Tesi: "Induced pluripotent stem cells IPSC"

1

Sartori, Chiara. "Generation of ovine induced pluripotent stem cells". Thesis, University of Edinburgh, 2012. http://hdl.handle.net/1842/6491.

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Abstract (sommario):

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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2

Ababneh, Nidaa. "Modelling of amyotrophic lateral sclerosis (ALS) using induced pluripotent stem cells (iPSC)". Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:b0e48523-2acc-4c1e-83a5-79696cbaf042.

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Abstract (sommario):

The hexanucleotide repeat expansion (HRE) mutation within C9orf72 gene is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Several hypotheses have been proposed for how the mutation contributes to pathogenicity, including the loss of C9orf72 gene function, RNA-mediate toxicity and the formation of toxic dipeptides by repeat-associated non-ATG (RAN) translation. Patient-specific iPSCs provide a promising tool for the study of the cellular and molecular mechanisms of human diseases in relevant cell types and discovering potential therapies. The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9-mediated homology directed repair (HDR) system represents an attractive approach for disease modelling and development of therapeutic strategies. In this thesis, iPSCs derived from ALS/FTD patient carrying the HRE mutation were generated and subsequently gene edited to remove a massive repeat expansion from the patient cells and replace it with the wild-type size of the repeats using HDR and a plasmid donor template. The successful genotypic correction of the mutation resulted in the normalization of the C9orf72 gene promoter methylation level and the gene variants RNA expression level. Removal of the mutation also resulted in abolition of sense and antisense RNA foci formation and reduction of DPRs accumulation. Furthermore, the repeat size correction also rescued the susceptibility of cells to Glutamate excitotoxicity, decreased the apoptotic cell death and stress granules formation under the baseline and stress conditions. This work provides a proof-of-principle that removal of the HRE can rescue ALS disease phenotypes and provides an evidence that HRE mutation is an attractive target for therapeutic strategies and drug screening, to block the underlying disease mechanisms.

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3

Chung, Julia. "Manipulating Somatic Cells to Remove Barriers in Induced Pluripotent Stem Cell Reprogramming". Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:10772.

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Abstract (sommario):

Development leads unidirectionally towards a more restricted cell fate that is usually stable. However, it has been proven that developmental systems are reversible by the success of animal cloning of a differentiated somatic genome through somatic cell nuclear transfer (SCNT). Recently, reprogramming of somatic cells to a pluripotent embryonic stem cell (ESC)-like state by introducing defined transcripton factor has been achieved, resulting in the generation of induced pluripotent stem cells (iPSCs), which resemble ESCs. iPSC reprogramming is of great medical interest, as it has the potential to generate a source of patient-specific cells. However, the dangerous delivery method, low efficiency, and slow kinetics of the reprogramming process have hampered progress with this technology.

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4

Zambon, Federico. "Studying α-Synuclein pathology using iPSC-derived dopaminergic neurons". Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:2856dcf3-0f38-4a37-9242-8c685d1c2c3a.

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Parkinson's disease (PD) is characterised by the loss of dopaminergic neurons in the Substantia Nigra pars compacta in the midbrain and the presence of intracellular aggregates, known as Lewy bodies (LBs), in the surviving neurons. The aetiology of PD is unknown but a causative role for α-Synuclein (SNCA) has been proposed. Although the function of αSyn is not well understood, a number of pathological mechanisms associated with αSyn toxicity have been proposed. In this study, nine induced pluripotent stem cells (iPSCs) lines from healthy individuals and PD patients carrying the A53T SNCA mutation or a triplication of SNCA were differentiated to dopaminergic neurons (iDAn). All iPSC lines differentiated with similar efficiency to iDAn, indicating that they could be used for phenotypic analysis. Quantification of αSyn expression showed increased αSyn intracellular staining and the novel detection of increased αSyn oligomerization in PD iDAn. Analysis of mitochondrial respiration found a decrease in basal respiration, maximal respiration, ATP production and spare capacity in PD iDAn, but not in undifferentiated iPSCs, indicating the cell-type specificity of these defects. Decreased phosphorylation of dynamin-1-like protein at Ser616 (DRP1^Ser616) and increased levels of Peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) in A53T SNCA iDAn suggest a new pathological mechanism linking αSyn to the imbalance in mitochondria homeostasis. Markers of endoplasmic reticulum (ER) stress were found to be up-regulated, along with increased β- Glucocerebrosidase (GBA) activity, perturbation of autophagy and decreased expression of fatty acids binding protein 7 (FAPB7) in PD iDAn. Lastly, lentiviral vectors for RNAi-mediated knockdown of αSyn were developed and these reduced αSyn protein levels in iDAn, resulting in increased expression of FABP7. These results describe a novel functional link between αSyn and FABP7. This work demonstrates that iDAn are a promising and relevant in vitro cell model for studying cellular dysfunctions in PD pathology, and the phenotypic analysis of A53T SNCA and SNCA triplication iDAn enabled the detection of novel pathological mechanisms associated with PD.

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5

Sendfeld, Franziska. "Modelling Brugada Syndrome using induced pluripotent stem cells". Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/19557.

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Abstract (sommario):

Objective: Brugada Syndrome is an autosomal dominant congenital heart disease that is responsible for 20% of sudden deaths of patients with structurally normal hearts. The majority of mutations involve the cardiac sodium channel gene SCN5A and give rise to classical symptoms, which include an abnormal electrocardiogram with ST segment elevation and a predisposition to ventricular fibrillation. To date, the implantation of a cardioverter defibrillator is the only proven effective treatment of the disease. The ability to reprogram dermal fibroblasts to induced pluripotent stem (iPS) cells and to differentiate these into cardiomyocytes with the same genetic background provides a novel approach to studying inherited cardiac channelopathies with advantages over existing model systems. Whilst this technique has enormous potential to model inherited channelopathies, such as Brugada Syndrome, the derived cells have not been fully characterised and compared to foetal and adult cardiomyocytes. Methods: Dermal fibroblasts from a patient with Brugada syndrome (SCN5A; c.1100G > A - pARG367HIS) and an age- and sex-matched control were reprogrammed using episomal vectors. All newly derived iPS cell lines were fully characterised using immunocytochemistry, flow cytometry, real-time quantitative reverse transcription PCR and single nucleotide polymorphism analysis and were compared to established human embryonic stem (hES) cell and in-house derived healthy control iPS cell lines. The same control cell lines were used to compare the efficiencies of several cardiac differentiation media. Spontaneously contracting areas, derived from control as well as patient iPS cell lines, were disaggregated and single cardiomyocytes were compared to foetal and adult cardiomyocytes isolated from primary human tissue using immunocytochemistry, transmission electron microscopy, membrane visualisation, calcium imaging and electrophysiology. Results: Comparison of cardiac differentiation protocols using healthy control hES and iPS cell lines found that despite significant inter-line variability with regard to efficiency of cardiac formation guided differentiation protocols could be used to reliably and efficiently generate beating bodies. Spontaneous contraction was observed in stem cell-derived cardiomyocytes and human foetal cardiomyocytes. Pluripotent stem cell-derived cardiomyocytes stained for markers of the cardiac contractile apparatus such as α-actinin, cardiac troponin I and cardiac troponin T. They also expressed functional voltage-activated sodium channels and exhibited action potential triggered calcium-induced calcium release. Stem cell-derived cardiomyocytes showed organisation of myofibrils, ultrastructure and calcium handling more similar to foetal than adult cardiomyocytes. Brugada Syndrome patient-specific cardiomyocytes were structurally indistinguishable from healthy control iPS cell line-derived cardiomyocytes. Electrophysiological analysis of sodium current density confirmed a ~50% reduction in patient-derived compared to healthy control-derived cardiomyocytes. Conclusion: Although iPS cells give rise to a mixture of immature and more mature cardiomyocytes, they all express typical cardiac proteins and have functional cardiac sodium channels. Results illustrate the ability of patient-specific iPS cell technology to model the abnormal functional phenotype of an inherited channelopathy that is independent of structural abnormalities and that the relative immaturity of iPS cell-derived cardiomyocytes does not prevent their use as an accurate model system for channelopathies affecting the cardiac sodium channel Nav1.5. This iPS cell based model system for classical Brugada Syndrome allows for the first time to study the mutation in its native environment and holds promise for further studies to investigate disease mechanisms of known and unknown mutations and to develop new therapies.

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6

Brightwell, Sara. "Identifying novel regulators of reprogramming using RNA interference". Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/16156.

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Abstract (sommario):

Since Yamanaka and Takahashi first described the isolation of induced pluripotent stem cells (iPSCs) in 2006, researchers have invested a vast amount of time and resources into trying to understand the process of reprogramming. However, the exact mechanisms underlying the induction of somatic cells to pluripotency is still incompletely understood. With this in mind, a screening approach was undertaken to identify shRNA that enhance the reprogramming process. A retrovirus based system was used to knock down candidate genes during reprogramming of mouse embryonic fibroblasts (MEF) containing doxycycline-inducible reprogramming factors and a Nanog-GFP reporter, which is activated when cells become iPSCs. The initial round of screening with over 150 shRNA vectors successfully identified several shRNAs that enhance reprogramming. One of these shRNA vectors exhibited both faster reprogramming kinetics as determined by activation of the Nanog-GFP reporter 2 to 3 days earlier and increased reprogramming efficiency giving rise to >5 fold more GFP+ colonies when compared with a control. Cell surface marker analysis with flow cytometry demonstrated that changes in CD44 and ICAM1 expression, which occur preceding Nanog-GFP expression, were also accelerated. Validation of this shRNA determined that the enhanced reprogramming phenotype is the result of an unknown off-target effect. Microarray and RNA-sequencing analysis was carried out to identify the off target gene with a view to investigate the functional importance of this knock down and its role in establishing the pluripotency transcriptional network during reprogramming.

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7

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.
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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8

Sharma, Ruchi. "Generation of equine induced pluripotent stem cells from keratinocytes". Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/17956.

