Advancing induced pluripotent stem cell (iPSC) technology by assessing genetic instability and immune response

[eng] 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.[cat] 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 teratomas 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

Certificates of quantum many-body properties assisted by machine learning

Computationally intractable tasks are often encountered in physics and optimization. Such tasks often comprise a cost function to be optimized over a so-called feasible set, which is specified by a set of constraints. This may yield, in general, to difficult and non-convex optimization tasks. A number of standard methods are used to tackle such problems: variational approaches focus on parameterizing a subclass of solutions within the feasible set; in contrast, relaxation techniques have been proposed to approximate it from outside, thus complementing the variational approach by providing ultimate bounds to the global optimal solution. In this work, we propose a novel approach combining the power of relaxation techniques with deep reinforcement learning in order to find the best possible bounds within a limited computational budget. We illustrate the viability of the method in the context of finding the ground state energy of many-body quantum systems, a paradigmatic problem in quantum physics. We benchmark our approach against other classical optimization algorithms such as breadth-first search or Monte-Carlo, and we characterize the effect of transfer learning. We find the latter may be indicative of phase transitions, with a completely autonomous approach. Finally, we provide tools to generalize the approach to other common applications in the field of quantum information processing.Comment: 22 pages (12.5 + appendices), 8 figure

Current Challenges in the Management of Infective Endocarditis

Infective endocarditis is a relatively rare, but deadly cause of sepsis, with an overall mortality ranging from 20 to 25% in most series. Although the classic clinical classification into syndromes of acute or subacute endocarditis have not completely lost their usefulness, current clinical forms have changed according to the profound epidemiological changes observed in developed countries. In this review, we aim to address the changing epidemiology of endocarditis, several recent advances in the understanding of the pathophysiology of endocarditis and endocarditis-triggered sepsis, new useful diagnostic tools as well as current concepts in the medical and surgical management of this disease. Given its complexity, the management of infective endocarditis requires the close collaboration of multidisciplinary endocarditis teams that must decide on the diagnostic approach; the appropriate initial treatment in the critical phase; the detection of patients needing surgery and the timing of this intervention; and finally the accurate selection of patients for out-of-hospital treatment, either at home hospitalization or with oral antibiotic treatment

Understanding and Managing Sepsis in Patients With Cancer in the Era of Antimicrobial Resistance

Sepsis is a frequent complication in immunosuppressed cancer patients and hematopoietic stem cell transplant recipients that is associated with high morbidity and mortality rates. The worldwide emergence of antimicrobial resistance is of special concern in this population because any delay in starting adequate empirical antibiotic therapy can lead to poor outcomes. In this review, we aim to address: (1) the mechanisms involved in the development of sepsis and septic shock in these patients; (2) the risk factors associated with a worse prognosis; (3) the impact of adequate initial empirical antibiotic therapy given the current era of widespread antimicrobial resistance; and (4) the optimal management of sepsis, including adequate and early source control of infection, optimized antibiotic use based on the pharmacokinetic and pharmacodynamics changes in these patients, and the role of the new available antibiotics

Myogenic Precursors from iPS Cells for Skeletal Muscle Cell Replacement Therapy.

The use of adult myogenic stem cells as a cell therapy for skeletal muscle regeneration has been attempted for decades, with only moderate success. Myogenic progenitors (MP) made from induced pluripotent stem cells (iPSCs) are promising candidates for stem cell therapy to regenerate skeletal muscle since they allow allogenic transplantation, can be produced in large quantities, and, as compared to adult myoblasts, present more embryonic-like features and more proliferative capacity in vitro, which indicates a potential for more self-renewal and regenerative capacity in vivo. Different approaches have been described to make myogenic progenitors either by gene overexpression or by directed differentiation through culture conditions, and several myopathies have already been modeled using iPSC-MP. However, even though results in animal models have shown improvement from previous work with isolated adult myoblasts, major challenges regarding host response have to be addressed and clinically relevant transplantation protocols are lacking. Despite these challenges we are closer than we think to bringing iPSC-MP towards clinical use for treating human muscle disease and sporting injuries

Alzheimer's disease: oral manifestations, treatment and preventive measures.

In the treatment of patients with dementia Alzheimer's type non-current and are facing tough situations. Treatment should be tailored to each stage of the disease and for each patient. In this type of disease is very important to involve families and caregivers to improve the quality of life of patients. The main goal with these patients is prevention. Patients should be all oral manifestations caused by the lack of inadequate oral hygiene, xerostomia and manifestations derived by taking drugs. The aim of this review is to describe the main oral manifestations that can result from this disease and the best treatment options taking into account the clinical stages in which patients are found.Keywords: Alzheimer, Dementia, Oral health, Disease, Prevention.Enfermedad de Alzheimer: manifestaciones orales, tratamiento y medidas preventivas.En el tratamiento a pacientes con demencias tipo Alzheimer se afrontan situaciones infrecuentes y comprometidas. El tratamiento debe personalizarse para cada estadio de la enfermedad y para cada paciente. En este tipo de enfermedades es muy importante involucrar a los familiares y cuidadores para mejorar la calidad de vida del enfermo. El  principal objetivo con estos pacientes es la prevención. Se deben controlar todas las manifestaciones orales provocadas por la falta de una inadecuada higiene oral, la xerostomía y las manifestaciones derivadas por los fármacos que consumen. El objetivo de esta revisión es describir cuáles son las principales manifestaciones orales que pueden derivar de esta enfermedad y las mejores opciones de tratamiento teniendo en cuenta las etapas clínicas en las que se encuentran los pacientes.Palabras clave: Alzehimer, Demencia, Salud oral, Enfermedad, Prevención.

