La sclérose latérale amyotrophique (SLA) est une maladie neurodégénérative incurable de l’adulte qui affecte principalement les motoneurones. Les mécanismes conduisant à la mort des motoneurones restent mal connus, notamment du fait de l'hétérogénéité de la maladie et du manque d'accès aux neurones humains affectés. La technologie des cellules souches pluripotentes induites humaines (iPSc) est un outil prometteur pour la modélisation de la SLA, car elle offre la possibilité unique d'obtenir et d’étudier des motoneurones humains.Des clones d’iPSc de deux sujets témoins ont été générés et nous avons comparé plusieurs protocoles afin de mettre au point un protocole efficace de différenciation des iPSc en motoneurones. Les cultures obtenues étaient hétérogènes et contenaient différents types de neurones et des précurseurs neuraux. Afin de pouvoir étudier des mécanismes intrinsèques aux motoneurones dans la SLA, nous avons développé une nouvelle technique pour purifier les motoneurones. Cette technique a consisté à trier les motoneurones par FACS en combinant l'utilisation d'un vecteur lentiviral rapporteur exprimant une protéine fluorescente sous le contrôle d'un promoteur spécifique des motoneurones, et d'un anticorps monoclonal dirigé contre le récepteur aux neurotrophines p75. Cette double sélection a permis l'isolement efficace de motoneurones purs. En parallèle, la technologie iPSc a été utilisée pour établir des modèles cellulaires de la SLA. Des clones de cellules iPS ont été générés à partir d’un patient avec une forme familiale de la SLA présentant une mutation dans le gène TARDBP (codant pour une protéine de liaison à l’ADN, TDP-43) et un patient atteint d’une forme sporadique de SLA. Afin de valider nos modèles, nous avons recherché des phénotypes caractéristiques de la maladie au cours de la différenciation des iPSc : i) la formation d’agrégats cytoplasmiques, ii) des altérations de génération et de survie des motoneurones, iii) des défauts de croissance neuritique. / Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disorder primarily affecting motor neurons. Mechanisms leading to motor neuron death in ALS are poorly understood mostly because of disease heterogeneity and lack of access to affected cells. The induced pluripotent stem cell (iPSc) technology provides the opportunity to obtain and study human motor neurons and is therefore a promising tool for ALS modeling.IPSc clones from control subjects were generated, and we compared several protocols in order to set up an efficient protocol for iPSc differentiation into motor neurons. The obtained cultures were heterogenous, comprising different neuron subtypes and neural precursors. To allow investigation of intrinsic disease mechanisms in ALS motor neurons, we developed a new technique to purify motor neurons by FACS sorting. By combining the use of a lentiviral vector expressing a fluorescent protein under control of a motoneuron-specific promoter and of a monoclonal antibody directed against the p75 neurotrophin receptor, isolation of exquisitely pure motor neurons was achieved. In parallel, iPSc technology was used to establish cellular models of ALS. IPSc were generated from one patient with familial ALS carrying a mutation in the TARDBP gene (encoding a DNA-binding protein, TDP-43) and one patient with sporadic ALS. To validate our models, we investigated characteristic disease phenotypes during iPSc differentiation, including i) cytoplasmic aggregate formation, ii) motor neuron generation and survival defects, iii) neurite growth alterations.