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Abstract (sommario):

Induced pluripotent stem cells (iPSCs) are generated by reprogramming somatic cells to an embryonic state. Therefore iPSCs represent an extremely valuable tool for modelling disease and organ toxicity, with enormous potential in veterinary medicine. Several equine diseases are currently untreatable and can result in euthanasia on medical grounds. In contrast to humans, in vitro models for cellular research in equine do not exist. Therefore it has been necessary to explore the use of stem cells in constructing cell based equine models. Pluripotent stem cell populations are of great interest in this field given their ability to form the three germ layers found in the developing embryo. While a promising notion, the isolation of equine embryonic stem cells has thus far proved elusive and therefore it has been necessary to explore other pluripotent stem cell populations. A very limited number of induced PSC lines have so far been generated from equine fibroblasts but studies in humans showed that other cell types such as keratinocytes were more amenable to reprogramming and generated iPSCs with much higher efficiency; whether this may be also the case in other species has not been investigated. Moreover, iPSC lines reported so far from domestic species, including the horse, depended on complex culture conditions for growth, including feeder layers and media supplementation with several growth factors. Although a promising alternative to fibroblast for generation of induced pluripotent stem cells there is dearth in literature on equine keratinocyte culture techniques. In this work I am reporting a novel approach to generate equine iPSCs lines from keratinocytes. Skin biopsies were used to derive keratinocyte cultures. The three dimensional culture systems were developed for robust culture of equine keratinocytes. These cells were then transduced with retroviral constructs coding for murine Oct-4, Sox-2, c-Myc and Klf-4 sequences, following the original Yamanaka protocol. Following transduction, tight cell colonies with sharp boundaries staining positive for alkaline phosphatase resembling previously reported human iPSCs were generated. The reprogrammed cells were successfully maintained in feeder free and serum free conditions with LIF supplementation. Immunochemistry and qPCR analyses revealed the equine iPSCs lines expressed pluripotency markers expressed in equine embryonic stages including, OCT4, SOX2, SSEA1, LIN 28, NANOG, REX1 and DNMT3B. Equine iPSCs were able to form embryoid bodies and differentiate into derivatives of the three germ layers in vitro. Equine iPSCs were pluripotent in vivo as demonstrated by the formation of teratoma consisting of tissue derivatives of all three lineages such as bone, cartilage, pulmonary epithelium and mature neurons in SCID mice. Importantly, equine iPSCs should not only have the ability to differentiate in a non-directed manner. Therefore, the ability for efficient and directed cellular differentiation was analysed. Equine iPSCs were successfully induced to differentiate into neurospheres forming extensive neuronal projections and synapses. Equine iPSCs were differentiated to neurons using a novel and robust approach. The neurons expressed FOXG1, TUBB3 at induction before ISL1 up regulation, a potent and specific inducer of motor neurons, during terminal differentiation. The neurons tested could fire multiple action potentials and also induce TTX –sensitive action potentials. The iPSC line that showed in vivo differentiation in bone and cartilage was tested for directed differentiation into bone and results were compared to equine mesenchymal stem cells. This study provides the first demonstration of the potential of iPSCs in equine biomedicine. The ability to derive iPSC cells capable of direct differentiation in vitro opens the way for new and exciting applications in equine regenerative medicine.

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9

GENOVA, ELENA. "Induced pluripotent stem cells as an innovative model for therapy personalization of inflammatory bowel disease". Doctoral thesis, Università degli Studi di Trieste, 2020. http://hdl.handle.net/11368/2961247.

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Crohn’s disease (CD) is a chronic relapsing inflammatory bowel disease that may affect any part of the gastrointestinal tract but most commonly the ileum and the colon. The inflammation extends through the entire thickness of the bowel wall from the mucosa to the serosa. Thiopurines are drugs commonly used in Crohn’s disease (CD) even if some adverse effects are reported. In particular, we focused on the study thiopurine-induced pancreatitis (TIP), a severe and idiosyncratic adverse reaction that affects around 3-5% of CD patients treated with azathioprine, that leads to therapy interruption and could require ad hoc therapy with significative associated costs. Molecular mechanism of TIP is unknown and no validated biomarker is available to assist clinicians in preventing it. Induced pluripotent stem cells (iPSCs) are stem cells obtained reprogramming somatic cells using specific reprogramming factors. iPSCs maintain the donor genetic heritage and have become a powerful technique to model drug adverse effects in a personalized way. iPSCs can differentiate under adequate stimuli into almost every somatic lineage, representing an innovative model to study mechanisms of adverse drug reactions in individual patients' tissues not easily obtainable from human probands. At IRCCS Burlo Garofolo (Trieste, Italy) 3 pediatric CD patients that developed TIP and 3 after azathioprine treatment and 3 CD controls were enrolled and iPSCs were obtained reprogramming peripheral blood mononuclear cells in collaboration with Prof. Gliliani (Brescia, Italy). CD iPSCs were differentiated in pancreatic exocrine cells using the 4 stage protocol developed by Prof. Sasaki (Shinshu University, Japan). Each differentiation stage presents characteristic genetic expression markers: OCT4 is characteristic of undifferentiated cells (iPSCs), FOXA2 and SOX17 of definitive endoderm (stage I), PDX1 of pancreatic progenitors (stage III) and amylase, in particular its pancreatic isoforms AMY2A and AMY2B of pancreatic exocrine cells (stage IV). Differentiation efficiency was analyzed by PCR-real time and immunofluorescence techniques. The sensitivity to thiopurines of TIP and no-TIP CD patient-specific iPSCs and differentiated cells were investigated by MTT assay exposing cells to azathioprine, mercaptopurine and thioguanine for 72 hours. TIP patients iPSCs and pancreatic progenitors resulted more sensitive to mercaptopurine and thioguanine (mercaptopurine p= 0.0162, thioguanine p= 0.0012; two way ANOVA no-TIP vs TIP patients iPSCs; mercaptopurine p = 0.0174; thioguanine p = 0.0144; two way ANOVA no-TIP vs TIP patients pancreatic progenitors). All patients resulted wild type for TPMT polymorphisms letting us to conclude that the different sensitivity between no-TIP and TIP iPSCs and pancreatic progenitors was not related to TPMT genetic variants but to other mechanisms. Thiopurine effect is strictly correlated to cell proliferation being these drugs cell cycle-specific agents interfering during the S phase. iPSCs resulted extremely sensitive to thiopurines in comparison to differentiated cells and to a panel of immortalized lines including the H6C7 ductal pancreatic line. Analysis of cell cycle showed an higher percentage of cells in the S phase in CD-iPSCs with respect to the H6C7 line but not to definitive endoderm or pancreatic progenitors. The faster proliferation of CD-iPSCs well explains their higher sensitivity to thiopurines with respect to H6C7 however, the lower sensitivity of definitive endoderm and pancreatic progenitors cannot be explained basing on the different proliferation of these cells in comparison to iPSCs. The in vitro model established has proven to be suitable for studying and investigating TIP predisposition in a personalized way in pediatric CD patients and could be further developed to study other drugs causing pancreatitis in other diseases.

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10

O'Malley, James. "Novel cell surface markers identify routes to iPS cells". Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/8883.

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Abstract (sommario):

The generation of induced pluripotent stem cells (iPSCs) presents a challenge to normal developmental processes. The low efficiency and heterogeneity of most methods have hindered understanding of the precise molecular mechanisms promoting, and roadblocks preventing, efficient reprogramming. While several intermediate populations have been described, it has proved difficult to characterize the rare, asynchronous transition from these intermediate stages to iPSCs. The rapid expansion of a minor population of reprogrammed cells can also obscure investigation of relevant processes. Understanding of the biological mechanisms essential for successful iPSC generation requires both accurate capture of cells undergoing the reprogramming process and identification of the associated global gene expression changes. Here we demonstrate that reprogramming follows an orderly sequence of stage transitions marked by changes in cell surface markers CD44 and ICAM1, and a Nanog-GFP reporter. RNA-sequencing (RNA-seq) analysis of these populations demonstrates two waves of pluripotency gene up-regulation, and unexpectedly, transient up-regulation of multiple epidermis-related genes, demonstrating that reprogramming is not simply the reversal of normal developmental processes. This novel high-resolution analysis enables the construction of a detailed reprogramming route map, and this improved understanding of the reprogramming process will lead to novel reprogramming strategies.

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11

McLaughlin, Heather Ward. "Modeling sporadic Alzheimer's disease using induced pluripotent stem cells". Thesis, Harvard University, 2014. http://nrs.harvard.edu/urn-3:HUL.InstRepos:13094355.

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Abstract (sommario):

Despite being the leading cause of neurodegeneration and dementia in the aging brain, the cause of Alzheimer's disease (AD) remains unknown in most patients. The terminal pathological hallmarks of abnormal protein aggregation and neuronal cell death are well-known from the post-mortem brain tissue of Alzheimer's disease patients, but research into the earliest stages of disease development is hindered by limited model systems. In this thesis, an in vitro human neuronal system was derived from induced pluripotent stem (iPS) cell lines reprogrammed from dermal fibroblasts of AD patients and age-matched controls. This allows us to investigate the cellular mechanisms of AD neurodegeneration in the human neurons of sporadic AD (SAD) patients, whose development of the disease cannot be explained by our current understanding of AD. We show that neural progenitors and neurons derived from SAD patients show an unexpected expression profile of enhanced neuronal gene expression resulting in premature differentiation in the SAD neuronal cells. This difference is accompanied by the decreased binding of the repressor element 1-silencing transcription/neuron-restrictive silencer factor (REST/NRSF) transcriptional inhibitor of neuronal differentiation in the SAD neuronal cells. The SAD neuronal cells also have increased production of \(amyloid-\beta\) and higher levels of tau protein, the main components of the plaques and tangles in the AD brain.

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12

Abdallah, Hussein(Hussein M. ). "The core mammalian pluripotency network in induced pluripotent stem cell (iPSC) formation : models for genetic and epigenetic reprogramming". Thesis, Massachusetts Institute of Technology, 2018. https://hdl.handle.net/1721.1/122910.

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This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Thesis: M. Eng., Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science, 2018
Cataloged from student-submitted PDF version of thesis. "February 2018."
Includes bibliographical references (pages 23-37).
In 2006, history was made in a seminal experiment that converted mouse fibroblasts to a pluripotent phenotype coined the 'induced pluripotent stem cell' (iPSC) state. Unhindered by ethical or immunogenic constraints, iPSCs potentially hold the keys to tremendous applications in therapeutic and regenerative medicine. Furthermore, on-demand iPSC generation has the capacity to revolutionize basic research in disease modeling and drug discovery. These promises notwithstanding, the economics of iPSC formation--which remains a slow, inefficient, expensive, and laborious process--still stand in the way of fully making use of this extraordinary technology. In this thesis, I present mathematical models aimed at understanding the theoretical reprogrammability of the core pluripotency gene regulatory network being awakened in iPSC reprogramming. Using these modeling insights, I discuss the merits of current reprogramming strategies, which can be viewed as open-loop perturbations in control theoretic terms. I then discuss an alternative paradigm of closed-loop reprogramming, which is theoretically shown to be far superior when it comes to the reprogrammability of the pluripotency network. Finally, I propose a reprogramming model that incorporates the eæect of DNA demethylation on the activation of the network, with attention given to the relationship between this epigenetic transformation and the cell proliferation barrier that somatic cells seemingly face on the road to pluripotency.
by Hussein Abdallah.
M. Eng.
M.Eng. Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science

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13

Kawaguchi, Takamasa. "Studies on induction of pluripotency in bovine somatic cells and generation of induced pluripotent stem cells". Kyoto University, 2016. http://hdl.handle.net/2433/215965.

