Towards Stem Cell Treatment for Duchenne Muscular Dystrophy-Related Cardiomyopathy

Duchenne muscular dystrophy (DMD) is a progressive muscular degenerative disease affecting 1 in 3,500 boys. Cardiomyopathy is observed in 95% of patients aged 18 and older, accounting for 30% of deaths. While innovative treatments have been employed to curb symptoms in skeletal muscle, few show success in the heart, and no cure for DMD exists. Recently, regenerative therapy using cardiac stem cells (CSCs) has shown very promising results, but efficacy in DMD remains undetermined. This dissertation sought to provide preliminary insight into the effects of murine Sca-1+ CSC therapy. Three-dimensional echocardiography was validated and used to gauge cardiomyopathy in the mdx:utrn-/- mouse model. CSCs were isolated from healthy mice and displayed differentiation potential. They were implanted into mdx:utrn-/- mice under ultrasound guidance, and showed preliminary trends towards functional improvement. These results support the notion that stem cells may be an excellent avenue of therapy that should be further investigated

Aldh1 Expression and Activity Increase during Tumor Evolution in Sarcoma Cancer Stem Cell Populations

Tumors evolve from initial tumorigenic events into increasingly aggressive behaviors in a process usually driven by subpopulations of cancer stem cells (CSCs). Mesenchymal stromal/stem cells (MSCs) may act as the cell-of-origin for sarcomas, and CSCs that present MSC features have been identified in sarcomas due to their ability to grow as self-renewed floating spheres (tumorspheres). Accordingly, we previously developed sarcoma models using human MSCs transformed with relevant oncogenic events. To study the evolution/emergence of CSC subpopulations during tumor progression, we compared the tumorigenic properties of bulk adherent cultures and tumorsphere-forming subpopulations both in the sarcoma cell-of-origin models (transformed MSCs) and in their corresponding tumor xenograft-derived cells. Tumor formation assays showed that the tumorsphere cultures from xenograft-derived cells, but not from the cell-of-origin models, were enriched in CSCs, providing evidence of the emergence of bona fide CSCs subpopulations during tumor progression. Relevant CSC-related factors, such as ALDH1 and SOX2, were increasingly upregulated in CSCs during tumor progression, and importantly, the increased levels and activity of ALDH1 in these subpopulations were associated with enhanced tumorigenicity. In addition to being a CSC marker, our findings indicate that ALDH1 could also be useful for tracking the malignant potential of CSC subpopulations during sarcoma evolution.This work was supported by the Plan Nacional de I + D + i 2008-2011 [ISC III/FEDER (Miguel Servet Program CP11/00024) and RTICC (RD12/0036/0015)], the Plan Nacional de I + D + i 2013-2016 [MINECO/FEDER (SAF-2013-42946-R)] and the Plan de Ciencia Tecnología e Innovación del Principado de Asturias (GRUPIN14-003) to R.R; and the Plan Nacional de I + D + i 2008-2011 [ISC III/FEDER RTICC (RD12/0036/0027)] to J.G.-C.S

The efficacy of multiparametric prostate magnetic resonance imaging in the diagnosis and treatment of prostate cancer

Aim: To investigate the accuracy of multiparametric prostate magnetic resonance imaging (mpMRI) in determining the diagnosis and treatment options of prostate cancer (PCa), and its pathology correlation. Methods: Between October 2017 and January 2018, 73 patients were subjected to an mpMRI at our clinic. Of these patients, 11 were radical prostatectomy (RP) after treatment, and four were post- radiation therapy (RT) follow-up. The remaining 58 patients were assigned to the PSA elevation and / or positive digital rectal examination (DRE) patient group in this study and their outcomes were evaluated. Results: Of the 58 patients included in the study, 13 were found to have a PI-RADS 5 on mpMRI and in 9 (90%) of 10 patients undergoing simultaneous biopsy, PCa was detected. The biopsy results of all cases evaluated as PI-RADS 1 were benign. All of the patients who were ISUP 3 and above had a PI-RADS 5. Patients with a PI-RADS score of 4 and above being ISUP 2 and above was statistically significant (p=0.011). A case had undergone a previous radical prostatectomy assessment revealed that tPSA increased to 2 ng/ml during the follow-up, and so RT was added to the treatment; although LAP was identified in the left iliac region on an mpMRI performed upon the continued increase of tPSA. During the follow-ups of the patient who had regional RT, the tPSA dropped below 0.01 ng/ml. Conclusion: The results of our study show that mpMRI can gain a new and important place in urology due to the guidance it provides in biopsies, facilitating targeted biopsy, its effectiveness in determining treatment modalities and its importance in post-PCa treatment follow-ups

