Die Rolle von Connexinen und Gap Junction-vermittelter Kommunikation in pluripotenten Stammzellen sowie der frühen Embryonalentwicklung sind bis heute nicht vollständig aufgeklärt. Mutationen in humanen Connexinen verursachen eine Vielzahl von Krankheiten. Connexin-defiziente iPS Zellen stellen eine gute Basis für die Erforschung der Rolle von Connexinen während der Embryonalentwicklung und bei der Krankheitsentstehung dar. Das Ziel der vorliegenden Arbeit war es, das CRISPR/Cas9-System in pluripotenten Stammzellen erfolgreich anzuwenden und ein Protokoll zur Erstellung verschiedener Cx43-Defektmutanten zu entwerfen. Nach der Etablierung der CRSIPR/Cas9-Methode in HEK293T-Zellen konnte in der vorliegenden Arbeit darüber hinaus erfolgreich eine Cx43-Defizienz in FSiPS-Zellen erzeugt werden. Weiterhin wurden mehrere Cx43-Mutanten geschaffen und initial auf Pluripotenzmarker und ihr Differenzierungspotential untersucht. Diese Arbeit bildet die Basis für weitere Untersuchungen des Cx43 in iPS-Zellklonen und davon abgeleiteten Zelltypen sowie artifiziellen 3D-Gewebekulturen. Darüber hinaus bildet sie die Grundlage für die Bildung weiterer Connexin-Defektmutanten sowie von iPS-Zellen mit krankheitsrelevanten Mutationen. / The roles of connexins and gap junction-mediated communication in pluripotent stem cells and early embryonic development have not been fully elucidated to date. Mutations in human connexins cause a variety of diseases. Connexin-deficient iPS cells provide a good basis for studying the role of connexins during embryonic development and in disease development. The aim of the present work was to successfully apply the CRISPR/Cas9 system in pluripotent stem cells and to design a protocol to generate different Cx43 defective mutants. Furthermore, after establishing the CRSIPR/Cas9 method in HEK293T cells, a Cx43 deficiency in FSiPS cells was successfully generated. Furthermore, several Cx43 mutants were created and initially screened for pluripotency markers and their differentiation potential. This work forms the basis for further studies of Cx43 in iPS cell clones and derived cell types as well as artificial 3D tissue cultures. Furthermore, it forms the basis for the generation of further connexin defect mutants as well as iPS cells with disease-relevant mutations.

Motor neuron diseases (MNDs) encompass a variety of clinically and genetically heterogeneous disorders, which lead to the degeneration of motor neurons (MNs) and impaired motor functions. MNs coordinate and control movement by transmitting their signal to a target muscle cell. The synaptic endings of the MN axon and the contact site of the muscle cell thereby form the presynaptic and postsynaptic structures of the neuromuscular junction (NMJ). In MNDs, synaptic dysfunction and synapse elimination precede MN loss suggesting that the NMJ is an early target in the pathophysiological cascade leading to MN death. In this study, we established new experimental strategies to analyze human MNDs by patient derived induced pluripotent stem cells (iPSCs) and investigated pathophysiological mechanisms in two different MNDs. To study human MNDs, specialized cell culture systems that enable the connection of MNs to their target muscle cells are required to allow the formation of NMJs. In the first part of this study, we established and validated a human neuromuscular co-culture system consisting of iPSC derived MNs and 3D skeletal muscle tissue derived from myoblasts. We generated 3D muscle tissue by culturing primary myoblasts in a defined extracellular matrix in self-microfabricated silicone dishes that support the 3D tissue formation. Subsequently, iPSCs from healthy donors and iPSCs from patients with the progressive MND Amyotrophic Lateral Sclerosis (ALS) were differentiated into MNs and used for 3D neuromuscular co-cultures. Using a combination of immunohistochemistry, calcium imaging, and pharmacological stimulations, we characterized and confirmed the functionality of the 3D muscle tissue and the 3D neuromuscular co-cultures. Finally, we applied this system as an in vitro model to study the pathophysiology of ALS and found a decrease in neuromuscular coupling, muscle contraction, and axonal outgrowth in co-cultures with MNs harboring ALS-linked superoxide dismutase 1 (SOD1) mutation. In summary, this co-culture system presents a human model for MNDs that can recapitulate aspects of ALS pathophysiology. In the second part of this study, we identified an impaired unconventional protein secretion (UPS) of Sod1 as pathological mechanisms in Pleckstrin homology domain-containing family G member 5 (Plekhg5)-associated MND. Sod1 is a leaderless cytosolic protein which is secreted in an autophagy-dependent manner. We found that Plekhg5 depletion in primary MNs and NSC34 cells leads to an impaired secretion of wildtype Sod1, indicating that Plekhg5 