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Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第19899号
農博第2182号
新制||農||1043(附属図書館)
学位論文||H28||N5003(農学部図書室)
32976
京都大学大学院農学研究科応用生物科学専攻
(主査)教授今井裕, 教授久米新一, 教授廣岡博之
学位規則第4条第1項該当

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14

Minami, Takahito. "Novel hybrid three-dimensional artificial liver using human induced pluripotent stem cells and a rat decellularized liver scaffold". Kyoto University, 2020. http://hdl.handle.net/2433/253138.

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15

Jung, Laura. "Optimisation de protocoles de reprogrammation de cellules somatiques humaines en cellules souches à pluripotence induite (hiPSC)". Thesis, Strasbourg, 2013. http://www.theses.fr/2013STRAJ066.

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En 2006 et 2007, les équipes de Yamanaka et Thomson réalisent la reprogrammation de cellules somatiques murines et humaines en cellules souches pluripotentes à partir de deux cocktails de gènes : OCT4, SOX2, KLF4, cMYC (OSKM) et OCT4, NANOG, SOX2, LIN28 (ONSL). Les cellules souches à pluripotence induite générées (iPS) partagent les propriétés fondamentales des cellules souches embryonnaires : l’auto-renouvèlement, le maintien de la pluripotence et la capacité de différenciation. Ces cellules laissent entrevoir des applications considérables tant en recherche fondamentale (compréhension des mécanismes de développement et de pathologies, développement de modèles) qu’en recherche appliquée (médecine régénérative, toxicologie prédictive, criblage de médicaments). L’avantage majeur de l’utilisation des iPS réside dans leur origine non embryonnaire. Les contraintes d’ordre éthique sont écartées et une grande diversité de types cellulaires à partir de n’importe quel donneur a priori est disponible pour une reprogrammation. L’établissement d’une banque d’hiPS issus de donneurs sains ou de patients, serait d’une grande utilité pour la communauté scientifique se consacrant à l’étude des mécanismes physiopathologiques ou de développement et une source considérable pour la dérivation à des fins de thérapie cellulaire. Dans le but de mettre en place une telle banque, nous avons développé avec la société Vectalys des rétrovirus de reprogrammation contenant les cassettes polycistroniques ONSL et OSKM. Dans un premier temps, nous avons établi un protocole de reprogrammation robuste à l’aide des rétrovirus RV-ONSL. Nous avons ensuite mis en évidence la synergie entre les facteurs ONSL et OSKM, la combinaison équimolaire de RV-ONSL et RV-OSKM permettant d’atteindre 2% d’efficacité de reprogrammation. Nous avons également entrepris la reprogrammation propre par transfections d’ARNm polycistroniques ONSL et OKM mettant à profit cette incroyable synergie
In 2006 and 2007, Yamanaka and Thomson teams achieved the reprogramming of mouse and human somatic cells into pluripotent stem cells through the transfection of two cocktails of genes: OCT4, SOX2, KLF4, cMYC (OSKM) and OCT4, NANOG, SOX2, LIN28 (ONSL). The generated cells, called induced Pluripotent Stem Cells (iPSC) share the same fundamental properties of ESC : self-renewing, pluripotency maintenance and capacity of differentiation into the three germ layers and suggest the same application potential in basic research (developmental and epigenetic biology) as well as in therapy (regenerative medicine, disease modeling for drug development). One of the major advantages of iPSC lies in their non-embryonic origin. Indeed, the use of iPSC resolves the ethical constraints and offers the possibility to work with extensive cell types directly from the patient to treat. Stéphane Viville’s research team aims to develop a hiPSC bank from patient suffering from genetic or other diseases which will be available for the scientific community. We are experienced in human primary fibroblasts reprogramming especially with the use of two polycistronic cassettes: ONSL encoding Thomson’s cocktail and OSKM encoding Yamanaka’s cocktail separated with 2A peptides. Thanks to the combination of RV-ONSL and RV-OSKM retroviral vectors (developed with Vectalys) we are yielding more than 2% of reprogramming efficiency in a highly reproducible way. Indeed, we demonstrated the reprogramming synergy of ONSL and OSKM combination. We are now focusing our effort on non-integrative strategies (ie mRNA) which are more appropriate for clinical usage

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16

Bova, Wesley Adam. "Analysis of the Commercial Potential of the Cell X Technologies, Inc. Cell Picker in the Induced Pluripotent Stem Cell Market". Case Western Reserve University School of Graduate Studies / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=case1607711405382538.

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17

Choompoo, Narawadee. "Induced pluripotent stem (iPS) cells for cell replacement therapy in Huntington's disease (HD)". Thesis, Cardiff University, 2015. http://orca.cf.ac.uk/73191/.

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Huntington’s disease (HD) is a neurodegenerative disease caused by a mutation in the huntingtin gene (HTT). The extended CAG repeat ultimately leads to loss of medium spiny neurons (MSNs) in the striatum of the HD brain. Cell replacement therapy using primary human fetal tissue as a source of “genuine” MSNs has shown ‘proof of principle’ as a strategy to treat this genetically inherited disease1. However, renewable cell sources need to be identified to overcome the ethical and logistical issues that are associated with using human fetuses. Here we attempted to generate iPS cells by introducing reprogramming factors using the piggyBac Transposon2 transduction system in human fetal fibroblasts and fetal neural stem cells. We wish to test the hypothesis that these cells are more easily reprogrammable and/or are more readily directed towards an MSN phenotype. The established iPS cell lines were similar to human embryonic stem (ES) cells in terms of their morphology, surface antigen, and proliferation. These iPS cells lines have been successfully manipulated to differentiate into MSNs in culture according to their expression of standard molecular markers of premature and mature MSNs - Ctip2 and Darrp32. Differentiation following transplantation into the quinolinic acid (QA) lesion model showed that grafts of these striatal progenitors derived from human fetal iPS cells could differentiate into neural progenitors according to expression of human nuclei marker (HuNu) and nestin.

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18

Yang, Chian. "Derivation of purified smooth muscle cells from mouse induced pluripotent stem (iPS) cells". Thesis, Boston University, 2013. https://hdl.handle.net/2144/12250.

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Thesis (M.S.)--Boston University
Cardiac and vascular disease syndromes and abnormalities have long been the leading causes of death in the United States, but the cause of congenital defects remain unclear due to the lack of appropriate model systems for scientific investigation. Moreover, the predominance of cardiovascular disease has stimulated scientists to focus on developing tissue-engineered blood vessels (TEBV) for vascular reconstruction and replacement. Major challenges remain in generating large amounts of epithelial cells (EC) and vascular smooth muscle cells (VSMC) for vascular reconstruction and in reducing the immunoresponse in patients after replacement. To address both issues of disease model generation and vascular tissue engineering, we can use induced pluripotent stem (iPS) cells discovered by Shinya Yamanaka in 2006: iPS cells generated from adult tissue and organs through the forced expression of two to four transcription factors are phenotypically indistinguishable from embryonic stem (ES) cells. First, by creating iPS from cardiovascular patients, we can generate patient-specific cell lines for scientific research investigation (i.e. elucidate molecular mechanisms and potential drug targets). Second, EC and VSMC derived from patient-specific iPS cell lines can be used for vascular reconstruction with cells of the patient's own genetic background, which should overcome many of the immunological limitations that currently impede vascular replacement (i.e. provide therapeutic interventions). The goal of this project is to study the differentiation capacity of iPS cells into smooth muscle cells (SMC). This project aims to develop a protocol that can maximize the derivation of purified smooth muscle cells from mouse induced pluripotent stem (iPS) cells through three linear developmental stages: induction of a posterior primitive- streak (PS) like population, formation of Flk1+ mesoderm, and induction of smooth muscle cells. Low dosage of Activin A and Wnt was used to differentiate iPS into a PS-like population, while the administration of BMP4 differentiates the cells to mesoderm via a posterior PS intermediate. Smooth muscle cells were induced from mesoderm by the addition of platelet-derived growth factor (PDGF-BB) and transforming growth factor b (TGF-β). The linear developmental progression from PS formation through mesoderm induction to smooth muscle cells were tracked by RT-qPCR and FACS for the expression of genes indicative of each individual stage, Flk1, and SMαA respectively. The results of this project can be used as a basis for in vitro derivation of purified mammalian smooth muscle cells from a mouse model system that can be further modified. Moreover, the differentiated SMCs can be further used in cell sheet construction as vascular patches for drug testing.

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19

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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20

Buchrieser, Julian. "Understanding human mononuclear phagocyte ontogeny using human induced pluripotent stem cells (iPSCs)". Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:aaf18203-5f30-4d6a-8f51-3096b29af252.