Protein misfolding and aggregation underlie several neurodegenerative proteinopathies including amyotrophic lateral sclerosis (ALS). Superoxide dismutase 1 (SOD1) was the first gene found to be associated with familial ALS. Overexpression of human mutant or wild type SOD1 in transgenic mouse models induces motor neuron (MN) degeneration and an ALS-like phenotype. SOD1 mutations, leading to the destabilization of the SOD1 protein is associated with ALS pathogenesis. However, how misfolded SOD1 toxicity specifically affects human MNs is not clear. The aim of this thesis was to develop patient-derived, cellular models of ALS to help understand the pathogenic mechanisms underlying SOD1. To understand which cellular pathways impact on the level of misfolded SOD1 in human cells, we established a model using patient-derived fibroblasts and quantified misfolded SOD1 in relation to disturbances in several ALS-related cellular pathways. Misfolded SOD1 levels did not change following reduction in autophagy, inhibition of the mitochondrial respiratory chain, or induction of endoplasmic reticulum (ER)-stress. However, inhibition of the ubiquitin-proteasome system (UPS) lead to a dramatic increase in misfolded SOD1 levels. Hence, an age-related decline in proteasome activity might underlie the late-life onset that is typically seen in SOD1 ALS. To address whether or not SOD1 misfolding is enhanced in human MNs, we used mixed MN/astrocyte cultures (MNCs) generated in vitro from patient-specific induced pluripotent stem cells (iPSCs). Levels of soluble misfolded SOD1 were increased in MNCs as well as in pure iPSC-derived astrocytes compared to other cell types, including sensory neuron cultures. Interestingly, this was the case for both mutant and wild type human SOD1, although the increase was enhanced in SOD1 FALS MNCs. Misfolded SOD1 was also found to exist in the same form as in mouse SOD1 overexpression models and was identified as a substrate for 20S proteasome degradation. Hence, the vulnerability of motor areas to ALS could be explained by increased SOD1 misfolding, specifically in MNs and astrocytes. To investigate factors that might promote SOD1 misfolding, we focussed on the stability of SOD1 mediated by a crucial, stabilizing C57-C146 disulphide bond and its redox status. Formation of disulphide bond is dependent on oxidation by O2 and catalysed by CCS. To investigate whether low O2 tension affects the stability of SOD1 in vitro we cultured fibroblasts and iPSC-derived MNCs under different oxygen tensions. Low oxygen tension promoted disulphide-reduction, SOD1 misfolding and aggregation. This response was much greater in MNCs compared to fibroblasts, suggesting that MNs may be especially sensitive to low oxygen tension and areas with low oxygen supply could serve as foci for ALS initiation. SOD1 truncation mutations often lack C146, and cannot adopt a native fold and are rapidly degraded. We characterized soluble misfolded and aggregated SOD1 in patient-derived cells carrying a novel SOD1 D96Mfs*8 mutation as well as in cells fom an unaffected mutation carrier. The truncated protein has a C-terminal fusion of seven non-native amino acids and was found to be extremely prone to aggregation in vitro. Since not all mutation carriers develop ALS, our results suggested this novel mutation is associated with reduced penetrance. In summary, patient derived cells are useful models to study factors affecting SOD1 misfolded and aggregation. We show for the first time that misfolding of a disordered and disease associated protein is enhanced in disease-related cell types. Showing that misfolded SOD1 exists in human cells in the same form as in transgenic mouse models strengthens the translatability of results obtained in the two species. Our results demonstrate disulphide-reduction and misfolding/aggregation of SOD1 and suggest that 20S proteasome could be an important therapeutic target for early stages of disease. This model provides a great opportunity to study pathogenic mechanisms of both familial and sporadic ALS in patient-derived models of ALS. / Varje år insjuknar omkring 5300 personer i världen i motorneuronsjukdomen Amyotrofisk lateralskleros (ALS). Sjukdomen kännetecknas av degeneration av motorneuron i hjärnan och ryggmärgen, de nervceller som styr kroppens muskler, vilket leder till musklerförtvining och gradvis förlamning. ALS-patienter avlider oftast till följd av andningssvikt när sjukdomen når andningsmuskulaturen. I de allra flesta fall uppkommer ALS sporadiskt (SALS), det vill säga utan känd genetisk orsak, medan ärftliga fall (FALS) drabbar omkring 10 % och beror på mutationer i ett antal kända gener. Upp till 6 % av alla ALS fall kan härledas till mutationer i genen superoxid dismutas 1 (SOD1). SOD1 är ett enzym som ansvarar för att omvandla och oskadliggöra fria syreradikaler som bildas vid normal ämnesomsättning. 206 olika SOD1 mutationer har identifierats, alla orsakar inte ALS men många leder till att den tredimensionella proteinstrukturen förändras, vilket ökar proteinets benägenhet att felveckas. Initialt trodde man att SOD1 mutationer förhindrade proteinets normalfunktion och följaktligen orsakade ALS. Studier har emellertid visat att den enzymatiska funktionen ofta bevaras, även hos muterade proteiner. Däremot kan små mängder felveckat SOD1 störa andra viktiga cellulära funktioner. Felveckat SOD1 har en benägenhet att klumpa ihop sig och bilda aggregat i det centrala nervsystemet (CNS). Dessa aggregat återfinns hos patienter med såväl FALS som SALS vilket tyder på att även vildtyps-SOD1 kan felveckas och vara involverat i sjukdomsutvecklingen. De flesta studier är baserade på transgena musmodeller som uttrycker extremt stora mängder av muterat humant SOD1. Det är dock oklart hur väl studier i möss överensstämmer med sjukdomsutvecklingen hos ALS-patienter, där mängden SOD1 är betydligt lägre. En central fråga som fortfarande står obesvarad är varför just motorneuron degenererar i ALS, trots att SOD1 uttrycks i alla kroppens celler. Det övergripande syftet med den här avhandlingen har varit att karakterisera felveckat SOD1 i patientceller för att studera dess roll i ALSrelaterade sjukdomsmekanismer med fysiologiskt relevanta nivåer av SOD1. Samtliga studier är gjorda in vitro med celler från friska donatorer med vildtyps-SOD1, celler från patienter med SOD1-FALS, FALS som bär andra ALS-associerade gener, samt SALS. I de allra flesta fallen har vi analyserat både lösligt felveckat SOD1 samt aggregerade former av SOD1 proteinet.