Influence of the Metastatic Microenvironment on Stem-like Human Breast Cancer Cells

Ninety percent of breast cancer-related mortalities result from metastasis. We have previously demonstrated that stem-like ALDHhiCD44+ breast cancer cells are critical for metastasis, and preferentially target the lung and bone marrow (BM). We hypothesize organ tropism occurs through promotion of the ALDHhiCD44+ phenotype. Using a 2D ex vivo model, lung and BM conditioned media (CM) were utilized to assess their influence on stem-like phenotype and behavior. Exposure of human breast cancer cells to lung-CM significantly decreased the proportion of cells with a stem-like ALDHhiCD44+ phenotype, decreased expression of cancer stem cell (CSC)-related genes, and increased gene expression related to migration (

Artificial Intelligence Based Deep Bayesian Neural Network (DBNN) Toward Personalized Treatment of Leukemia with Stem Cells

The dynamic development of computer and software technology in recent years was accompanied by the expansion and widespread implementation of artificial intelligence (AI) based methods in many aspects of human life. A prominent field where rapid progress was observed are high‐throughput methods in biology that generate big amounts of data that need to be processed and analyzed. Therefore, AI methods are more and more applied in the biomedical field, among others for RNA‐protein binding sites prediction, DNA sequence function prediction, protein‐protein interaction prediction, or biomedical image classification. Stem cells are widely used in biomedical research, e.g., leukemia or other disease studies. Our proposed approach of Deep Bayesian Neural Network (DBNN) for the personalized treatment of leukemia cancer has shown a significant tested accuracy for the model. DBNNs used in this study was able to classify images with accuracy exceeding 98.73%. This study depicts that the DBNN can classify cell cultures only based on unstained light microscope images which allow their further use. Therefore, building a bayesian‐based model to great help during commercial cell culturing, and possibly a first step in the process of creating an automated/semiautomated neural network‐based model for classification of good and bad quality cultures when images of such will be available

Automatic Spine Curvature Estimation from X-ray Images of a Mouse Model

Automatic segmentation and quantification of skeletal structures has a variety of applications for biological research. Although solutions for good quality X-ray images of human skeletal structures are in existence in recent years, automatic solutions working on poor quality X-ray images of mice are rare. This paper proposes a fully automatic solution for spine segmentation and curvature quantification from X-ray images of mice. The proposed solution consists of three stages, namely preparation of the region of interest, spine segmentation, and spine curvature quantification, aiming to overcome technical difficulties in processing the X-ray images. We examined six different automatic measurements for quantifying the spine curvature through tests on a sample data set of 100 images. The experimental results show that some of the automatic measures are very close to and consistent with the best manual measurement results by annotators. The test results also demonstrate the effectiveness of the curvature quantification produced by the proposed solution in distinguishing abnormally shaped spines from the normal ones with accuracy up to 98.6%

Deriving an in vitro source of canine corneal stromal cells for future studies of corneal disease and therapeutic applications

