zugrundeliegenden Prozesse und Mechanismen zu vertiefen. Durch neue Datengewinnungsmethoden und stetig wachsende biologische Komplexität hat sich der Denkansatz der Systembiologie in den letzten Jahrzehnten stark etabliert und erfährt eine fortwährende Entwicklung seiner Anwendbarkeit auf komplexe biologische und biochemische Zusammenhänge. Verschiedene mathematische Modellierungsmethoden werden auf den Reprogrammierungsprozess angewendet um Engpässe und mögliche Effizienz-Optimierungen zu erforschen. Es werden topologische Merkmale eines Pluripotenznetzwerkes untersucht, um Unterschiede zu zufällig generierten Netzen und so topologische Einschränkungen des biologisch relevanten Netzwerkes zu finden. Die Optimierung eines Booleschen Modells aus einem selbst kuratierten Netzwerk in Bezug auf Genexpressionsdaten aus Reprogrammierungsexperimenten gewährt tiefgreifende Einblicke in die ersten Schritte und wichtigsten Faktoren des Prozesses. Der Transkriptionsfaktor SP1 spielt hierbei eine wichtige Rolle zur Induktion eines intermediären, transkriptionell inaktiven Zustands. Ein probabilistisches Boole''sches Modell verdeutlicht das Zusammenspiel epigenetischer und transkriptioneller Kontrollprozesse zusammen, um Pluripotenz- und Zelllinien-Entscheidungen in Reprogrammierung und Differenzierung zu treffen. Erklärungen für die geringe Effizienz werden versucht. / Somatic Cell Reprogramming has emerged as a powerful technique for the generation of induced pluripotent stem cells (iPSCs) from terminally differentiated cells in recent years. Although holding great promises for future clinical development, especially in patient specific stem cell therapy, the barriers on the way to a human application are manifold ranging from low technical efficiencies to undesirable integration of oncogenes into the genome. It is thus indispensable to further our understanding of the underlying processes involved in this technique. With the advent of new data acquisition technologies and an ever-growing complexity of biological knowledge, the Systems Biology approach has seen an evolution of its applicability to the elaborate questions and problems of researchers. Using different mathematical modeling approaches the process of somatic cell reprogramming is examined to find out bottlenecks and possible enhancements of its efficiency. I analyze the topological characteristics of a pluripotency network in order to find differences to randomly generated networks and thus deduce constraints of the biologically relevant network. The optimization of a Boolean model from a curated network against early reprogramming gene expression profiles reveals profound insights into the first steps and most important factors of the process. The transcription factor SP1 emerges to play an important role in the induction of an intermediate, transcriptionally inactive state. A probabilistic Boolean network (PBN) illustrates the interplay of transcriptional and epigenetic regulatory processes in order to explain pluripotency and cell lineage decisions in reprogramming and differentiation. Explanations for the low reprogramming efficiencies are tried.
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