Les dystrophies rétiniennes héréditaires (DRH) sont des maladies qui conduisent à une perte de la vision au cours de leur évolution. Les premiers essais cliniques utilisant la thérapie génique pour traiter ces maladies ont été réalisés et apportent des résultats encourageants. En amont de telles études, les essais précliniques s'effectuent le plus souvent sur modèle animal. Cependant, pour un certain nombre de DRH, il n'existe pas de modèle animal approprié ce qui compromet l'arrivée d'un traitement à un stade clinique. C'est le cas de la Choroïdérémie, qui représente 2% des DRH. La choroïdérémie est caractérisée par une perte de la vision nocturne dès la petite enfance et conduit à la cécité autour des 40-50 ans. Son diagnostic précoce et son évolution lente résultent en une grande fenêtre thérapeutique qui fait de la choroïdérémie une bonne candidate pour la thérapie génique. Sur le plan génétique, la maladie est causée par une mutation dans le gène CHM qui est localisé sur le chromosome X et code pour la Rab Escort Protein 1 (REP1). Cette protéine est impliquée dans le processus de prénylation de petites protéines GTPases, les protéines Rab. Afin de pallier au manque de modèle animal, nous avons généré au cours de ce travail de thèse, un modèle cellulaire humain de la choroïdérémie pour évaluer l'efficacité d'un protocole de thérapie génique sur le tissu réellement atteint in vivo. Pour cela, nous avons reprogrammé des fibroblastes de patient CHM-/y en cellules souches pluripotentes induites (iPS), que nous avons ensuite différenciées en Epithélium Pigmentaire Rétinien (EPR). Nous avons caractérisé cet EPR, montrant que c'est une couche monocellulaire polarisée possédant une morphologie et une expression de marqueurs caractéristiques. De plus, ce tissu est fonctionnel, sur le plan du transport de fluide et de la phagocytose, et possède le même phénotype biochimique que celui observé chez les patients. Dans un but de thérapie génique et afin d'évaluer le vecteur viral le plus efficace sur nos cellules, j'ai testé un panel de 5 sérotypes d'AAV et démontré que l'AAV2/5 est le plus efficient pour transduire un EPR dérivé de cellules iPS humaines. J'ai ensuite utilisé un AAV2/5-CAG-CHM afin d'évaluer l'efficacité fonctionnelle du vecteur et j'ai pu montrer qu'outre une expression correcte du transgène, le traitement de cellules de patients déficientes pour REP1 avec ce vecteur permet de restaurer une activité normale de prénylation. Nous avons donc démontré la supériorité d'efficacité de transduction de l'AAV2/5 dans des cellules d'EPR humain et soulignons le potentiel d'un modèle d'EPR pathologique dérivé de cellules iPS pour apporter une preuve de concept de thérapie génique en absence d'un modèle animal approprié. / Inherited retinal dystrophies (IRDs) lead to a progressive vision loss. The first clinical trials using gene transfer to treat such diseases have been performed with positive results. Prior to clinical trials, preclinical studies are usually performed on animal models. However, for many IRDs, appropriate animal models do not exist, which compromises their progress towards a clinical trial. An example of an IRD that lacks an appropriate model is choroideremia, which represents 2% of IRD patients. It is characterized by night blindness in childhood, followed by progressive loss of the visual field resulting in blindness by 40–50 years of age. Its early diagnosis and slow evolution result in a large therapeutic window making choroideremia a good candidate for gene therapy. Genetically, the disease is caused by a mutation in the CHM gene located on the X chromosome and encoding the Rab Escort Protein 1 (REP1). This protein is involved in the prenylation of small GTPases, the Rab proteins. To palliate the lack of an animal model, we generated a human cellular model of choroideremia in order to evaluate the efficacy of a gene therapy approach in the tissue that is affected in vivo.Towards this aim, we reprogrammed REP1-deficient fibroblasts from a CHM-/y patient into induced pluripotent stem cells (iPScs), which we differentiated into retinal pigment epithelium (RPE). We characterized the iPSc-derived RPE that is a polarized monolayer with a classic morphology, expresses