drives the UPS of Sod1 in vitro. By interfering with different steps during the biogenesis of autophagosomes, we could show that Plekhg5-regulated Sod1 secretion is determined by autophagy. To analyze our findings in a clinically more relevant model we utilized human iPSC MNs from healthy donors and ALS patients with SOD1 mutations. We observed reduced SOD1 secretion in ALS MNs which coincides with reduced protein expression of PLEKHG5 compared to healthy and isogenic control MNs. To confirm this correlation, we depleted PLEKHG5 in control MNs and found reduced extracellular SOD1 levels, implying that SOD1 secretion depends on PLEKHG5. In summary, we found that Plekh5 regulates the UPS of Sod1 in mouse and human MNs and that Sod1 secretion occurs in an autophagy dependent manner. Our data shows an unreported mechanistic link between two MND-associated proteins. / Motoneuronerkrankungen (MNE) umfassen eine Vielzahl klinisch und genetisch heterogener Erkrankungen, die zur Degeneration von Motoneuronen (MN) und zu beeinträchtigten motorischen Funktionen führen. MN koordinieren und steuern Muskelbewegungen, indem sie ihr Signal an eine Zielmuskelzelle übertragen. Die synaptischen Endungen des MN-Axons und die Kontaktstelle der Muskelzelle bilden dabei die präsynaptischen und postsynaptischen Strukturen der neuromuskulären Endplatte (NME). Bei MNE zeichnen sich synaptische Dysfunktion und Synapseneliminierung bereits vor dem Verlust von MN ab, was darauf hindeutet, dass die NME ein frühes Ziel in der pathophysiologischen Kaskade ist, die zum MN-Tod führt. In dieser Studie haben wir neue experimentelle Strategien zur Analyse humaner MNE mithilfe von humanen induzierten pluripotenten Stammzellen (iPSZ) entwickelt und pathophysiologische Mechanismen bei zwei verschiedenen MNE untersucht. Um humane MNE zu untersuchen sind Zellkultursysteme erforderlich, die die Verbindung von MN mit ihren Zielmuskelzellen ermöglichen, um NME zu bilden. Im ersten Teil dieser Studie haben wir ein humanes neuromuskuläres Co-Kultursystem etabliert und validiert, das aus iPSZ abgeleiteten MN und 3D Skelettmuskelgewebe aus Myoblasten besteht. Wir haben 3D Muskelgewebe erzeugt, indem wir primäre Myoblasten in einer definierten extrazellulären Matrix in selbst gefertigten Silikonschalen kultivierten, die die 3D-Gewebebildung unterstützen. Anschließend wurden iPSZ von gesunden Spendern und iPSZ von Patienten mit der MNE Amyotrophe Lateralsklerose (ALS) in MN differenziert und für neuromuskuläre 3D Co-Kulturen verwendet. Mithilfe von immunhistochemischen Untersuchungen, Calcium-Imaging und pharmakologischen Stimulationen konnten wir die Funktionalität des 3D Muskelgewebes und neuromuskulären 3D Co-Kulturen charakterisieren und validieren. Anschließend wurde das System als in vitro Modell zur Untersuchung der Pathophysiologie von ALS verwendet. ALS Co-Kulturen mit MN, die eine Superoxid Dismutase 1 (SOD1)-Genmutation aufwiesen, zeigten eine Abnahme der neuromuskulären Verbindung, der Muskelkontraktion und des axonalen Wachstums. Zusammenfassend stellt dieses Co-Kultursystem ein humanes Modell für die Untersuchung von MNE dar, das Aspekte der ALS-Physiologie rekapitulieren kann. Im zweiten Teil dieser Studie konnten wir eine Beeinträchtigung der unkonventionellen Proteinsekretion (UPS) von Sod1 als pathologischen Mechanismus bei Pleckstrin homology domain-containing family G member 5 (Plekhg5)-assoziiertem MNE identifizieren. Sod1 ist ein cytosolisches Protein ohne Signalsequenz für konventionelle Sekretion. Stattdessen wird die UPS über sekretorische Autophagie-Mechanismen reguliert. Unsere Ergebnisse zeigen, dass Plekhg5-Depletion in primären MN und NSC34-Zellen zu einer beeinträchtigten Sekretion von Wildtyp-Sod1 führt, was darauf hinweist, dass die UPS von Sod1 Plekgh5 abhängig ist. Indem verschiedene Schritte während der Biogenese von Autophagosomen gestört wurden, konnten wir nachweisen, dass die Plekhg5-regulierte Sod1-Sekretion Autophagie abhängig ist. Um unsere Ergebnisse in einem klinisch relevanteren Modell zu analysieren, wurden humane iPSZ-MN von gesunden Spendern und ALS-Patienten mit SOD1-Mutationen untersucht. Hier fand sich, dass die Sekretion von mutiertem SOD1 in ALS-MN im Vergleich zu gesunden und isogenen Kontrollen verringert ist. Dabei konnten wir zeigen, dass eine verringerte SOD1 Sekretion in ALS-MNs mit einer verringerten Expression von PLEKHG5 einhergeht. Um diese Korrelation zu bestätigen, wurden Kontroll-MN nach PLEKHG5-Depletion untersucht und eine verminderte SOD1-Sekretion dokumentiert, was auf eine PLEKHG5 Abhängigkeit hindeutet. Zusammenfassend konnten wir zeigen, dass Plekh5 die UPS von Sod1 in Maus MN und humanen MN reguliert und dass die Sod1-Sekretion Autophagie abhängig erfolgt. Unsere Daten belegen eine bislang noch nicht gezeigte mechanistische Verknüpfung zwischen zwei MNE-assoziierten Proteinen.