The cornea is the transparent tissue located at the front of the eye, that transmits and refracts light onto the retina. Despite great advances in corneal stem cell biology in human and laboratory animal research, no information is available in dogs. Corneal pathology, as corneal crystalline dystrophy has a prevalence of up to 15% and has been described in eight different canine breeds. Cholesterol and phospholipids are deposited in the stroma, similar to Schnyder’s dystrophy (SCD) in humans. Chronic corneal fibrosis is one of the leading causes of visual impairment in veterinary ophthalmology. Similar to the situation in human ophthalmology, there is a shortage of corneal donor tissue. Therefore, the overall aim was first to investigate whether corneal stromal stem cells exist in the canine cornea., The second aim was to determine the potential of deriving an in vitro source of corneal stroma cells from corneal stromal stem cells, adipose derived mesenchymal stromal cells (adMSC) and canine induced pluripotent stem cells (ciPSC), to provide a resource for studies investigating the pathogenesis of inherited stromal dystrophies, and for the development of novel cell-based therapies for dogs. First, a canine corneal stromal cell (CSC) population was characterised that demonstrated mesenchymal stromal cell properties, they differentiated into keratocyte-like cells (KDCs) in vitro and appeared to be immune privileged. Second, canine adMSC were differentiated into KDCs, but expressed high levels of a myofibroblastic marker, similar to those found in fibrotic tissue. Third, a modified protocol was established whereby ciPSCs were induced into neural crest (stem) cell lineages and then into KDCs. This led to the successful expression of some keratocyte associated markers in absence of a myofibroblastic expression. Taken together, a novel cell population was characterised in the canine corneal stroma. The differentiation protocols of adMSC and ciPSC led to preliminary results and built a basic foundation for future studies

Advanced Algorithms for 3D Medical Image Data Fusion in Specific Medical Problems

Fúze obrazu je dnes jednou z nejběžnějších avšak stále velmi diskutovanou oblastí v lékařském zobrazování a hraje důležitou roli ve všech oblastech lékařské péče jako je diagnóza, léčba a chirurgie. V této dizertační práci jsou představeny tři projekty, které jsou velmi úzce spojeny s oblastí fúze medicínských dat. První projekt pojednává o 3D CT subtrakční angiografii dolních končetin. V práci je využito kombinace kontrastních a nekontrastních dat pro získání kompletního cévního stromu. Druhý projekt se zabývá fúzí DTI a T1 váhovaných MRI dat mozku. Cílem tohoto projektu je zkombinovat stukturální a funkční informace, které umožňují zlepšit znalosti konektivity v mozkové tkáni. Třetí projekt se zabývá metastázemi v CT časových datech páteře. Tento projekt je zaměřen na studium vývoje metastáz uvnitř obratlů ve fúzované časové řadě snímků. Tato dizertační práce představuje novou metodologii pro klasifikaci těchto metastáz. Všechny projekty zmíněné v této dizertační práci byly řešeny v rámci pracovní skupiny zabývající se analýzou lékařských dat, kterou vedl pan Prof. Jiří Jan. Tato dizertační práce obsahuje registrační část prvního a klasifikační část třetího projektu. Druhý projekt je představen kompletně. Další část prvního a třetího projektu, obsahující specifické předzpracování dat, jsou obsaženy v disertační práci mého kolegy Ing. Romana Petera.Image fusion is one of today´s most common and still challenging tasks in medical imaging and it plays crucial role in all areas of medical care such as diagnosis, treatment and surgery. Three projects crucially dependent on image fusion are introduced in this thesis. The first project deals with the 3D CT subtraction angiography of lower limbs. It combines pre-contrast and contrast enhanced data to extract the blood vessel tree. The second project fuses the DTI and T1-weighted MRI brain data. The aim of this project is to combine the brain structural and functional information that purvey improved knowledge about intrinsic brain connectivity. The third project deals with the time series of CT spine data where the metastases occur. In this project the progression of metastases within the vertebrae is studied based on fusion of the successive elements of the image series. This thesis introduces new methodology of classifying metastatic tissue. All the projects mentioned in this thesis have been solved by the medical image analysis group led by Prof. Jiří Jan. This dissertation concerns primarily the registration part of the first project and the classification part of the third project. The second project is described completely. The other parts of the first and third project, including the specific preprocessing of the data, are introduced in detail in the dissertation thesis of my colleague Roman Peter, M.Sc.