characteristic markers, is functional for fluid transport and phagocytosis, and mimics the biochemical phenotype of patients. In terms of gene therapy and to evaluate the most efficient viral vector, I assayed a panel of 5 adeno-associated virus (AAV) vector serotypes and showed that AAV2/5 is the most efficient at transduce the iPSc-derived RPE. I then transduced the iPSc-derived RPE of a choroideremia patient with an AAV2/5-CAG-CHM and demonstrated that this vector is able to restore a normal prenylation function to the cells.To conclude, I demonstrated the superiority of the transduction efficiency of AAV2/5 in the iPSc-derived RPE and highlight the potential of a diseased RPE model derived from iPS cells to provide a proof of concept of gene therapy in the absence of a suitable animal model.

The aim of this dissertation is an analysis of the regulatory framework for the procurement and distribution of stem cells in South Africa. This research includes aspects of the law of obligations, medical law and human rights law as found in the Bill of Rights. More specifically however, this dissertation attempts to bring to attention the shortcomings of chapter 8 of the National Health Act. An examination is undertaken according to the multilayered approach and therefore the proposed regulatory framework is examined within a constitutional framework, an ethical framework, the framework as established by common law, in this case the doctrine of informed consent and lastly within the national legislation framework as found in the National Health Act of 2003 and the regulations made in terms of the Act. This dissertation further entails a brief comparative study of the regulatory mechanisms of the United Kingdom as entrenched in the Human Fertilisation and Embryology Act of 2008 and the Human Tissue Act of 2004 and as practiced by the Human Fertilisation and Embryology Authority and the Human Tissue Authority. The analysis in this dissertation firstly provides an overview of the clinical manifestations and science of stem cell technology. Secondly, the impact of the Constitution of the Republic of South Africa is discussed with particular reference to the Bill of Rights on stem cell research and therapy. The most noteworthy conclusion to be made in this regard is that the embryo is not the bearer of constitutional rights. The ethical guidelines which act as regulatory tools in this field are then discussed with attention to general ethical principles as provided for by the Health Professions Council of South Africa as well as the Medical research Council. The doctrine of informed consent further enjoys attention as it is discussed in context of medical research and key issues are addressed regarding the process of obtaining consent in context of stem cell technologies. Certain recommendations are then made pertaining to the minimum scope required for lawful consent. Lastly a critical analysis is made of chapter 8 of the National Health Act. The findings which are made here lead to further recommendations regarding the regulation of stem cells. / Dissertation (LLM)--University of Pretoria, 2011. / Public Law / unrestricted

Les réactions immunitaires de la peau sont initiées par les cellules dendritiques cutanées (dendritic cells, DCs). L'effet potentiellement sensibilisateur d'un composé peut être prédit in vitro en utilisant des monocytes humains différenciés en DCs (MonoDCs). Cependant, ces modèles simplistes restent imprécis car l'activation des DCs cutanés par les sensibilisateurs peut être déclenchée ou modulée par des interactions microenvironnementales avec de multiples types de cellules non immunitaires. Notre objectif est de développer une peau immunocompétente qui combinera des MonoDCs avec tous les éléments structurels et fonctionnels de la peau, c'est-à-dire une barrière épidermique posée sur un derme contenant une pseudo-vascularisation et des neurones nociceptifs. Une matrice de collagène a été ensemencée avec des fibroblastes et des cellules endothéliales, puis avec des précurseurs de fibres nerveuses dérivées soit de l'iPSC humaine, soit de la DRG embryonnaire murins. Enfin, nous avons introduit les MonoDC et les kératinocytes. Nous avons observé que les