Neisseria meningitidis (the meningococcus) is one of the major causes of bacterial meningitis, a life-threatening inflammation of the meninges. Traversal of the meningeal blood-cerebrospinal fluid barrier (mBCSFB), which is composed of highly specialized brain endothelial cells (BECs), and subsequent interaction with leptomeningeal cells (LMCs) are critical for disease progression. Due to the human-exclusive tropism of N. meningitidis, research on this complex host-pathogen interaction is mostly limited to in vitro studies. Previous studies have primarily used peripheral or immortalized BECs alone, which do not retain relevant barrier phenotypes in culture. To study meningococcal interaction with the mBCSFB in a physiologically more accurate context, BEC-LMC co-culture models were developed in this project using BEC-like cells derived from induced pluripotent stem cells (iBECs) or hCMEC/D3 cells in combination with LMCs derived from tumor biopsies. Distinct BEC and LMC layers as well as characteristic expression of cellular markers were observed using transmission electron microscopy (TEM) and immunofluorescence staining. Clear junctional expression of brain endothelial tight and adherens junction proteins was detected in the iBEC layer. LMC co-culture increased iBEC barrier tightness and stability over a period of seven days, as determined by sodium fluorescein (NaF) permeability and transendothelial electrical resistance (TEER). Infection experiments demonstrated comparable meningococcal adhesion and invasion of the BEC layer in all models tested, consistent with previously published data. While only few bacteria crossed the iBEC-LMC barrier initially, transmigration rates increased substantially over 24 hours, despite constant high TEER. After 24 hours of infection, deterioration of the barrier properties was observed including loss of TEER and altered expression of tight and adherens junction components. Reduced mRNA levels of ZO-1, claudin-5, and VE-cadherin were detected in BECs from all models. qPCR and siRNA knockdown data suggested that transcriptional downregulation of these genes was potentially but not solely mediated by Snail1. Immunofluorescence staining showed reduced junctional coverage of occludin, indicating N. meningitidis-induced post-transcriptional modulation of this protein, as previous studies have suggested. Together, these results suggest a potential combination of transcellular and paracellular meningococcal traversal of the mBCSFB, with the more accessible paracellular route becoming available upon barrier disruption after prolonged N. meningitidis infection. Finally, N. meningitidis induced cellular expression of pro-inflammatory cytokines and chemokines such as IL-8 in all mBCSFB models. Overall, the work described in this thesis highlights the usefulness of advanced in vitro models of the mBCSFB that mimic native physiology and exhibit relevant barrier properties to study infection with meningeal pathogens such as N. meningitidis. / Neisseria meningitidis (der Meningokokkus) ist einer der Hauptursachen bakterieller Meningitis, einer lebensbedrohlichen Entzündung der Hirnhäute. Entscheidend für das für das Voranschreiten der Krankheit ist die Fähigkeit des Erregers, die meningeale Blut-Liquor-Schranke (mBCSFB), bestehend aus spezialisierten Hirnendothelzellen (BECs) und leptomeningealen Zellen (LMCs), zu überwinden und in den submeningealen Raum einzudringen. Da es sich bei N. meningitidis um ein rein humanes Pathogen handelt, beschränkt sich die Erforschung dieser speziellen Interaktion primär auf die Verwendung von in vitro Modellen. Bisher wurden hierfür hauptsächlich periphere oder immortalisierte BECs verwendet, welchen jedoch wichtige Barriere-Eigenschaften fehlen. Um die Interaktion von N. meningitidis mit der mBCSFB in einem physiologisch relevanteren Umfeld zu untersuchen, wurden in dieser Arbeit neuartige BEC-LMC Kokulturmodelle entwickelt. Dabei wurden sowohl BEC-ähnliche Zellen, die aus induzierten pluripotenten Stammzellen generiert wurden (iBECs), als auch hCMEC/D3 Zellen verwendet und zusammen mit LMCs aus Tumorbiopsien kultiviert. Mittels Transmissions-Elektronenmikroskopie und Immunfluoreszenzfärbung