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Tissue-resident macrophages (MΦ) such as microglia, Kupffer and Langerhans cells derive from Myb-independent yolk sac (YS) progenitors generated before the emergence of hematopoietic stem cells (HSCs). Myb-independent YS-derived resident MΦ self-renew locally, independently of circulating adult monocytes and HSCs. In contrast, adult blood monocytes as well as infiltrating, gut and dermal MΦ derive from Myb-dependent HSCs and are less proliferative. These findings are derived from the mouse, using gene knock-outs and lineage tracing, but their applicability to human development has not been formally demonstrated. Here I use a human pluripotent stem cell (hPSC) differentiation model of hematopoiesis, capable of monocyte/MΦ production over prolonged periods of time, as a tool to investigate human mononuclear phagocyte ontogeny. Using a transcriptomic approach I showed that hiPSC-derived monocytes/MΦ (iPS-Mo/MΦ) produced early in differentiation (first weeks) are more proliferative and less immunologically mature than iPS-Mo/MΦ produced at a later time point. I therefore hypothesised either that iPS-Mo/MΦ only become fully mature after several weeks of differentiation or that there are two developmentally distinct waves of MΦ produced over time. By comparing the transcription profile of iPS-Mo/MΦs to that of primary adult blood monocytes and fetal microglia I then showed that early and late iPS-Mo/MΦs were transcriptionally closer to fetal microglia than to adult blood monocytes. To further investigate if iPS-Mo/MΦs are indeed of the same developmental origin as MYB-independent MΦ such as microglia, I used a CRISPR-Cas9 knock-out strategy to show for the first time, that human iPS-Mo/MΦs develop in a MYB-independent, RUNX1 and SPI1 (PU.1)-dependent fashion. This result makes human iPS-Mo/MΦs developmentally related to, and a good model for, MYB-independent tissue-resident \Macros such as alveolar and kidney MΦs, microglia, Kupffer and Langerhans cells. Interestingly, while MYB was not required for the generation of iPS-Mo/MΦs, its knock-out resulted in an increase in iPS-Mo/MΦ production. To investigate this increase I developed two methods for quantifying the differentiation bottleneck occurring during hiPSC differentiation to iPS-Mo/MΦs. Those techniques highlighted a potential increase in progenitor cell generation in MYB KO cells and thus lay foundation to improve our technical understanding of EB differentiation and will enable enhanced manipulation of the EB model.

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21

MONTAGNA, ANNA. "Induced pluripotent stem cells (IPSCS) for modelling mucopolysaccharidosis type I (Hurler syndrome)". Doctoral thesis, Università degli Studi di Milano-Bicocca, 2016. http://hdl.handle.net/10281/113869.

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Mucopolysaccharidosis type I (MPS-IH or Hurler syndrome) is a rare lysosomal storage disease caused by mutations in the IDUA gene, resulting in the deficiency of alpha-L-iduronidase (IDUA) enzyme activity with a consequent intracellular accumulation of glycosaminoglycans (GAGs). Among a broad spectrum of clinical manifestations, MPS-IH is characterized by a range of skeletal abnormalities known as dysostosis multiplex. To date, the skeletal pathogenesis of the MPSs has been assumed to be directly related to the progressive storage of GAGs. It is now clear that more complex cellular and molecular mechanisms underlie the patient clinical symptoms. Therefore, an appropriate humanized in vitro model is highly recommended to highlight these mechanisms. Compared to mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs) represent a useful tool to achieve this purpose, due to their high proliferation capability in culture and, mostly, to their ability to mimic development. Thus, they demonstrate great potential for investigating the osteogenic differentiation process. In this study, we generated MPS-IH patient-specific iPS cells (MPS-IH iPSCs) which maintained the genetic mutation in the IDUA gene and, as a consequence, reduced IDUA enzyme activity and GAGs intracellular accumulation. In order to assess if the osteogenic differentiation phenotype is already compromised in MPS-IH iPSCs cell, we focused on their bone differentiation capability. Thus, we developed an osteogenic differentiation protocol through the generation of mesenchymal stromal cells from iPSC (hereafter named MSCs-like cells). We designed a robust, multistep differentiation method to isolate MSCs-like cells, both from wild-type iPSCs (WT-iPSCs) and MPS-IH iPSCs. The process included: embryoid body (EB) formation, cell outgrowth from EBs, monolayer culture of sprouted cells from EBs, and a serial of passages in culture until they reached a fibroblast-like morphology and the full expression of mesenchymal surface markers. Firstly, we characterized WT and patient derived-MSCs-like cells in terms of morphology, phenotype, proliferation kinetics and differentiation capacity in mesodermal tissues. WT and patient derived-MSCs-like cells showed the capacity to differentiate in adipocytes, as confirmed by Oil Red O staining. Moreover, MSCs-like cells derived-chondrogenic pellets exhibited a spherical, compact morphology. Histological analysis revealed an initial chondrogenic differentiation, as confirmed by q-RT-PCR for key early chondrogenic markers, such as SOX9 and COLII. Subsequently, we developed an osteogenic differentiation protocol for the obtained MSC-like cells. In order to verify if the differentiation process was accomplished, we performed Alizarin Red staining and quantified the hydroxyapatite production by colorimetric detection at 405 nm both on WT and MPS-IH iPSCs-derived osteoblasts. At the same time, we examined the expression for key osteogenic markers, such as OPN, RUNX2 OTC, OTN, ALP and COL 1A2, through q-RT-PCR. Recently, our group isolated MSCs from bone marrow (BM-MSCs) of both healthy donors and MPS-IH patients, studying a possible involvement of MSCs in the skeletal abnormalities affecting Hurler patients. We previously observed the ability of WT, MPS-IH BM-MSCs and MSCs-derived osteoblasts to stimulate osteoclastogenesis in vitro by measuring the molecular levels of receptor activator of nuclear factor-Kb ligand (RANKL) and osteoprotegerin (OPG), two key partners of the system directly regulating osteoclast differentiation. MPS-IH MSCs and osteoblasts derived from MPS-IH MSCs, expressed a higher level of RANKL compared to HD-MSCs and osteoblasts. OPG level, instead, was similar. In the present study, the osteogenic differentiation protocol developed allowed us to assess if this altered phenotype is already evident in both MSCs-like cells MSCs-like derived osteoblasts, by evaluating the OPG and RANKL expression levels.

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22

Di, Stefano Bruno 1984. "C/EBPα poises B cells for rapid reprogramming into induced pluripotent stem cells". Doctoral thesis, Universitat Pompeu Fabra, 2014. http://hdl.handle.net/10803/283484.

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One of the major goals of current stem cell research is understanding the mechanism of somatic cell reprogramming by Oct4, Sox2, Klf4 and Myc (OSKM) into induced pluripotent stem cells (iPSCs). However, the finding that only a small proportion of the cells become reprogrammed, typically requiring >12 days, has hampered progress towards this goal. C/EBPα is a transcription factor specifically expressed in myelomonocytic cells within the hematopoietic system whose forced expression in B cells efficiently induces transdifferentiation into macrophages. We have now found that an 18-hour pulse of C/EBPα expression followed by OSKM activation induces an approximately 100-fold increase in the iPSC reprogramming efficiency, involving up to 95% of the cells within a week. Concomitantly, the cells undergo an epithelial-mesenchymal transition and pluripotency genes become upregulated to levels comparable to embryonic stem and iPS cells. In serum-free conditions the process is further accelerated, with 60% of the poised and OSKM induced B cells becoming Oct4-GFP positive within 2 days. These results are consistent with the idea that the C/EBPα pulse helps to overcome the stochastic phase of iPSC reprogramming. In addition, our work shed new light on the role of C/EBPα in induced pluripotency. Our data indicate that C/EBPα acts as a pathbreaker, at least in part mediated by the dioxygenase Tet2. C/EBPα binds to the Tet2 gene, induces its expression and translocates the protein to the nucleus. Here Tet2 binds to regulatory regions of pluripotency genes and converts methylated cytosine residues into hydroxymethylated cytosines. The pulse also renders the chromatin at regulatory sites of pluripotency genes accessible to DNase I digestion and, following OSKM induction, leads to local demethylation and to the binding of Oct4, correlating with the observed rapid upregulation of pluripotency genes. In line with an important role of Tet2 as a mediator of reprogramming, coexpression of the gene with OSKM enhanced B cell reprogramming substantially. The rapid and highly efficient iPSC reprogramming approach described herein should help to fully elucidate the early events of reprogramming to pluripotency and, if applicable to human cells, could have potential clinical applications.
Actualmente uno de los principales objetivos de la investigación con células madre es la comprensión de los mecanismos por los cuales las células somáticas se pueden reprogramar a células madre pluripotentes inducidas (iPSCs) por la acción de los factores de transcripción Oct4, Sox2, Klf4 y Myc (OSKM). Sin embargo, la baja eficiencia de este proceso, que tiene lugar sólo en un pequeño porcentaje de células y que típicamente requiere más de 12 días para llevarse a cabo, ha impedido la consecución de grandes avances en este campo en los últimos años. C/EBPα es un factor de transcripción específico de células del linaje mielo-monocítico del sistema hematopoyético. La expresión ectópica de esta proteína en células B puede inducir su transdiferenciación a macrófagos. En nuestro estudio de investigación hemos descubierto que la exposición de C/EBPα durante 18 horas seguida de la activación de OSKM, aumenta en 100 veces la eficiencia de reprogramación de las iPSC, resultando en la reprogramación del 95% de las células después de una semana. En detalle, durante este proceso de reprogramación las células experimentan una transición epitelio-mesénquima y los genes de pluripotencia se expresan en niveles comparables a los expresados en células madre embrionarias y iPSC. Cuando la reprogramación se lleva a cabo en medio de cultivo sin suero el proceso es aún más rápido, de tal modo que el 60% de las células B inducidas por C/EBPα y OSKM son positivas para Oct4-GFP en tan sólo dos días. Estos resultados apoyan la idea de que una exposición transitoria de C/EBPα ayuda a superar la fase estocástica de la reprogramación de las iPSC. Además, nuestros descubrimientos aclaran el papel de C/EBPα en el proceso de pluripotencia inducida, indicando que actúa como un catalizador, mediado en parte por la actividad de la dioxigenasa Tet2. De tal modo, que C/EBPα se une a regiones reguladoras del locus de Tet2, induciendo de esta manera su expresión y translocando la proteína al núcleo. Una vez en el núcleo, Tet2 se une a las regiones regulatorias de los genes de pluripotencia y convierte los residuos de citosinas metilados existentes en estas regiones en citosinas hidroximetiladas. Además, la exposición transitoria de C/EBPα deja la cromatina más accesible a la digestión con DNasa I alrededor de las regiones regulatorias de los genes de pluripotencia y, tras la inducción con OSKM, desencadena una demetilación local favoreciendo la posterior unión de Oct4 a estas regiones. Todo ello finalmente promueve la expresión concomitante de los genes de pluripotencia. Adicionalmente, en nuestro estudio se demuestra que la coexpresión de Tet2 y OSKM aumenta significativamente la reprogramación de las células B, lo cual se encuentra en línea con un papel importante de Tet2 en la reprogramación. En resumen, en este estudio se presenta el sistema de reprogramación de iPSC más rápido y eficiente descrito a día de hoy. El cual, facilitará la comprensión de los eventos precoces en el proceso de reprogramación a pluripotencia y, en el caso de que se pueda extrapolar a células humanas, podrá tener aplicaciones clínicas relevantes en el campo de la medicina regenerativa.