Development, characterization and evaluation of advanced therapies for the treatment of cardiac pathologies

Cardiovascular diseases (CVDs) are the leading cause of disease burden and mortality in the world, as well as a major cause of disability and health care costs. With the average lifespan of the human population continuously increasing, it is expected that the problem of CVDs will only continue to grow in the following years. Current pharmacological treatments for age-associated cardiac pathologies such as heart failure and atrial fibrillation present severe clinical efficacy and safety problems and are not regarded as definitive cures. This makes it necessary to develop new treatment strategies that target the involved molecular pathways and trigger endogenous reparative responses. Contrary to current molecular treatments, advanced therapy medicinal products (ATMPs) such as stem cells, extracellular vesicles (EVs) and biomaterials such as hydrogels could have the potential to treat cardiac aging-associated pathologies from a more fundamental level. However, many problems and unknowns still need to be solved before they can reach the clinical scenario. Some of the most highlighted limitations we focus on in this work are: (i) the lack of deep understanding of their mechanism of action (MoA), (ii) their large variability and lack of standardization (including inadequate potency tests) and (iii) low in vivo retention at the site of interest. Therefore, the main objective of this thesis is to develop, characterize and evaluate advanced therapies for the treatment of cardiac pathologies solving some of their current limitations to enhance their therapeutic potential. To achieve this aim, we first focus on improving standardization and development of potency assays. We describe the main characteristics and challenges for a cell therapy based potency test in the cardiovascular field and we review and propose different types of assays that could be taken into consideration based on the product’s expected MoA and the target cardiovascular disease. Secondly, as cardiosphere-derived cells (CDCs) and their secreted EVs (CDC-EVs) have previously reported to have anti-senescent effects and this is considered important in aging-related cardiac diseases, we explore potential predictors of rejuvenating potency with a special focus on the chronological age of the CDC-donors and CDC-senescence, among others. Multiple in vitro tests allow us to conclude that more than cell particular biological markers or characteristics, the cell bioactivity relative to the expected MoA should be a better predictor for the ATMP potency. Thus, we evaluate if the in vitro anti-senescent and pro-angiogenic effect of the CDC-EVs, scored with a matrix assay, can be used to predict the in vivo potency of the CDC-EVs in an animal model of cardiac aging. Our results show that EVs classified in vitro as potent with the matrix assay have more cardiac reparative potential in vivo than EVs classified as non-potent. After further validation, the matrix assay proposed here could be a suitable in vitro potency test for discerning suitable allogenic biological products in the cardiac aging clinical scenario. Next, with the purpose of improving EV retention at the site of interest, we develop an optimized product combining hydrogels from cardiac extracellular matrix (cECM), polyethylene glycol and EVs to overcome some of their individual limitations: long gelation time of the cECM and poor retention of the EVs. We conclude that the combined product rapidly gels at physiological temperature and presents improved mechanical properties while maintaining the injectability, the biodegradability, and the bioactivity of its individual components. In addition, it serves to better retain the EVs on-site in vivo. Finally, we explore the electrophysiological modifications induced by CDC-EVs on arrhythmogenic tissue to better understand the mechanisms behind their antiarrhythmic effect. We found that CDC-EVs reduce spontaneous activation complexity and increase conduction velocity of cardiomyocytes leading to a less arrhythmogenic profile. If validated in other cellular models, CDC-EVs may be used specifically as antiarrhythmic agents in a wide range of cardiac pathologies. Although future work should aim to further validate these results both at preclinical and clinical level, these findings together partially overcome some of the main challenges for the therapeutic use of cellular therapies and open a new horizon for the treatment of cardiac-aging related pathologies, some still considered as unmet medical needs.Programa de Doctorado en Ciencia y Tecnología Biomédica por la Universidad Carlos III de MadridPresidenta: Eva Delpón Mosquera.- Secretaria: Marta García Díez.- Vocal: Javier Bermejo Thoma