konnten die unterschiedlichen Zellschichten und deren Expression charakteristischer zellulärer Marker dargestellt werden. Durchgängige Expression von wichtigen Bestandteilen Barriere-formender Zellverbindungen, sogenannter Tight und Adherens Junctions, wurde in der iBEC-Schicht beobachtet. Die Integrität der zellulären Barriere wurde mittels transendothelialer elektrischer Resistenz (TEER) und Permeabilität gegenüber Natrium-Fluorescein (NaF) bestimmt. Erhöhte TEER-Werte und verringerte NaF-Permeabilität, gemessen über einen Zeitraum von sieben Tagen, zeigten eine durch die Kokultur mit LMCs ausgelöste Steigerung der Dichtigkeit und Stabilität der iBEC-Barriere. Infektionsexperimente mit N. meningitidis zeigten in allen Modellen vergleichbare bakterielle Adhäsion und Invasion der BEC-Schicht. Bakterielle Transmigration durch die gesamten Zellbarriere war im iBEC-LMC Modell kurz nach Infektion nur in geringem Maße detektierbar, nahm jedoch innerhalb von 24 Stunden deutlich zu. Interessanterweise wurde bis zu 24 Stunden nach Infektion noch eine hohe Integrität der Barriere gemessen, welche allerdings im weiteren Verlauf verloren ging. Neben signifikantem TEER-Verlust wurde eine verringerte Expression der Tight und Adherens Junction Proteine ZO-1, claudin-5, und VE-cadherin mittels qPCR festgestellt. qPCR und siRNA Knockdown Experimente deuteten darauf hin, dass dies möglicherweise, aber nicht ausschließlich, auf den Transkriptionsfaktor Snail1 zurückzuführen war. Zusätzlich zu den beobachteten Effekten auf die zelluläre Transkription von Tight Junction Genen, zeigten Immunfluoreszenzfärbungen eine verringerte Expression von Occludin an den Zell-Zell-Verbindungen, was auf eine post-translationale Modulation schließen lässt. Zusammen deuten die Ergebnisse dieser Infektionsstudien auf eine mögliche Kombination aus trans- und parazellulärer bakterieller Transmigration der mBCSFB hin. Zuletzt wurden in dieser Arbeit noch die Immunaktivierung von BECs nach N. meningitidis Infektion in den neuen BEC-LMC Kokulturmodellen untersucht. Hierbei wurde eine erhöhte Expression von Zytokinen, insbesondere Interleukin-8, beobachtet. Insgesamt konnten in dieser Arbeit neue, fortschrittlicher in vitro Modelle der mBCSFB entwickelt werden, welche die humane Physiologie besser widerspiegeln und daher für Infektionsstudien mit Meningitis-verursachenden Erregern wie N. meningitidis von besonderem Nutzen sind.

In dieser Studie wurden nicht-integrative Vektorkonstrukte zur Reprogrammierung von zwei menschlichen Zelllinien (HFF1, HUVEC) verwendet, um integrations-freie, episomal generierte iPSC Zelllinien (E-iPSCs) zu generieren. Darüber hinaus wurden diese iPSCs zu sogenannten Leberzell-ähnlichen Zellen (HLCs) differenziert. Hierzu konnten die verschiedenen Stufen der Hepatogenese und die potentielle Reifung zu Leberzellen untersucht sowie mit fötalen und ausgereiften menschlichen Leberzellen verglichen werden. Diese Studie konnte Gen-regulierende Netzwerke aufdecken, welche eine pi-potentiale Vorläuferpopulation in den HLCs präsentieren. Zusätzlich deckte das Transkriptions-Profil auf, dass die iPSC-generierten HLCs unreif und ähnlicher den fötalen Leberzellen sind. Dennoch weisen die HLCs typische funktionelle Charakteristika von Leberzellen auf, z.B. Glykogen-Einlagerung, Aufnahme und Abgabe von Substanzen wie ICG und CDFDA, Sekretierung von Gallensäure und Harnstoff. Zusätzlich konnten typische Leber-Strukturen wie Gallenkanälchen mit Mikrovilli, Fettspeicherung und sogenannte tight junctions, Verbindungsgänge zwischen den Zellen nachgewiesen werden. Um die potentielle Reifung dieser HLCs voranzutreiben, wurde eine Langzeit-Kultivierung von HUVEC-iPSC-generierten HLCs durchgeführt. Dies sollte zugleich zeigen, ob die HLCs länger kultiviert und gleichzeitig reifen können. Ein zweiter Teil dieser Studie befasst sich mit der Generierung von endodermalen Vorläuferzellen (EPs). Es wurden HFF1-iPSCs zu EPs differenziert um die endodermale Entwicklung vor der Entstehung der Gallenwege und des Hepatoblasten zu untersuchen. Die EPs zeigen Merkmale dafür, dass sie sowohl in Hepatozyten, Cholangozyten und auch Pankreaszellen differenziert werden können. Mit