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23

Bartish, Margarita. "Establishing iPSCs as a method to model neurodevelopment in Down’s syndrome". Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-182353.

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Abstract (sommario):

The derivation of pluripotent stem cells (now termed induced pluripotent stem cells, iPSC) from mature somatic cells was a finding of seminal importance to fundamental cell biology. Thus established iPSC technology has been predicted to advance fields that previously relied on the ethically disputed use of embryonic stem cells. Being pluripotent (able to differentiate into every cell type present in the human body) and sharing most other characteristics with embryonic stem cells, but being much readier obtainable and their derivation free from ethical restraints, human induced pluripotent stem cells (hiPSC) provide access to cell types and insights into cell processes previously unattainable to researches. For this thesis, a hiPSC line was established from a skin biopsy donated by a Down’s syndrome patient. Most of what is known today about the molecular neurobiology behind this disease has been gathered from mice models or human post mortem studies, but this has a limited extrapolation potential to early human brain development in DS patients, as Down’s syndrome is an inherently human disease whose defining phenotype is established early during embryonic development. Having access to human pluripotent cells able to recapitulate the events of early neurogenesis is thus invaluable to the understanding of the mechanisms of this disorder. In parallel, work has been performed on optimizing iPSC reprogramming protocol. By exchanging one of the transcription factors used for reprogramming with a reporter gene, genomic integration of reprogramming factors has become possible to be traced visually, enabling more efficient selection of reprogrammed iPSC colonies.

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24

MAZZARA, PIETRO GIUSEPPE. "TWO FACTOR BASED REPROGRAMMING OF FIBROBLASTS AND INDUCED PLURIPOTENT STEM CELLS INTO MYELINOGENIC SCHWANN CELLS". Doctoral thesis, Università degli Studi di Milano-Bicocca, 2018. http://hdl.handle.net/10281/199039.

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Le cellule di Schwann (SC) sono cellule derivate dalla cresta neurale (NC) in grado di produrre la guaina mielinica avvolgendo gli assoni neuronali nel sistema nervoso periferico (PNS). I trapianti di SC potrebbero diventare un'opportunità terapeutica interessante per il trattamento delle lesioni del midollo spinale, dei nervi periferici e delle malattie demielinizzanti del PNS. Tuttavia, questi approcci terapeutici sono fortemente limitati dall'attuale mancanza di una fonte rinnovabile di SC. Le strategie di riprogrammazione cellulare si sono rivelate efficaci nel fornire una varietà di cellule specifiche per la modellizzazione delle malattie e per le procedure di trapianto di cellule, ottenute mediante sovraespressione di fattori di trascrizione cardine nello sviluppo embrionale del tipo cellulare di interesse. Ho identificato i due fattori di trascrizione Sox10 ed Egr2 in grado di generare cellule di Schwann indotte (iSCs) ad alta efficienza quando co-espresse in fibroblasti murini. Le iSC assomigliano a SC primarie nel profilo globale di espressione genica ed esprimono marcatori specifici di SC, tra cui S100ß, O4 e MPZ. Quando co-coltivati con espianti di gangli della radice dorsale di topo (DRG), le iSCs hanno generato guaine di mielina compatte organizzate in internodi Mbp+ intervallati da domini paranodali caspr + e domini nodali con canali del sodio. Al contrario, le iSC derivate da topi Twitcher hanno mostrato una grave perdita del potenziale mielinogenico, indicando le iSC come un sistema attraente per la modellazione in vitro delle malattie del PNS. Quindi, ho derivato iSCs da ratti sottoposti ad assotomia del nervo mediano seguito da trapianto di condotti di chitosano precedentemente seminati con iSC autologhe. Questi condotti con le iSC supportano e accelerano la rigenerazione dei nervi con un migliorato contenuto di mielina. Allo stesso modo, Sox10 ed Egr2 sono sufficienti per convertire i fibroblasti umani in iSC. Inoltre, la loro espressione facilita fortemente la differenziazione in SC delle cellule staminali pluripotenti indotte umane (iPSC), includendo nella strategia di riprogrammazione pochi passaggi intermedi che forniscono diversi stimoli trofici alle cellule differenzianti. In particolare, dopo la trasduzione lentivirale con i lentivirus esprimenti Sox10 ed Egr2, ho aggiunto piccole molecole neuralizzanti (SB431542 e LDN193189 in terreno iPSC), insieme a un terreno di differenziazione della cresta neurale (B27, acido ascorbico e FGF2 in terreno neurobasico) e infine un medium specifico per la crescita delle cellule di Schwann (Forskoline, NRG1, FGF2 in DMEM 10% FBS), fornendo una semplice procedura per ottenere un gran numero di SC omogenee e ben differenziate. Complessivamente, Sox10 ed Egr2 sono una combinazione unica di fattori per la generazione efficace di iSC mielinogeniche da fibroblasti di roditori e umani e da iPSC. Il processo rapido e diretto per generare iSC faciliterà la modellazione in vitro e gli approcci di trapianto di cellule autologhe per le malattie del PNS.
Schwann cells (SCs) are neural crest (NC) derived cells able to produce the myelin sheaths, wrapping neuronal axons in the peripheral nervous system (PNS). Transplantations of SCs might become an interesting therapeutic opportunity for the treatment of spinal cord and peripheral nerves injuries and demyelinating diseases of the PNS. However, these therapeutic approaches are strongly limited by the current lack of a renewable source of SCs. Cell reprogramming strategies have proven to be effective in providing a variety of tissue-specific cells for disease modelling, and cell transplantation procedure by over expression of cardinal developmental transcription factors of the interest cell type. I have identified the two transcription factors Sox10 and Egr2 able to generate induced Schwann Cells (iSCs) when co-expressed in murine fibroblasts with high efficiency. iSCs resembled primary SCs in global gene expression profiling and expressed cardinal markers of SCs including S100ß, O4 and MPZ. When co-cultured with mouse dorsal root ganglion (DRG) explants, iSCs generated compact myelin sheaths organized in Mbp+ internodes spaced by Caspr+ paranodal and Na+ channel nodal domains. Conversely, iSCs from Twitcher mice showed a severe loss in the myelinogenic potential, indicating iSCs as an attractive system for in vitro modeling of PNS diseases. Then, I derived iSCs from rats that were subjected to median nerve axotomy followed by transplantation of chitosan conduits previously seeded with autologous iSCs. These iSC-seeded conduits supported accelerated nerve regeneration with improved myelin content. Similarly, Sox10 and Egr2 are sufficient to convert human fibroblasts into iSCs. Moreover, their expression strongly facilitate the SC differentiation of human induced pluripotent stem cells (iPSCs), including in the reprogramming strategy few intermediate steps that provide different trophic stimuli to the differentiating cells. In particular, after the lentiviral transduction with the Sox10 and Egr2 expressing lentiviruses, I added neuralizing small molecules (SB431542 and LDN193189 in hiPS medium), together with a neural crest differentiation medium (B27, Ascorbic Acid and FGF2 in neurobasal medium), and finally a specific medium for Schwann cell growth (Forskoline, NRG1, FGF2 in DMEM 10% FBS), providing a simple procedure for obtaining a large number of homogeneous and well-differentiated SCs. Altogether, Sox10 and Egr2 is a unique combination of factors for the effective generation of myelinogenic iSCs from rodent as well as human fibroblasts and iPSCs. The fast and straightforward process to generate iSCs will facilitate in vitro disease modeling and autologous cell transplantation approaches for PNS diseases.

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Chai, Yi Wen. "Understanding the Cellular Mechanisms of the Leukocyte Adhesion Deficiency Type III Disorder with the Use of Patient Induced Pluripotent Stem Cells". University of Cincinnati / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1417784194.

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26

Beevers, Joel Edward. "Investigating the function of microtubule-associated protein tau (MAPT) and its genetic association with Parkinson's using human iPSC-derived dopamine neurons". Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:7a94919a-73a1-4a9f-b04d-cdf5b9c64be7.

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Abstract (sommario):

Parkinson's disease (PD) primarily manifests as loss of motor control through the degeneration of nigrostriatal dopaminergic neurons. The microtubule-associated protein tau (MAPT) locus is highly genetically associated with PD, wherein the H1 haplotype confers disease risk and the H2 haplotype is protective. As this haplotype variation does not alter the amino acid sequence, disease risk may be conferred by altered gene expression, either of total MAPT or of specific isoforms, of which there are six in adult human brain. To investigate haplotype-specific control of MAPT expression in the neurons that die in PD, induced pluripotent stem cells (iPSCs) from H1/H2 heterozygous individuals were differentiated into dopaminergic neuronal cultures that expressed all six mature isoforms of MAPT after six months' maturation. A reporter construct using the human tyrosine hydroxylase locus was also generated to identify human dopaminergic neurons in mixed cultures. Haplotype-specific differences in the inclusion of exon 3 and total MAPT were observed in iPSC-derived dopaminergic neuronal cultures and a novel variant in MAPT intron 10 increased the inclusion of exon 10 by two-fold. RNA interference tools were generated to knockdown total MAPT or specific isoforms, wherein knockdown of the 4-repeat isoform of tau protein increased the velocity of axonal transport in iPSC-derived neurons. MAPT knockdown also reduced p62 levels, suggesting an impact of tau on macroautophagy, likely through altered axonal transport. These results demonstrate how variation at a disease susceptibility locus can alter gene expression, thereby impacting on neuronal function.

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27

Raykova, Doroteya. "Genetics of Two Mendelian Traits and Validation of Induced Pluripotent Stem Cell (iPSC) Technology for Disease Modeling". Doctoral thesis, Uppsala universitet, Medicinsk genetik och genomik, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-246228.