neurones différenciés in situ innervent l'épiderme comme observé habituellement dans la peau humaine normale. De plus, les neurones dérivées d’iPSCs, expriment neuropeptides et canaux calcique spécifiques des fibres nociceptives. Enfin, les Mono-DC intégrés au modèle restent stable pendant toute la durée nécessaire à la formation de l’épiderme et peuvent être stimulé. Le modèle sera utilisé pour prédire le potentiel irritant des composés chimiques et l'impact de l’innervation nociceptive sur l'activation des DCs. / Immune reactions in the skin are initiated by the cutaneous dendritic cells (DCs). The potential sensitizing effect of a compound can be predicted in vitro using human monocytes differentiated into DCs (Mono-DCs). However, these simplistic models remain inaccurate because the activation of cutaneous DCs by sensitizers may be triggered or modulated by microenvironmental interactions with multiple types of non-immune cells. Our goal is to develop an immunocompetent human tissue-engineered skin that will combine DCs with all structural and functional element of the skin, i.e. an epidermal barrier laid upon a dermis containing a pseudo-vascularization and nociceptive neurons. Collagen matrix was seeded with fibroblasts and endothelial cells, then with precursors of nerve fibers derived from either human iPSC or murine embryonic DRG. Finally, we introduced Mono-DCs and keratinocytes. We observed that in situ differentiated neurons grow axons towards the epidermis as usually observed in normal human skin. What's more, the neurons derive from iPSC, express neuropeptides and calcium channel as normal nociceptive fibers. Moreover, Mono-DCs settled as expected beneath the epidermis and remained sessile to stimulation for several weeks. The model will be used to predict the irritant potential of chemical compounds, and the impact of nerves on DC activation.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22379号 / 医科博第109号 / 新制||医科||7(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 遊佐 宏介, 教授 武田 俊一, 教授 近藤 玄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Amyotrophe Lateralsklerose (ALS) ist eine neurodegenerative Erkrankung, bei welcher speziell erste (kortikospinal) und zweite (spinal) Motorneurone (MN) von Neurodegeneration betroffen sind. Gegenwärtig bleibt ALS eine unheilbare Erkrankung. Der Tod tritt durchschnittlich 2 bis 5 Jahre nach Auftreten der Symptome ein. Circa 90% der Fälle treten sporadisch auf (sALS), während 10% familiär sind (fALS). Es ist von großem Interesse monogenetische Formen der fALS zu untersuchen um zugrundeliegende Pathologien und Mechanismen zu verstehen. Bislang wurden über 20 Gene mit ALS in Verbindung gebracht, einschließlich Fused in sarcoma (FUS) und Chromsosome 9 open reading frame (C9ORF72). Circa 4% der fALS Fälle sind durch dominante Mutationen in FUS verursacht und repräsentieren damit die dritthäufigste Form der fALS in Deutschland. Die G4C2 hexanucleotide repeat expansion (HRE) in C9ORF72 ist die häufigste Ursache für ALS und Frontotemporale Demenz (FTD). ALS Patienten unterscheiden sich erheblich in der Präsentation ihrer klinischen Symptome wie Ausbruchsort, Progressionsrate und Auftreten kognitiver Störungen. Diese Faktoren sind auch stark abhängig von der zugrundeliegenden Mutation in fALS. Ziel dieser Doktorarbeit ist die Modellierung von FUS- und C9ORF72-assozierter ALS in einem krankheits-relevanten in vitro Model von speziell kortikaler Neuropathologie mit Hilfe von Patienten-spezifischen iPSZs. Die Hypothese der vorliegenden Arbeit ist das in einer Zelltyp-abhängigen Art und Weise zugrundeliegende Erkrankungsmechanismen in kortikalen vs. spinalen Neuronen unterschiedlich betroffen sind. Humane iPSZ, generiert von gesunden Kontrollen und ALS Patienten mit FUS oder C9ORF72 Mutation, wurden für die gerichtete kortikale und spinale Differenzierung genutzt. Zusätzlich wurden zwei neue FUS-WT- und FUS-P525L-EGFP-markierte isogene Linien mittels CRISPR/Cas9n Technik generiert. Methoden basierend auf Immunfluoreszenz Färbungen und Lebendzell-Mikroskopie wurden angewendet um Krankheits-relevante Proteine, DNA Schäden und axonale Organell-Mobilität zu analysieren. In diesem Projekt konnte ein deutlicher Zelltyp-abhängiger