Hilfe dieser multipotenten EPs könnte es möglich sein die endodermale Entwicklung des Darmes, der Lunge, Leber, Gallengänge und Gallenblase sowie der Bauchspeicheldrüse näher zu untersuchen. / This project generated and characterized integration-free, episomal-derived induced pluripotent stem cell lines (E-iPSCs) from human somatic cell lines of different origins. Two different somatic cell lines were used, the human fetal fibroblast cell line HFF1 and human umbilical vein endothelial cell line HUVEC. Both were reprogrammed into integration-free iPSCs and were comparable amongst themselves and to human embryonic stem cells, the gold standard of pluripotent stem cells. Furthermore, the iPSCs with different genetic background were differentiated to hepatocyte-like cells (HLCs). With the use of iPSC-derived hepatocytes different stages during hepatogenesis and the potential of maturation could be analyzed as well as compared to fetal liver and primary human hepatocytes (PHH). This study could uncover gene regulatory networks which presence bipotential progenitor populations in HLCs. Additionally, comparable transcriptome profile analyses revealed that the iPSC-derived HLCs are immature and more similar to fetal liver. However, the HLCs hold typical functionality characteristics of hepatocyte, e.g. glycogen storage, uptake and release of ICG and CDFDA, bile acid and urea secretion. Furthermore, typical structures of hepatocytes such as bile canaliculi with microvilli, lipid storage and tight junctions are visible. In order to analyze the maturation potential of HLCs a long-term culture experiment was performed using HUVEC-iPSC-derived HLCs which implies the possibility for long-term culture of HLCs while increasing maturation. Additionally, HFF1-derived iPSCs were differentiated to endodermal progenitors (EPs) to analyze the endodermal development before biliary tree and hepatoblast which can give rise to hepatocytes, cholangiocytes and pancreatic cells. The multipotent EPs hold a great potential to analyze the endodermal development of intestine, lung, liver, bile duct and gallbladder, as well as pancreas.

A síndrome de Prader Willi (PWS) é uma doença de neurodesenvolvimento; a principal hipótese de causa de PWS é a ausência da expressão de SNORD116. O SNORD116 fica na região 15q11-q13 que apresenta vários genes com imprinting genômico e é conhecida por ser controlada pela região de controle de imprinting PWS (PWS-IC) que se localiza sobreposta à região promotora e ao exon 1 do gene SNRPN. Em camundongos, uma proteína zinc finger (Zfp57) foi descrita como importante para o estabelecimento e manutenção do imprinting no Snrpn. Através de análise do ENCODE do Genome Browser, verificamos que outra proteína zinc finger (ZNF274) se liga ao SNORD116. ZNF274 é conhecida por formar um complexo com TRIM28 e SETDB1 que inibe a expressão através da trimetilação da lisina 9 na histona 3 (H3K9me3). No atual estudo mostramos que ZNF274 se liga ao SNORD116 preferencialmente ao alelo materno nas células-tronco pluripotente induzidas (iPSCs). Adicionalmente, as proteínas TRIM28 e SETDB1, que formam um complexo com a ZNF274, estão presentes na região do SNORD116, e a modificação H3K9me3 ocorre preferencialmente no alelo materno nas iPSCs. Na análise funcional, mostramos que o knockdown de SETDB1 isoladamente ou combinado com o knockdown de ZNF274 causa aumento na expressão de SNRPN e SNORD116 nas iPSCs. Além disso, ocorre redução do H3K9me3 e aumento da modificação relacionada à ativação da transcrição, H3K4me2 (dimetilação da lisina 4 na histona 3), na PWS-IC. Os knockdowns também afetam a metilação de DNA, ocasionando o aumento de 5-hidroximetliação de citosinas na PWS-IC. Em outros tipos celulares estudados, neurônios derivados de iPSCs e SHEDs, ZNF274 e a modificação H3K9me3 ocorrem em ambos os alelos dentro do SNORD116. É possível que, nas iPSCs, este complexo proteja a região imprintada da desmetilação do DNA de proteína(s) que atue(m) nessa região somente em células pluripotentes. Nossos achados possibilitam melhor compreensão dos mecanismos envolvidos no imprinting da região 15q11-q13, principalmente do SNORD116, e, consequentemente, disponibiliza