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Novel technologies for genome analysis have provided almost unlimited opportunities to uncover structural gene variants behind human disorders. Whole exome sequencing (WES) is especially useful for understanding rare Mendelian conditions, because it reduces the requirements for a priori clinical data, and can be applied on a small number of patients. However, supporting functional data on the effect of specific gene variants are often required to power these findings. A variety of methods and biological model systems exists for this purpose. Among those, induced pluripotent stem cells (iPSCs), which are capable of self-renewal and differentiation, stand out as an alternative to animal models. In papers I and II we took advantage of WES to identify gene variants underlying autosomal recessive pure hair and nail ectodermal dysplasia (AR PHNED) as well as autosomal dominant familial visceral myopathy (FVM). We identified a homozygous variant c.821T>C (p.Phe274Ser) in the KRT74 gene as the causative mutation in AR PHNED, supported by the fact that Keratin-74 was undetectable in hair follicles of an affected family member. In a family segregating FVM we found a heterozygous tandem base substitution c.806_807delinsAA (p.(Gly269Glu)) in the ACTG2 gene in the affected members. This novel variant is associated with a broad range of visceral symptoms and a variable age of onset. In Paper III we explored the similarity between clonally derived iPSC lines originating from a single parental fibroblast line and we highlighted the necessity to use lines originating from various donors in disease modeling because of biological variation. Paper IV focused on how the genomic integrity of iPSCs is affected by the choice of reprogramming methods. We described several novel cytogenetic rearrangements in iPSCs and we identified a chromosome 5q duplication as a candidate aberration for growth advantage. In summary, this doctoral thesis brings novel findings on unreported disease-causing variants, as supported by extensive genetic analysis and functional data. A novel molecular mechanism behind AR PHNED is presented and the phenotypic spectrum associated with FVM is expanded. In addition, the thesis brings novel understanding of benefits and limitations of the iPSC technology to be considered for disease modeling.

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

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

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

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30

Buccini, Stephanie M. "Cardiogenic differentiation of induced pluripotent stem cells for regeneration of the ischemic heart". University of Cincinnati / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1382373160.

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31

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

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32

Louçã, Mathilde. "Functional impacts of Huntingtin lowering on the synaptic maturation and activity of neuronal networks derived from human induced pluripotent stem cells". Electronic Thesis or Diss., université Paris-Saclay, 2024. http://www.theses.fr/2024UPASL054.

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La maladie de Huntington (MH) est une maladie neurodégénérative causée par la mutation de la Huntingtine (HTT). La réduction de l'expression de la HTT mutante est une piste thérapeutique évidente en cours d’exploration chez les patients. Le ciblage de la HTT mutante s’accompagne cependant le plus souvent d’une réduction concomitante de la HTT non mutée. Les conséquences de la perte de cette protéine sur la santé des neurones restent mal connues.Mon travail de thèse traite cette question en utilisant des modèles in vitro de réseaux neuronaux humains différenciés à partir de cellules souches induites à la pluripotence. Mes travaux démontrent que la perte de HTT induit des anomalies de développement et d’homéostasie de ces réseaux. Mes résultats suggèrent que les thérapies ciblant indifféremment la HTT mutante et non mutante pourraient compromettre la santé des circuits neuronaux ciblés
Huntington's disease (HD) is a neurodegenerative disorder caused by a mutation in the Huntingtin gene (HTT). Reducing the expression of mutant HTT is an obvious therapeutic approach explored in patients. However, targeting mutant HTT often leads to a simultaneous reduction in non-mutant HTT. The consequences of losing this protein on neuronal health remain poorly understood.My doctoral work addresses this question using in vitro models of human neuronal networks differentiated from induced pluripotent stem cells. My research demonstrates that HTT loss induces developmental and homeostatic abnormalities in these networks. My results suggest that therapies targeting both mutant and non-mutant HTT indiscriminately could compromise the health of targeted neuronal circuits

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Yoshimatsu, Masayoshi. "In vivo regeneration of rat laryngeal cartilage with mesenchymal stem cells derived from human induced pluripotent stem cells via neural crest cells". Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/265189.

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京都大学
新制・課程博士
博士(医学)
甲第23417号
医博第4762号
新制||医||1052(附属図書館)
京都大学大学院医学研究科医学専攻
(主査)教授松田秀一特定拠点, 教授妻木範行, 教授安達泰治
学位規則第4条第1項該当
Doctor of Medical Science
Kyoto University
DFAM

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34

Chokesuwattanaskul, S. "The use of induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSCs) to study the genetic basis of human diseases". Thesis, University of Liverpool, 2017. http://livrepository.liverpool.ac.uk/3006683/.

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Objectives: The aim of this thesis was to evaluate the potential of new technologies, including two stem cell technologies, mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), to understand the molecular basis of human diseases. These technologies were evaluated for their ability to restore diabetes-induced defects in wound tissue repair (MSCs) and to generate mature neutrophils after in vitro differentiation of iPSCs. The latter used iPSCs from a patient with abnormal function due to impaired WASp (Wiskott Aldrich syndrome protein) signalling. Other techniques evaluated were differentiation of the myeloid cell line, PLB-985 (expressing exogenous genes) into mature neutrophils and new advances in metabolomics, to identify altered neutrophil function in human diseases. Methods: The potential of MSCs and oral vitamin C to generate factors that could promote healing of diabetic wounds, was measured by RT-PCR for eight genes associated with either angiogenesis or extracellular matrix production, after incubation under normoglycaemic and hyperglycaemic conditions with and without vitamin C. The angiogenic effects of the MSC secretome on wound healing was measured using a tubular formation assay (in vitro) and a nude mice diabetic wound model (in vivo). The bilateral full-skin thickness wounds were created in an in vivo wound model using diabetic nude mice. Oral vitamin C (1.5 g/L) was administered in combination with topical MSC treatment (MSCs 1x 106 cells per wound). Diabetic wound models were divided into five groups; control (CON; n=6), diabetes (DM; n=12), diabetes treated with MSCs (DM+MSCs; n=12), diabetes treated with VitC (DM+VitC; n=6), and diabetes treated with MSCs and VitC (DM+MSCs+VitC; n=12). The capillary density was measured under in vivo fluorescent microscopy, and the tissue VEGF levels were measured. WAS dermal fibroblasts were reprogrammed using retrovirus transfection, and the corrected-WAS-iPSCs were differentiated into the neutrophil-like cells via the formation of iPS-sacs (the sac-like structure containing haematopoietic progenitor cells derived from iPSCs). Neutrophil (from WAS patients and healthy controls) chemotaxis was measured using transwell migration towards N-formylmethionine-leucyl-phenylalanine (fMLP). PLB-985 and KCL-22 cells were differentiated into neutrophil-like cells using RPMI-1640 media containing N,N-dimethyl formamide, sodium pyruvate, all-trans retinoic acid, human AB serum and dimethyl sulfoxide (with penicillin/streptomycin). Morphology was assessed by cytospin. PLB-985 cells were transduced with enhanced green fluorescent protein (EGFP)-tagged Myeloid Cell Leukaemia-1 (Mcl-1), sub-cloned into a pLVX-TetOne-Puro system. 1H NMR metabolomics was carried out using protocols optimised for neutrophils as part of this thesis. An intracellular metabolite extraction method was developed to minimise the loss of neutrophil metabolites and to avoid contaminants arising during the extraction procedure. The NMR analyses were also optimised to identify neutrophil metabolites and allow the comparison from resting and activated states and in health and disease (rheumatoid arthritis patients). Results: Upregulation of angiogenic genes, vascular endothelial growth factor-α (mVEGF-α) and platelet-derived growth factor-BB (mPDGF-BB), in response to TGF-β1 in MSCs was lower following incubation under hyperglycemia (compared to normoglycaemic controls), but vitamin C treatment re-sensitised the MSC response to TGF-β1. A diabetic mouse model showed that administration of oral vitamin C, as an adjunct to MSC therapy, resulted in accelerated wound healing that was associated with increased capillary density. Preliminary experiments with WAS neutrophils showed significantly lower rates of chemotaxis towards fMLP compared to healthy controls. iPSCs from WAS fibroblasts were cultured and differentiated into neutrophil-like cells. The efficiency of both PLB-985 and KCL-22 cells to differentiation into neutrophil-like cells was evaluated and PLB-985 cells differentiated more efficiently into neutrophil-like cells than the KCL-22 cells. PLB-985 cells, transfected with Mcl-1:EGFP in pLVX-TetOne-Puro system were generated. Nuclear magnetic resonance (NMR) metabolomics identified metabolites and pathways altered during in vitro activation with PMA (including metabolites of NADPH synthesis and inhibitors of reactive oxygen species (ROS)) and in vivo activation in rheumatoid arthritis identified metabolites of the ketosis pathway, citrullination pathway and tryptophan metabolism. Conclusions: A number of technologies have been evaluated to study the molecular basis of human disease, including metabolic (diabetes mellitus) and genetic (WAS) diseases. Vitamin C modulated the secretome of MSCs, increasing angiogenesis and accelerating wound healing, providing a potential new approach for designing adjuncts to existing therapies. Neutrophils from WAS patients demonstrated chemotactic defects, and the potential of WAS-iPSCs to differentiate into neutrophil-like cells was demonstrated. The approach could be applied in further studies to study genetic defects of leukocyte function. PLB-985 cells transduced with EGFP-tagged Mcl-1 in an inducible expression vector, was developed as a cell-line model of neutrophil differentiation, to facilitate further studies into the role of the Mcl-1 gene in regulating neutrophil survival. Protocols for human neutrophil metabolomics, using 1H NMR spectroscopy were developed and applied to the study of in vitro and in vivo activated neutrophils. The results demonstrated the potential of metabolomics for future studies of human diseases.

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Manzar, Gohar Shahwar. "Generation and function of glucose-responsive insulin producing cells derived from human induced pluripotent stem cells". Diss., University of Iowa, 2015. https://ir.uiowa.edu/etd/5808.

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Type I diabetes (T1D) is caused by autoimmune destruction of pancreatic β-cells. Immediate consequences of T1D are severe weight loss, ketoacidosis and death unless insulin is administered. The long-term consequences of T1D are dysregulation of metabolism leading to cardiovascular complications, neuropathy and kidney insufficiency. It is estimated that 3 million Americans have T1D, and its prevalence among young individuals is progressively rising. Islet transplantation is the most effective way to treat T1D. Unfortunately, there is a chronic shortage of cadaveric organ donors to treat all of the patients on the waiting list. Thus, an alternative source of insulin producing cells (IPCs) could significantly improve patient treatment. Our lab seeks to establish human induced pluripotent stem (iPS) cells as a novel source of IPCs that are patient tailored. The aim of this thesis was to 1) compare the differentiation of T1D and nondiabetic (ND) patient-derived iPS cells into IPCs, and 2) devise an effective protocol for differentiating skin fibroblast-derived T1D iPS cells into functional, glucose-responsive IPCs. Initially, T1D iPS cells were differentiated into IPCs. However, the yield was very poor. We hypothesized that epigenetic barriers were prevalent in T1D iPS cells, limiting their differentiation into IPCs. To address this problem, we utilized 5-aza-2’-deoxycytidine (5-aza-DC), a potent demethylating agent that inhibits the DNA methyltransferase (Dnmt). We reasoned that the use of a demethylation agent might induce a more labile, permissive state, allowing for greater cell responses to differentiation stimuli. Typically, after the differentiation of T1D iPS cells, several cell cluster types are obtained, namely compact cell clusters and hollow cysts. 5-aza-DC treatment appeared to convert all of the cell clusters into characteristic islet-like compact structures. In contrast, in untreated T1D IPC cultures, we observed the dominant presence of many hollow cysts with only a few tight spheroids. The hollow cysts stained negative for insulin whereas the rare solid spheroids highly expressed insulin. Flow cytometry analysis indicated a much greater percentage of Pdx1+ and insulin+ cells in 5-Aza-DC-treated cultures. These cells express markers typical of pancreatic β-cells, possessed insulin granules in similar quantities as islets, and were glucose-responsive. When transplanted in immunodeficient mice that had developed streptozotozin-induced diabetes, there was a dramatic decrease of hyperglycemia within 28 days. These mice effectively managed glucose challenge by recovering to normoglycemia, whereas nontransplanted mice did not. Altogether, our data for the first time reveal a very high yield of functional IPCs derived from human iPS cells derived from a patient with T1D, which presents a novel alternative source of IPCs that could be used to treat T1D.