Effekt auf analysierte Phänotypen beobachtet werden, während ALS-assoziierte Mutationen scheinbar nur geringfügige Effekte zeigten. Dementsprechend wurde ein Zelltyp-abhängiger Anstieg des basalen DNA Schadens in kortikalen Astrozyten vs. Neuronen und spinalen vs. kortikalen Neuronen detektiert. Jedoch konnte in FUS oder C9ORF72 mutierten kortikalen Zellen kein erhöhter DNA Schaden nachgewiesen werden, wie es zuvor in spinalen MN beobachtet wurde. Des Weiteren beeinflussen FUS Mutationen die Rekrutierung von FUS zu DNA-geschädigten Stellen, die Organell-Mobilität und die zytoplasmatische Fehllokalisation des Proteins in Abhängigkeit vom Zelltyp. In kortikalen Neuronen wurde in Bezug auf die Rekrutierung von mutiertem FUS und Organell-Mobilität nur leichte Mutations-abhängige und wesentlich schwächer ausgeprägte Effekte beobachtet als in spinalen MN. Zusammenfassend kann gesagt werden, dass Patienten-spezifische Zellmodelle ein wichtiges Instrument in der ALS Forschung sind und das vor allem Unterschiede zwischen kortikalen und spinalen MN weiter untersucht werden müssen, um zugrundeliegende Krankheits-relevante Mechanismen zu entschlüsseln und wie diese zum Fortschreiten der Erkrankung beitragen / Amyotrophic lateral sclerosis (ALS) is a of neurodegenerative diseases, in which neurodegeneration specifically affects upper (corticospinal) and lower (spinal) motor neurons (MNs). At present, ALS remains an incurable disease. Death occurs on average 2 to 5 years after symptom onset. About 90% are sporadic cases (sALS) and 10% are familial cases (fALS). It is of great interest to investigate monogenetic forms causing fALS to understand its underlying disease pathologies and mechanisms. Over 20 genes have been linked to ALS until now, including Fused in sarcoma (FUS) and Chromosome 9 open reading frame 72 (C9ORF72). About 4% of fALS cases are caused by dominant mutations within FUS, representing the third most common fALS form in Germany. The G4C2 hexanucleotide repeat expansion (HRE) in the C9ORF72 gene is the most common cause for ALS and Frontotemporal dementia (FTD). ALS patients differ significantly in their presentation of clinical symptoms, including site of onset, rate of progression, and presence of cognitive dysfunction. Those factors were also shown to highly depend on the underlying mutation in fALS cases. Aim of this thesis work is the modeling of FUS- and C9ORF72-associated ALS in a disease-related in vitro model of particularly cortical neuropathology using patient-derived iPSCs. The hypothesis of the current work is that underlying disease mechanisms do differentially affect cortical vs. spinal neurons and act in a cell type-dependent manner. Human iPSCs derived from healthy controls and ALS patients carrying mutations within FUS or C9ORF72 were used for directed cortical and spinal differentiation. Additionally, two new FUS-WT- and FUS-P525L-EGFP-tagged isogenic iPSC lines were generated by CRISPR/Cas9n gene editing. Immunofluorescence staining and live cell imaging approaches were implemented to analyze disease-associated proteins, DNA damage, and axonal trafficking. Within this project, a clear cell type-dependent effect on analyzed phenotypes was observed, while ALS-associated mutations seemed to have only minor effects. Accordingly, cell type-dependent increased basal DNA damage levels in cortical astrocytes vs. neurons and spinal vs. cortical neurons were detected. However, FUS or C9ORF72 mutant cortical cells do not recapitulate increased DNA damage levels as they have been observed in spinal MNs. Furthermore, FUS mutation affected recruitment to DNA damage sites, axonal trafficking, and cytoplasmic mislocalization differentially, depending on the analyzed cell type. In cortical neurons, recruitment and trafficking of mutant FUS showed only slight mutation-dependent effects and also less pronounced phenotypes than observed in spinal MNs. In conclusion, patient-specific cellular models are an important tool in ALS research and particularly differences between cortical and spinal MNs need to be further investigated to decipher underlying disease mechanisms, the interplay of cell types affected by the disease, and how they participate in disease progression.