novas ferramentas para o desenvolvimento de futuras terapias para PWS. / Prader-Willi syndrome (PWS) is a neurodevelopmental disorder. Loss of paternal copies of the cluster of SNORD116 C/D box snoRNAs and their host transcript, 116HG, on human chromosome 15q11-q13 imprinted region is considered to be the major responsible for PWS. PWS-imprinting center (PWS-IC) regulates 15q11-q13 imprinting. PWS-IC is located upstream and in the exon 1 of SNURF-SNRPN gene. In mice, Zfp57 plays an important role in establishment and maintenance of Snrpn imprinting. In human, ENCODE database indicates that ZNF274 binds to SNORD116. Moreover, ZNF274 are C2H2/KRAB zinc finger proteins as Zfp57. We have investigated the mechanism of repression of the maternal SNORD116. Here, we report that the ZNF274, in association with the histone H3 lysine 9 (H3K9) methyltransferase SETDB1, is part of a complex that binds to the silent maternal but not to the active paternal alleles in induced pluripotent stem cells (iPSCs). Knockdown of SETDB1 in PWS-specific iPSCs causes a decrease in the accumulation of H3K9 trimethylation (H3K9me3) at SNORD116. We also show that upon knockdown of SETDB1 in PWS-specific iPSCs, expression of maternally silenced 116HG RNA is partially restored. SETDB1 knockdown in PWS iPSCs also disrupts DNA methylation at the PWS-IC where a decrease in 5-methylcytosine is observed in association with a concomitant increase in 5-hydroxymethylcytosine. In iPSCs-derived neurons and stem cells from human exfoliated teeth (SHEDs) ZNF274/SETDB1 complex binding and H3K9me3 modification occur in both alleles. These observations suggest that the ZNF274/SETDB1 complex bound to the SNORD116 cluster may protect the PWS-IC from DNA demethylation during early development, as indicated by iPSCs. Our findings reveal novel epigenetic mechanisms that function to repress the maternal 15q11-q13 region. The better understanding of epigenetic mechanisms provides new tools for future therapy research.

The discovery of resetting human somatic cells via introduction of four transcription factors into an embryonic stem cell-like state that enables the generation of any cell type of the human body has revolutionized the field of medical science. The generation of patient-derived iPSCs and the subsequent differentiation into the cells of interest has been, nowadays, widely used as model system for various inherited diseases. The aim of this thesis was to generate iPSCs and to subsequently derive NPCs which can be differentiated into neurons in order to model the two most common forms of the NCLs: LINCL which is caused by mutations within the TPP1 gene, encoding a lysosomal enzyme, and JNCL which is caused by mutations within the CLN3 gene, affecting a lysosomal transmembrane protein. It was shown that patient-derived fibroblasts can be successfully reprogrammed into iPSCs by using retroviral vectors that introduced the four transcription factors POU5F1, SOX2, KLF4 and MYC. The generated iPSCs were subsequently differentiated into expandable NPCs and finally into mature neurons. Phenotype analysis during the different stages, namely pluripotent iPSCs, multipotent NPCs and finally differentiated neurons, revealed a genotype-specific progression of the disease. The earliest events were observed in organelle disruption such as mitochondria, Golgi and ER which preceded the accumulation of subunit c of the mitochondrial ATPase complex that was only apparent in neurons. However, none of these events led to neurodegeneration in vitro. The established disease models recapitulate phenotypes reported in other NCL disease models such as mouse, dog and sheep model systems. More importantly, the hallmark of the NCLs, accumulation of subunit c in neurons, could be reproduced during the course of disease modeling which demonstrates the suitability of the established system. Moreover, the derived expandable NPC populations can be used for further applications in drug screenings. Their robust phenotypes such as low levels of TPP1 activity in LINCL patient-derived NPCs or cytoplasmic vacuoles, containing storage material, observed in CLN3 mutant NPCs, should serve as possible phenotypic read-outs.