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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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Espinha, Nuno Miguel Moura. "Bioprocess engineering of induced pluripotent stem cells for application in cell therapy and pre-clinical research". Master's thesis, Faculdade de Ciências e Tecnologia, 2014. http://hdl.handle.net/10362/11551.

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38

Gumede, Dimakatso B. "Investigating the role of a FAM111B mutation in hereditary fibrosing poikiloderma (POIKTMP) using induced pluripotent stem cell (iPSC) model". Doctoral thesis, Faculty of Health Sciences, 2019. http://hdl.handle.net/11427/31070.

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Hereditary fibrosing poikiloderma is an autosomal dominant disorder that is characterised by mottled pigmentation and telangiectasia, accompanied by tendon contractures, myopathy and pulmonary fibrosis (POIKTMP). Mutations in POIKTMP cases have been shown to harbour the Family with sequence similarity 111B (FAM111B) gene. However, its function is unknown. The aim of this study was to investigate the causative role of the FAM111B mutation (c.1861T>G) in the multi-systemic fibrosis affecting the South African kindred with POIKTMP. Dermal fibroblasts from two affected siblings and a familial control were reprogrammed into induced pluripotent stem cells (iPSCs) via the Sendai virus vector (SeVdp) packaged with pluripotency transgenes (OCT4; SOX2; KLF4; C-MYC). The derived iPSCs successfully showed a) endogenous expression of pluripotency markers (OCT4; NANOG; TRA-1-60), b) in vitro differentiation into the three germ layers (endoderm; mesoderm; ectoderm) and c) normal karyotyping. Next, the iPSCs from two patients, a Familial control and a Non-familial control were differentiated into mesenchymal stem/stromal cells (iPSC-MSCs) as a cell model in this study. Characterisation of derived iPSC-MSCs indicated positive expression of MSC markers (CD73; CD90; α-SMA). Differentiation of iPSC-MSCs demonstrated adequate osteogenicity but limited adipogenicity. Patient-derived iPSC-MSCs were thereafter analysed by qPCR and collagen staining to determine whether the FAM111B mutation alters endogenous expression of pro-fibrotic markers as well as collagen synthesis in patient cells compared to controls. Messenger RNA expression of pro-fibrotic markers (COL1A1; COL3A1; α-SMA) was similar between patient and control iPSC-MSCs. Collagen staining and quantification also showed no statistical differences between patient and control cells. These results suggest that FAM111B does not directly alter the expression of these profibrotic genes in this in vitro model system. Growth curves were then carried out to investigate if the FAM111B mutation modulates cell proliferation and it was found that patient cells proliferated at a higher rate compared to controls. To explore the mechanisms underlying the rate change, analyses of FAM111B expression during cell cycle progressions were conducted. Extensive optimization experiments using the double thymidine block approach were necessary to establish the appropriate synchronization protocol, keeping in mind the extended doubling time of iPSCMSCs. The results revealed that FAM111B mRNA expression was temporally regulated, with a peak at the S-phase and low at the G2/M phase. While there were no pattern differences between patient and control cells, FAM111B mRNA expression was significantly higher in the patient cells compared to controls at the G1- and S-phase. These results suggest that the mutation in FAM111B might affect the stability or perdurance of the mRNA. Unfortunately, analysis of the FAM111B protein data was inconclusive. Problems related to synchronization of the cells and the specificity of the antibody would have to be rectified in order to follow this further. The overall findings in this in vitro study reveal that the FAM111B mutation does not alter expression of pro-fibrotic markers but does affect the cell proliferation rate of patient cells compared to controls. Future work will focus on further optimisation of iPSC-MSCs synchronisation to determine correlation of FAM111B mRNA and protein expression during cell cycle progression in the patient cells. Furthermore, 3D in vitro cellular models that recapitulate some parts of the POIKTMP phenotype will need to be created. Future work will also explore the gain-of-function hypothesis to further understand the role of FAM111B in fibrosis and cancer phenotype in POIKTMP.

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Serio, Andrea. "Using induced pluripotent stem cells to model glial-neuronal interactions in TDP-43 proteinopathies". Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/9557.

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Amyotrophic Lateral Sclerosis (ALS) is an incurable late onset neurodegenerative disorder characterised by the specific loss of motor neurones (MNs). It has been recently demonstrated that Transactive response DNA-binding protein (TDP-43) is the dominant disease protein in both ALS and a sub-group of frontotemporal lobar degeneration (FTLDTDP). Moreover, the identification of TARDBP mutations in familial ALS confirms a mechanistic link between the observed mis-accumulation of TDP-43 and neurodegeneration but also provides an opportunity to establish an in vitro platform to model these diseases, based on patient-derived induced pluripotent stem cells (iPSCs). This study presents the optimization of an iPSC-based platform to study the consequences of TDP-43 M337V mutation in human functional populations of MNs and astrocytes in isolation as well as in co-culture. To develop this platform, two protocols to differentiate patient-derived iPSCs into functional MNs and astrocytes were first optimized, and the obtained cellular populations were then used to characterize the behaviour of mutant TDP-43 and its effect on the different cell types. This study show that it is possible to use iPSC-based platforms to recapitulate in vitro key aspects of TDP-43 proteinopathies such as MN cell autonomous toxicity and TDP-43 accumulation, but they can also be used to highlight previously unrecognised disease specific mechanisms and to test novel therapeutic approaches. Moreover, by performing co-culture experiments it was possible to evaluate the effects of M337V astrocytes on the survival of wild-type and M337V TDP-43 motor neurons, showing that mutant TDP-43 astrocytes do not adversely affect survival of co-cultured neurons. This iPSC-based platform represents an in vitro model to study both the effect of somatic mutations on isolated patient-specific cultures, but also to investigate cellular autonomy and neurodegeneration in the context of TDP-43 proteinopathies.

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

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Ribeiro, Fernandes Hugo José. "Elucidating the role of GBA in the pathology of Parkinson's disease using patient derived dopaminergic neurons differentiated from induced pluripotent stem cells". Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:7027574c-dda4-4752-9010-4c573bd0b2aa.

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Heterozygous mutations in the glucocerebrosidase (GBA) gene represent the most common risk factor for Parkinson’s disease (PD), a disease in which midbrain dopaminergic neurons are preferentially vulnerable. However, the mechanisms underlying this association are still unknown, mostly due to the lack of an appropriate model of study. In this thesis, we aimed at elucidating the role of heterozygous GBA mutations in PD using a specific human induced pluripotent stem cell (hiPSC)-based model of disease. First we developed a protocol for the efficient differentiation of hiPSCs into dopaminergic cultures, and extensively characterized the derived dopaminergic neurons which expressed multiple midbrain relevant markers and produced dopamine. Next we screened a clinical cohort of PD patients to identify carriers of GBA mutations of interest. Using for the first time hiPSCs generated from PD patients heterozygous for a GBA mutation (together with idiopathic cases and control individuals) we were able to efficiently derive dopaminergic cultures and identify relevant disease mechanisms. Upon differentiation into dopaminergic neuronal cultures, we observed retention of mutant glucocerebrosidase (GCase) protein in the endoplasmic reticulum (ER) with no change in protein levels, leading to upregulation of ER stress machinery and resulting in increased autophagic demand. At the lysosomal level, we found a reduction of GCase activity in dopaminergic neuronal cultures, and the enlargement of the lysosomal compartment in identified dopaminergic neurons suggesting a decreased capacity for protein clearance. Together, these perturbations of cellular homeostasis resulted in increased release of α-synuclein and could likely represent critical early cellular phenotypes of Parkinson's disease and explain the high risk of heterozygous GBA mutations for PD.

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

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Chopra, Karishma. "Improved Cryopreservation of Induced Pluripotent Stem Cells Using N-aryl Glycosidic Small Molecule Ice Recrystallization Inhibitors". Thesis, Université d'Ottawa / University of Ottawa, 2021. http://hdl.handle.net/10393/42323.

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Induced pluripotent stem cells (iPSCs) are an attractive cell source for various applications in regenerative medicine and cell-based therapies given their unique capability to differentiate into any cell type of the human body. However, human iPSCs are highly vulnerable to cryopreservation with post-thaw survival rates of 40-60%; this is due to cryoinjury resulting from ice recrystallization when using conventional slow cooling protocols. Ice recrystallization is a process where the growth of large ice crystals occurs at the expense of small ice crystals. Ice recrystallization inhibitors (IRIs) are designed to inhibit the growth of intracellular ice crystals, increasing post-thaw viability. In this study, we tested a panel of four IRIs to determine if the inhibition of ice recrystallization can decrease cellular damage during freezing and improve viability post-thaw of iPSC colonies. We supplemented commercially available and serum-free cryopreservation medium mFreSR, routinely used for the cryopreservation of iPSCs, with a class of N-aryl-D-ß-gluconamide IRIs. A 2-fold increase in post-thaw viability was observed, in a dose dependent response, for N-(4-methoxyphenyl)-D-gluconamide (PMA) at 15 mM, N-(2-fluorophenyl)-D-gluconamide (2FA) at 10 mM, and N-(4-chlorophenyl)-D-gluconamide (4ClA) at 0.5 mM over mFreSR controls. After testing the panel of four IRIs, 2FA frozen iPSCs showed an increase in cell viability, proliferation, and recovery. The addition of ROCK inhibitor (RI), commonly used to increase iPSC viability post thaw, further enhanced the survival of the iPSCs frozen in the presence of 2FA and is used routinely in research. This additive effect increased cell recovery and colony formation post thaw, resulting in increased proliferation with no adverse effects on iPSC pluripotency or differentiation capabilities. The development of improved cryopreservation strategies for iPSCs is key to establishing master clonal cell banks and limiting cell selection pressures, all while maintaining high post-thaw viability and function. This will help ensure sufficient supplies of high-quality iPSC required to meet the cell demands for cell and regenerative based therapies. Since iPSCs hold promise as a potentially unlimited cell source for a plethora of cell-based therapies, improving cryopreservation is essential to the successful deployment of iPSC-derived therapeutic cell products in the future.