Heart failure is the main cause of death for both men and women in the United States. The only proven treatment for patients with heart failure is heart transplantation. The goal of this research is to create patches of tissue that could mimic the function of the native heart to repair the damaged portions of the heart. In this study, whole porcine hearts were decellularized to create a 3D construct that was recellularized with cardiomyocytes (CM) differentiated from human induced pluripotent stem (IPS) cells. At day 4 of differentiation, IPS-derived CMs were implanted onto cardiac extracellular matrix (cECM) and ten days after recellularization, the cells started to beat spontaneously. After implantation, the progenitor CMs continued to proliferate and populate the cECM. A live/dead assay showed the potential of the cECM as a scaffold suitable for recellularization. Confocal microscopy images were taken to evaluate the organization of the cells within the matrix and the impact of the cECM on the growth and maturation of the CMs. Representative cardiac Troponin T (cTNT) and vimentin immunostaining images of CMs derived from iPSCs, on cECM and on standard cell culture plates showed that the cECM allowed the cells to organize and form fibrils with the fibroblasts, compared with CMs cultured in regular culture plates. The timeline of implantation of the cells was a key factor for the development of the heart tissue constructs. Progenitor CMs seeded onto cECM showed better organization and the ability to penetrate 96 µm deep within the collagen fibers and align to them. However, mature CMs seeded onto the matrix showed a disorganized network with very reduced interaction of CMs with fibroblasts, forming two different layers of cells; CMs on top of fibroblasts. In addition, the depth of penetration of the mature CMs within the matrix was only 20 µm. To evaluate the impact of the addition of support cells to the CM monolayer cultures, CMs were co-cultured with human umbilical vein endothelial cells (HUVEC) and it was demonstrated that at ratios of 2:1 HUVEC:CM the beating rate of the CMs was improved from 20 to 112 bpm, additionally, the CM monolayer cultures showed a more synchronized beating pace after the addition of HUVECs. Pharmacological stimulation was performed on CM monolayer cultures using norepinephrine as a stimulator and the results showed that the beating pace of the CMs was improved to 116 bpm after 5 minutes of drug exposure. For future studies, inosculation of the tissue constructs could be performed with the incorporation of membrane proteins to understand the mechanotransduction of the cells. As a preliminary study, the action of dual claudins was evaluated with HUVEC cultures and the results showed the potential of these membrane proteins in the healing of the damaged cell membrane.

Cellular cardiomyoplasty is a cell therapy using stem cells or progenitor cells for myocardial regeneration to improve cardiac function and mitigate heart failure. Since we first published cellular cardiomyoplasty in 1989, this procedure became the innovative method to treat damaged myocardium other than heart transplantation. A significant improvement in cardiac function, metabolism, and perfusion is generally observed in experimental and clinical studies, but the improvement is mild and incomplete. Although safety, feasibility, and efficacy have been well documented for the procedure, the beneficial mechanisms remain unclear and optimization of the procedure requires further study. This chapter briefly reviews the stem cells used for cellular cardiomyoplasty and their clinical outcomes with possible improvements in future studies.

Cellular cardiomyoplasty is a cell therapy using stem cells or progenitor cells for myocardial regeneration to improve cardiac function and mitigate heart failure. Since we first published cellular cardiomyoplasty in 1989, this procedure became the innovative method to treat damaged myocardium other than heart transplantation. A significant improvement in cardiac function, metabolism, and perfusion is generally observed in experimental and clinical studies, but the improvement is mild and incomplete. Although safety, feasibility, and efficacy have been well documented for the procedure, the beneficial mechanisms remain unclear and optimization of the procedure requires further study. This chapter briefly reviews the stem cells used for cellular cardiomyoplasty and their clinical outcomes with possible improvements in future studies.