A síndrome de Prader Willi (PWS) é uma doença de neurodesenvolvimento; a principal hipótese de causa de PWS é a ausência da expressão de SNORD116. O SNORD116 fica na região 15q11-q13 que apresenta vários genes com imprinting genômico e é conhecida por ser controlada pela região de controle de imprinting PWS (PWS-IC) que se localiza sobreposta à região promotora e ao exon 1 do gene SNRPN. Em camundongos, uma proteína zinc finger (Zfp57) foi descrita como importante para o estabelecimento e manutenção do imprinting no Snrpn. Através de análise do ENCODE do Genome Browser, verificamos que outra proteína zinc finger (ZNF274) se liga ao SNORD116. ZNF274 é conhecida por formar um complexo com TRIM28 e SETDB1 que inibe a expressão através da trimetilação da lisina 9 na histona 3 (H3K9me3). No atual estudo mostramos que ZNF274 se liga ao SNORD116 preferencialmente ao alelo materno nas células-tronco pluripotente induzidas (iPSCs). Adicionalmente, as proteínas TRIM28 e SETDB1, que formam um complexo com a ZNF274, estão presentes na região do SNORD116, e a modificação H3K9me3 ocorre preferencialmente no alelo materno nas iPSCs. Na análise funcional, mostramos que o knockdown de SETDB1 isoladamente ou combinado com o knockdown de ZNF274 causa aumento na expressão de SNRPN e SNORD116 nas iPSCs. Além disso, ocorre redução do H3K9me3 e aumento da modificação relacionada à ativação da transcrição, H3K4me2 (dimetilação da lisina 4 na histona 3), na PWS-IC. Os knockdowns também afetam a metilação de DNA, ocasionando o aumento de 5-hidroximetliação de citosinas na PWS-IC. Em outros tipos celulares estudados, neurônios derivados de iPSCs e SHEDs, ZNF274 e a modificação H3K9me3 ocorrem em ambos os alelos dentro do SNORD116. É possível que, nas iPSCs, este complexo proteja a região imprintada da desmetilação do DNA de proteína(s) que atue(m) nessa região somente em células pluripotentes. Nossos achados possibilitam melhor compreensão dos mecanismos envolvidos no imprinting da região 15q11-q13, principalmente do SNORD116, e, consequentemente, disponibiliza novas ferramentas para o desenvolvimento de futuras terapias para PWS. / Prader-Willi syndrome (PWS) is a neurodevelopmental disorder. Loss of paternal copies of the cluster of SNORD116 C/D box snoRNAs and their host transcript, 116HG, on human chromosome 15q11-q13 imprinted region is considered to be the major responsible for PWS. PWS-imprinting center (PWS-IC) regulates 15q11-q13 imprinting. PWS-IC is located upstream and in the exon 1 of SNURF-SNRPN gene. In mice, Zfp57 plays an important role in establishment and maintenance of Snrpn imprinting. In human, ENCODE database indicates that ZNF274 binds to SNORD116. Moreover, ZNF274 are C2H2/KRAB zinc finger proteins as Zfp57. We have investigated the mechanism of repression of the maternal SNORD116. Here, we report that the ZNF274, in association with the histone H3 lysine 9 (H3K9) methyltransferase SETDB1, is part of a complex that binds to the silent maternal but not to the active paternal alleles in induced pluripotent stem cells (iPSCs). Knockdown of SETDB1 in PWS-specific iPSCs causes a decrease in the accumulation of H3K9 trimethylation (H3K9me3) at SNORD116. We also show that upon knockdown of SETDB1 in PWS-specific iPSCs, expression of maternally silenced 116HG RNA is partially restored. SETDB1 knockdown in PWS iPSCs also disrupts DNA methylation at the PWS-IC where a decrease in 5-methylcytosine is observed in association with a concomitant increase in 5-hydroxymethylcytosine. In iPSCs-derived neurons and stem cells from human exfoliated teeth (SHEDs) ZNF274/SETDB1 complex binding and H3K9me3 modification occur in both alleles. These observations suggest that the ZNF274/SETDB1 complex bound to the SNORD116 cluster may protect the PWS-IC from DNA demethylation during early development, as indicated by iPSCs. Our findings reveal novel epigenetic mechanisms that function to repress the maternal 15q11-q13 region. The better understanding of epigenetic mechanisms provides new tools for future therapy research.

Here we describe some of the crucial steps to generate induced pluripotent stemcells (iPSCs) usingmRNA transfection. Our approach uses a V. virus-derived capping enzyme instead of a cap-analog, ensuring 100% proper cap orientation for in vitro transcribedmRNA. V. virus\'' 2′-O-Methyltransferase enzymecreates a cap1 structure found in higher eukaryotes and has higher translation efficiency compared to other methods. Use of the polymeric transfection reagent polyethylenimine proved superior to other transfection methods. The mRNA created via this method did not trigger an intracellular immune response via human IFN-gamma (hIFN-γ) or alpha (hIFN-α) release, thus circumventing the use of suppressors. Resulting mRNA and protein were expressed at high levels for over 48 h, thus obviating daily transfections. Using this method, we demonstrated swift activation of pluripotency associated genes in human fibroblasts. Low oxygen conditions further facilitated colony formation. Differentiation into different germ layers was confirmed via teratoma assay. Reprogramming with non-synthetic mRNA holds great promise for safe generation of iPSCs of human origin. Using the protocols described herein we hope to make this method more accessible to other groups as a fast, inexpensive, and non-viral reprogramming approach.