LA ECONOMÍA DEL CONOCIMIENTO EN ESPAÑA: UNA COMPARACIÓN INTERNACIONAL DE SU DESARROLLO

SIN RESUME

E-Learning and Labour Market: Wage-premium Analysis

The link between ICT and the demand for high-skilled labour is due to the fact that the introduction of digital technologies alters the skill requirements of occupations in three main ways (Spitz, 2003): 1) ICT capital substitutes repetitive manual and repetitive cognitive activities, 2) ICT capital is complementary to analytic and interactive activities, and 3) ICT capital increases the requirement for computing skills. Within this framework, we have analysed the determinants of labour productivity of individuals that have taken higher education programmes online to test how occupational skill requirements and the degree of ICT adoption by the industry matches skills of online students. In order to do this, we have assumed an implicit relationship between education and ability (Griliches and Mason, 1972), recognizing that online students may acquire specific skills, such as computing skills and abilities related to ICT use. For the empirical analysis we have used a database of degree students from the UOC (Universitat Oberta de Catalunya). The results from our model based on Mincerian equations show three important facts: 1) schooling is not a significant variable to explain wage differentials; 2) experience, understood as previous productivity and production losses avoided, is the most important variable explaining improvement of wages; and 3) ICT skills have a positive and significant effect on wage levels

E‐learning y desarrollo de competencias: la micronización de contenidos en economía y empresa

Entre las nuevas necesidades de formación y de aprendizaje surgidas de la definición de un Espacio Europeo de Enseñanza Superior (EEES) común, destaca la necesidad de profundizar en una mejor enseñanza científica y técnica, así como en el desarrollo de competencias genéricas y transversales de cara a un aprendizaje permanente a lo largo de la vida. De acuerdo con este marco general, en este artículo se define una metodología educativa adaptada a la formación virtual, y aplicada a la enseñanza y aprendizaje en el área de conocimiento de economía y empresa, que permita la integración de las principales competencias genéricas al currículum docente. Así, después de establecer en la primera parte del artículo cuales son estas competencias, que clasificamos en instrumentales, personales, sistémicas y en transferibles, se propone una metodología que permita encajarlas en cada uno de los objetivos de aprendizaje propuestos para los grados de economía y empresa. La micronización de los contenidos, es decir, la división sucesiva de contenidos en unidades menores, permitirá en la parte central del artículo definir lo que llamamos el mapa de competencias y de contenidos para la formación virtual, concretando en donde se trabaja una u otra competencia. Se definen estas unidades menores, producto de la micronización de contenidos, de forma que contemplen la posibilidad de que haya diferentes niveles de dificultad, y diferentes niveles de profundidad. Asimismo se propone que contengan ejercicios y actividades, elementos que permitan la evaluación de su aprendizaje, y la relación de contenidos relacionados. En la parte final del artículo, se desarrolla una aplicación práctica de la metodología propuesta alrededor de un objetivo de formación concreto en economía y empresa, como es el estudio de las estructuras de mercado. Esta implementación, nos ha permitido crear un ejemplo de un nuevo material de aprendizaje, identificando tanto los diferentes niveles de dificultad como de profundidad, así como aquellas competencias concretas que se desarrollan con su trabajo.One of the formation and learning priorities that has appeared from the definition of the European Space for Higher Education, is the need to study in depth a better scientific and technical education, as well as the development of generic and transversal competences based on the lifelong learning. In this article an educative methodology adapted to e‐learning is defined and applied to the area of economic and business knowledge, which allows the integration of the main generic competences to the educational curriculum. After setting these competences and its classification on the first part of the article, we propose a methodology that allows fitting each of them with the learning objectives proposed for the Economy and Business Degrees. Micronization or the successive division of contents in smaller units will allow us to define the map of competences and contents for e‐learning, indicating where each competence is developed. These small units, outcomes of the micronization, are defined in order to consider different levels of difficulty and depth. It also contains exercises and activities, elements that permit us to relate contents and evaluate its learning and performance. Finally, a practical application of the proposed methodology about a formative objective is developed, as it is the study of the market structures. This implementation has permitted us to create an example of a new learning material, identifying the different levels of difficulty and depth, as well as the development of competences

NoMeplot: analysis of DNA methylation and nucleosome occupancy at the single molecule

Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-44597-2.We are very grateful to Peter A. Jones for sharing protocols and advice and we thank Serafin Moral for constructive and useful discussion.Recent technical advances highlight that to understand mammalian development and human disease we need to consider transcriptional and epigenetic cell-to-cell differences within cell populations. This is particularly important in key areas of biomedicine like stem cell differentiation and intratumor heterogeneity. The recently developed nucleosome occupancy and methylome (NOMe) assay facilitates the simultaneous study of DNA methylation and nucleosome positioning on the same DNA strand. NOMe-treated DNA can be sequenced by sanger (NOMe-PCR) or high throughput approaches (NOMe-seq). NOMe-PCR provides information for a single locus at the single molecule while NOMe-seq delivers genome-wide data that is usually interrogated to obtain population-averaged measures. Here, we have developed a bioinformatic tool that allow us to easily obtain locus-specific information at the single molecule using genome-wide NOMe-seq datasets obtained from bulk populations. We have used NOMePlot to study mouse embryonic stem cells and found that polycomb-repressed bivalent gene promoters coexist in two different epigenetic states, as defined by the nucleosome binding pattern detected around their transcriptional start site.This study was supported by the Spanish ministry of economy and competitiveness (SAF2013-40891-R; BFU2016-75233-P) and the andalusian regional government (PC-0246-2017). David Landeira is a Ramón y Cajal researcher of the Spanish ministry of economy and competitiveness (RYC-2012- 10019)