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GIAGNORIO, ELEONORA. "Revealing the involvement of MALAT1, NEAT1, HOTTIP lncRNAs in Amyotrophic Lateral Sclerosis (ALS) via an induced pluripotent stem cell (iPSC)-derived muscle cell model". Doctoral thesis, Università degli Studi di Milano-Bicocca, 2022. http://hdl.handle.net/10281/385034.

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Abstract (sommario):

La SLA è una malattia neurodegenerativa caratterizzata da una progressiva degenerazione dei MN, con conseguente atrofia muscolare, paralisi e morte del paziente. Esistono due forme di SLA, la forma sporadica, nel 90% dei pazienti, e la forma familiare, nel restante 10% dei casi. Diversi geni sono associati alla SLA, come C9ORF72 che è il gene più comunemente associato alla forma familiare di SLA, seguito da TARDBP, SOD1 e FUS. Questi geni influiscono su diverse funzioni cellulari, tra cui lo stress ossidativo, il metabolismo dell’RNA, l’organizzazione del citoscheletro e l’autofagia. I geni associati alla SLA sono espressi in modo ubiquitario e quindi diversi tipi cellulari possono subire alterazioni nella struttura e metabolismo e insieme contribuiscono ai pathways degenerativi della SLA. Oltre ai MN, studi recenti dimostrano che il muscolo scheletrico è coinvolto precocemente durante la patogenesi della SLA. Ad oggi non esistono cure e uno degli obiettivi della ricerca è lo sviluppo di terapie, ottenute tramite una conoscenza specifica degli eventi molecolari che portano alla degenerazione dei MN e del tessuto muscolare. La deregolazione dell’RNA ha un contributo chiave nella patogenesi della SLA. Nel campo dell’RNA non coding, i long non coding RNA (lncRNA) emergono come contribuenti alla patofisiologia della SLA. I lncRNA, lunghi dalle 300 alle centinaia di nucleotidi, sono regolatori dell’espressione di geni muscolari e neuronali, ma il loro contributo alla patogenesi della SLA è ancora ignoto. In questo lavoro abbiamo analizzato i livelli di espressione di MALAT1, NEAT1 e HOTTIP lncRNA coinvolti nello sviluppo e omeostasi del muscolo scheletrico, nel modello di cellule pluripotente indotte umane (iPSC) derivate da pazienti SLA e controlli sani, e differenziate verso un destino miogenico tramite un protocollo basato sulle small molecules. Abbiamo analizzato l’espressione di marcatori dello sviluppo del muscolo scheletrico tramite qPCR. Inoltre, abbiamo predetto in silico e poi validato gli mRNA target dei lncRNA. Abbiamo riportato un diverso pattern di espressione dei lncRNA e target mRNA nelle colture cellulari SLA, rispetto ai controlli, in particolare allo stadio di progenitore mesodermico, miociti e miotubi. Tramite un’analisi di clustering gerarchico abbiamo identificato cluster specifici di lncRNA/geni target che caratterizzano le linee SLA, il che suggerisce che un’alterata espressione di queste molecole può contribuire alla patogenesi della malattia. Le nostre scoperte sulla deregolazione di MALAT1, NEAT1 e HOTTIP e dei loro geni target offre nuovi spunti riguardo le basi molecolari della SLA, suggerendo la possibilità che un alterato sviluppo del muscolo scheletrico, dipendente da queste molecole, possa portare a un’alterazione della massa e funzionalità muscolare durante malattia. Ulteriori studi sono necessari per indagare maggiormente l’effetto sinergico di MALAT1, NEAT1 e HOTTIP sull’insorgenza e/o progressione della malattia, con lo scopo di sviluppare strategie terapeutiche contro la SLA o altre malattie del motoneurone che siano paziente specifiche, basate sui lncRNA
Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative and fatal disease characterized by progressive cortical, bulbar and spinal motor neuron (MN) degeneration, leading to progressive muscle weakness, atrophy, paralysis and, ultimately, death. ALS can occur in two different forms: sporadic ALS (sALS) in ∼90% of individuals and familial ALS (fALS). Different genes have been associated with fALS and/or sALS; C9ORF72–SMCR8 complex subunit (C9ORF72) is the gene most commonly linked to inherited ALS, followed by TAR DNA-binding protein 43 (TARDBP), superoxide dismutase 1 (SOD1) and FUS RNA-binding protein (FUS). Such genes affect several cellular functions, including oxidative stress (SOD1), RNA metabolism (C9ORF72, TARBDP and FUS), cytoskeletal organization [e.g. tubulin alpha-4a (TUBA4A) and profilin 1 (PFN1)] and autophagy [e.g. TANK-binding kinase 1 (TBK1) and optineurin (OPTN). ALS-associated mutant genes are ubiquitously expressed, thus alterations in structure, metabolism and physiology occur in different cell types, synergistically contributing to ALS degenerative pathways. It is generally accepted that ALS is primarily caused by MN death. However, growing evidence has shown that muscle is active and plays a crucial role in the disease onset and progression. Currently, there are no effective treatments for ALS. Indeed, one of the major aims in ALS research is the development of successful therapies, by deepening the knowledge of the molecular events leading to the degeneration of both MNs and muscle tissue. It has become increasingly clear that RNA dysregulation is a key contributor to ALS pathogenesis. Among non-coding RNAs, long non-coding RNA (lncRNAs) are emerging as molecular contributors to ALS pathophysiology because of their role in regulating gene expression. LncRNAs, that are 300 to thousands nucleotides long, being more similar to mRNA than microRNAs, are key MN and muscle gene expression regulators. However, the exact contribution to ALS pathogenesis is still unknown. Here, we analysed the expression levels of MALAT1, NEAT1 and HOTTIP lncRNAs, known to be involved in the development and homeostasis of the skeletal muscle, in a human induced pluripotent stem cell (hiPSC) model differentiated towards a myogenic destiny through a small molecule-based protocol, obtained from ALS patients and healthy controls. The expression of key markers of skeletal muscle development was assessed by qPCR. Further, mRNA targets of the lncRNAs were predicted in silico, and validated by qPCR. We reported a differential lncRNA and mRNA target expression pattern in ALS-mutant cultures compared to controls, particularly at the mesodermal progenitor, early myocyte and myotube stages. Specifically, through hierarchical clustering analysis we identified specific clusters of lncRNA/target gene defining ALS cell lines, suggesting that an altered expression of these molecules might contribute to the disease pathogenesis. Our findings on dysregulation of MALAT1, NEAT1, HOTTIP and their target genes in the iPSC-based ALS in vitro model provide new insights into ALS molecular basis, pointing out the possibility that altered muscle differentiation processes, depending on these lncRNAs, could eventually lead to an altered availability of muscle mass and function in the disease. Further studies in genetically defined, or not defined, ALS patients, and in other motor neuron diseases (MNDs), could help to deeply understand the synergistic effect of MALAT1, NEAT1 and HOTTIP in disease onset and/or progression, towards future development of patient-specific lncRNA-based therapeutic strategies for ALS and other MNDs.

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46

VOLPE, CLARA. "EPIGENETIC MARKS AND PATHOLOGICAL FEATURES ASSOCIATED TO MUTANT C9ORF72 GENE IN AMYOTROPHIC LATERAL SCLEROSIS: AN IN VITRO STUDY IN PATIENT-DERIVED INDUCED PLURIPOTENT STEM CELLS AND MOTOR NEURONS". Doctoral thesis, Università degli Studi di Milano, 2020. http://hdl.handle.net/2434/704314.

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Abstract (sommario):

The expansion of the hexanucleotide repeat sequence GGGGCC (>30 repeats) in the first intron of C9ORF72 gene is the main genetic cause of two neurodegenerative diseases: amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The 5’ promoter of C9ORF72 gene has been found hypermethylated in 30% of C9ORF72 positive (C9+) ALS/FTLD patients and never in unexpanded patients and healthy controls. Promoter methylation seems to have a neuroprotective role from RNA toxicity and RAN translated dipeptide repeats (DPRs). The aim of this study has been to characterize C9+ ALS patient-derived induced pluripotent stem cells (iPSC) and differentiated motor neurons (iPSC-MN) correlating epigenetic marks of gene promoter and of the GC-rich repeat expansion (HRE) to C9ORF72-related pathological features (gene expression, RNA foci and DPRs). We initially studied 3 different C9+ iPSC lines which were cultured for up to 40 passages in vitro and, at each timepoint (10th, 20th, 30th and 40th passage), DNA was extracted for genetic and epigenetic characterization, RNA was obtained for Q-PCR analyses and slides were fixed for C9ORF72 RNA foci count. C9+ iPSCs were also differentiated into motor neurons for three times and iPSC-MNs were harvested for the same molecular characterization as for iPSCs. We observed a down-regulation of the two HRE-harboring mRNA isoforms (V1 and V3) both in iPSCs and iPSC-MNs when the promoter was methylated, while RNA foci number showed no correlation with methylation state. Moreover, we found that the epigenetic pattern of promoter methylation could change after C9+ iPSC reprogramming and through differentiation into iPSC-MNs. When we extended our analysis to a cohort of 8 different C9+ iPSC lines, we observed that both epigenetic marks and HRE length may influence RNA foci formation. Our study reports for the first time in C9+ iPSC and iPSC-MNs that promoter methylation can be considered a possible therapeutic target and corroborates that patient-derived cells represent a suitable model for further studies on C9ORF72-related neurodegeneration.

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47

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

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48

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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Abstract (sommario):

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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49

Duong, Khanh Linh. "Molecular and cellular basis of hematopoietic stem cells maintenance and differentiation". Diss., University of Iowa, 2014. https://ir.uiowa.edu/etd/1448.

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Abstract (sommario):

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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50

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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