The discovery of resetting human somatic cells via introduction of four transcription factors into an embryonic stem cell-like state that enables the generation of any cell type of the human body has revolutionized the field of medical science. The generation of patient-derived iPSCs and the subsequent differentiation into the cells of interest has been, nowadays, widely used as model system for various inherited diseases. The aim of this thesis was to generate iPSCs and to subsequently derive NPCs which can be differentiated into neurons in order to model the two most common forms of the NCLs: LINCL which is caused by mutations within the TPP1 gene, encoding a lysosomal enzyme, and JNCL which is caused by mutations within the CLN3 gene, affecting a lysosomal transmembrane protein. It was shown that patient-derived fibroblasts can be successfully reprogrammed into iPSCs by using retroviral vectors that introduced the four transcription factors POU5F1, SOX2, KLF4 and MYC. The generated iPSCs were subsequently differentiated into expandable NPCs and finally into mature neurons. Phenotype analysis during the different stages, namely pluripotent iPSCs, multipotent NPCs and finally differentiated neurons, revealed a genotype-specific progression of the disease. The earliest events were observed in organelle disruption such as mitochondria, Golgi and ER which preceded the accumulation of subunit c of the mitochondrial ATPase complex that was only apparent in neurons. However, none of these events led to neurodegeneration in vitro. The established disease models recapitulate phenotypes reported in other NCL disease models such as mouse, dog and sheep model systems. More importantly, the hallmark of the NCLs, accumulation of subunit c in neurons, could be reproduced during the course of disease modeling which demonstrates the suitability of the established system. Moreover, the derived expandable NPC populations can be used for further applications in drug screenings. Their robust phenotypes such as low levels of TPP1 activity in LINCL patient-derived NPCs or cytoplasmic vacuoles, containing storage material, observed in CLN3 mutant NPCs, should serve as possible phenotypic read-outs.

L’hyperoxalurie primitive de type 1 (ou HP1) est une maladie héréditaire du métabolisme liée à un déficit en enzyme hépatocytaire AGT (alanine:glyoxylate aminotransférase), codée par le gène AGXT. Ce déficit entraîne, chez les patients atteints d’HP1, une excrétion hépatique accrue d’oxalate ; celui-ci est ensuite éliminé dans les urines où il se complexe avec le calcium pour former des néphrolithiases oxalo-calciques massives, pouvant conduire à une insuffisance rénale chronique. Le seul traitement curatif disponible pour cette pathologie est la greffe allogénique combinée hépatorénale, actuellement limitée par la disponibilité des donneurs de greffons, une morbi-mortalité significative et la nécessité d’un traitement immunosuppresseur au long cours. L’objectif du projet de recherche est de développer une thérapie génique de l’HP1 par greffe de cellules hépatiques autologues génétiquement corrigées. La faible disponibilité et la difficulté d’amplification in vitro des hépatocytes adultes nous a conduit à explorer la piste des cellules souches pluripotentes induites (iPSCs) pour produire des cellules hépatiques humaines utilisables en médecine régénérative. Nous avons dérivé et caractérisé des lignées de cellules iPSCs à partir de fibroblastes de patients atteints d’HP1, après expression transitoire des facteurs de reprogrammation par des vecteurs Sendai. Nous avons développé deux stratégies de thérapie génique additive par insertion d’un minigène codant une séquence optimisée de l’ADNc AGXT au moyen (1) d’un vecteur lentiviral à expression hépato-spécifique et (2) d’un processus de recombinaison homologue au locus AAVS1 facilité par le système de clivage ciblé de l’ADN « CRISPR/Cas9 ». Enfin, nous avons mis en évidence l’expression de la cassette thérapeutique après différenciation hépatocytaire des iPSCs génétiquement corrigées. Ces résultats ouvrent de nouvelles perspectives de médecine régénérative pour l’HP1 par transplantation de cellules hépatocytaires autologues génétiquement corrigées dérivées d’iPSCs de patients. / Primary hyperoxaluria type 1 (or PH1) is an inherited metabolic disorder related to the deficiency of the hepatic AGT enzyme (alanine:glyoxylate aminotransferase), which is encoded by the AGXT gene. In PH1 patients, this deficiency leads to oxalate overexcretion by liver, followed by urine filtration and complexation with calcium to form massive calcium-oxalate nephrolithiasis potentially leading to chronic renal failure. The only available curative treatment is combined hepatorenal allogeneic engraftment, which is currently limited by the availability of transplant donors, significant morbidity and mortality, and the need for long-term immunosuppressive treatment. The aim of our research project is to develop gene therapy for PH1, consisting in engraftment of genetically corrected autologous liver cells. Considering that adult hepatocytes are hardly available and expandable in vitro, we chose to explore the use of induced pluripotent stem cells (iPSCs) to produce human liver cells for application in regenerative medicine. We derived and characterized iPSC lines from PH1 patient fibroblasts after transient expression of reprogramming factors delivered by Sendai virus vectors. We developed two additive gene therapy strategies by inserting a minigene encoding an optimized AGXT cDNA sequence using (1) a lentiviral vector designed for liver-specific expression and (2) homologous recombination process at the AAVS1 locus favoured by the targeted DNA cutting system “CRISPR/Cas9”. Finally, we highlighted therapeutic cassette expression after hepatic differentiation of genetically corrected iPSCs. These results pave the way for regenerative medicine for PH1 by transplantation of genetically modified autologous hepatocyte-like cells derived from patient-specific iPSCs.