京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20788号 / 医博第4288号 / 新制||医||1025(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙橋 良輔, 教授 高橋 淳, 教授 山下 潤 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Human induced pluripotent stem cell (iPSCs)-derived brain organoids have potential to recapitulate the earliest stages of brain development, serving as an effective in vitro model for studying both normal brain development and disorders. In this study, we demonstrate a straightforward approach of generating multiple cerebral organoids from iPSCs on a pillar plate platform, eliminating the need for labor-intensive, multiple transfer and encapsulation steps to ensure the reproducible generation of cerebral organoids. We formed embryoid bodies (EBs) in an ultra-low attachment (ULA) 384-well plate and subsequently transferred them to the pillar plate containing Matrigel, using a straightforward sandwiching and inverting method. Each pillar on the pillar plate contains a single spheroid, and the success rate of spheroid transfer was in a range of 95 - 100%. Using this approach, we robustly generated cerebral organoids on the pillar plate and demonstrated an intra-batch coefficient of variation (CV) below 9 – 19% based on ATP-based cell viability and compound treatment. Notably, our spheroid transfer method in combination with the pillar plate allows miniaturized culture of cerebral organoids, alleviates the issue of organoid variability, and has potential to significantly enhance assay throughput by allowing in situ organoid assessment as compared to conventional organoid culture in 6-/24-well plates, petri dishes, and spinner flasks.

Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease or Lou Gehrig’s disease, is characterized by progressive and selective loss of the upper motor neurons in the motor cortex and lower motor neurons in the brainstem and spinal cord. ALS is one of the most common motor neuron disorders worldwide, and death occurs on average within three to five years of the disease onset; unfortunately, there is no effective cure available. Over 1000 mutations in about 40 genes have been associated with ALS pathology, including superoxide dismutase 1 (SOD1), transactive response (TAR)-DNA binding protein (TARDBP), fused in sarcoma (FUS), and chromosome 9 open reading frame 72 (C9orf72). FUS mutations induce an early onset (juvenile) form of ALS and are associated with rapid disease progression. A molecular mechanism of pathogenesis common to many forms of ALS is the disruption of the synthesis, folding, trafficking, and degradation of proteins (proteostasis). Therefore, enhancing protein quality control, for example, via autophagy, might be able to protect motor neurons against ALS pathogenesis. One strategy for enhancing protein homeostasis and protecting neurons is the induction of autophagy. BECN1 is a master regulator of autophagy, but it is repressed by direct interaction with BCL2 via a BH3 domain. In this work, we identify a small molecule BH3 mimetic that disrupts the BECN1-BCL2 interaction using an induced pluripotent stem cell model of ALS with mutant P525L FUS. We identified obatoclax, a brain-penetrant drug candidate that rescued neurons at nanomolar concentrations by reducing cytoplasmic FUS levels, restoring protein homeostasis, and reducing degeneration. Proteomics data suggest that obatoclax protects neurons via multiple mechanisms. Thus, obatoclax is a candidate for repurposing as a possible ALS therapeutic and, potentially, for other disorders linked to defects in protein homeostasis.:TABLE OF CONTENTS 1 Introduction 11 1.1 Amyotrophic lateral sclerosis 12 1.1.1 Clinical manifestations 12 1.1.2 ALS therapeutics 12 1.2 Introduction to ALS genetics 14 1.2.1 SOD1 14 1.2.2 C9orf72 17 1.2.3 TARDBP 19 1.3 FUS 21 1.3.1 Introduction to FUS 21 1.3.2 FUS domains 21 1.3.3 FUS protein and its physiological function 23 1.3.4 ALS-associated FUS mutations 26 1.3.5 Pathomechanisms of FUS-ALS 29 1.4 ALS models 31 1.4.1 In vivo ALS models using mice 31 1.4.2 In vitro models using human iPSCs 33 1.5 Protein homeostasis and ALS 36 1.5.1 Ubiquitin-proteasome system 37 1.6 Autophagy 39 1.6.1 Molecular mechanisms regulating autophagy 39 1.6.2 Defects in autophagy in ALS 41 1.6.3 Autophagy as a possible therapeutic target against ALS-RBP pathogenesis 42 1.6.4 BECN1/BCL2 complex as a possible therapeutic target 43 1.7 Project rationale and aims 48 2 Material 49 2.1 Chemicals 49 2.1.1 Table list of chemicals 49 2.2 Purchased kits 50 2.2.1 Table list of kits 50 2.3 Antibodies 50 2.3.1 Table list of primary antibodies 50 2.3.2 Table list of secondary antibodies for immunostaining 51 2.3.3 Table list of secondary antibodies for western blotting 51 2.4 Cell culture 52 2.4.1 Table list of cell culture media and reagents 52 2.4.2 Table list of small molecules 53 2.4.3 Table list of cell culture media in the small molecules-based neuronal differentiation protocol 54 2.5 BH3 mimetics 56 2.5.1 Table list of BH3 mimetics compounds 56 2.6 Proteomics 57 2.6.1 Table list SDS gel electrophoresis 57 2.6.2 Table list gel digestion 58 2.6.3 Table list instrumentation: Q-EXACTIVE HF - DIA 58 2.6.4 Table list instrumentation: THERMO DIONEX3000 RSLC 59 3 Methods 60 3.1 Licenses 60 3.2 Cell culture 60 3.2.1 Induced pluripotent stem cells (iPSCs) 60 3.2.2 Small molecules-based neuronal differentiation 60 3.3 BH3 mimetics screening and analysis 61 3.3.1 BH3 reconstitution 61 3.3.2 Cell viability 61 3.3.3 Stress granules amelioration 61 3.4 Obatoclax evaluation 62 3.4.1 Autophagic activity and efflux 62 3.4.2 Immunostaining 62 3.4.3 Proximity ligation assay 62 3.4.4 Protein isolation and quantification 62 3.4.5 Capillary electrophoresis 63 3.4.6 SDS-PAGE and Western blot 63 3.4.7 Proteomics 63 3.5 Senolytic activity evaluation 64 3.6 Statistical analysis 64 4 Results 65 4.1 Selection of BH3 mimetics for testing 65 4.2 Identifying BH3 mimetics that are non-toxic to iPSC-derived neurons 65 4.3 ABT-737, obatoclax and gambogic acid reduce aberrant P525L FUS-eGFP stress granules 66 4.4 Obatoclax reduces cytoplasmic P525L FUS-eGFP levels 68 4.5 Obatoclax promotes protein homeostasis in iPSC-derived neurons 69 4.6 Obatoclax ameliorates the degeneration of P525L-FUS iPSC-derived neurons 70 4.7 Obatoclax induces autophagy in iPSC-derived neurons 71 4.8 Long-term effects of obatoclax on LC3 are not due to compound instability 75 4.9 Obatoclax disrupts BECN1-BCL2 complex 77 4.10 Obatoclax contributes to neuroprotection via multiple mechanisms suggested by proteomics analysis 79 4.11 Obatoclax has senolytic activity in vitro 84 5 Discussion 88 5.1 The potential of autophagy as a therapeutic target in ALS and its present limitation 88 5.2 Identification of bh3 mimetics protecting mutant fus neurons against aberrant stress granules 88 5.2.1 ABT-737 89 5.2.2 Gambogic acid 89 5.3 Obatoclax is a candidate for drug repurposing for FUS-ALS 90 5.3.1 Obatoclax, a drug that induces autophagy 90 5.3.2 Obatoclax reduces neuronal cell death 91 5.3.3 Obatoclax as a candidate for clinical trials 91 5.4 Molecular mechanisms by which obatoclax protects neurons 92 5.4.1 Proteasome is affected in P525L-FUS but not in WT-FUS iPSC-derived neurons 92 5.4.2 Obatoclax may rescue protein homeostasis 92 5.4.3 Obatoclax may increase gangliosides biosynthesis 93 5.4.4 Obatoclax may rescue endosomal trafficking and the Golgi network 93 5.4.5 Obatoclax may reduce DNA damage 94 5.4.6 Obatoclax may increase chaperone activity 94 5.5 Beyond ALS: obatoclax as a possible anti-aging drug 95 5.6 Outlook and future experiments 95 5.6.1 Enhancing the neuroprotective effect of obatoclax 95 5.6.2 Obatoclax has an effect in mixed culture of neurons 95 5.6.3 Obatoclax interaction with other BCL2 proteins 96 5.6.4 Obatoclax could have an effect on other subtypes of ALS and other neurogenerative diseases. 96 5.6.5 Obatoclax could have potential as anti-aging drug 97 6 Bibliography 98 7 Appendix 121 8 Acknowledgments 126

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.

The blood system consists of two main lineages: myeloid and lymphoid. The myeloid system consists of cells that are part of the innate immune response while the lymphoid system consist of cells that are part of humoral response. These responses protect our bodies from foreign pathogens. Thus, malignancies in these systems often cause complications and mortality. Scientists world wide have been researching alternatives to treat hematologic disorders and have explored induced pluripotent stem cells (iPSCs) and the conversion of one cell type to another. First, iPS cells were generated by overexpression of four transcription factors: Oct4, Sox2, Klf4 an cMyc. These cells closely resemble embryonic stem cells (ESCs) at the molecular and cellular level. However, the efficiency of cell conversion is less than 0.1%. In addition, many iPS colonies can arise from the same culture, but each has a different molecular signature and potential. Identifying the appropriate iPS cell lines to use for patient specific therapy is crucial. Here we demonstrate that our system is highly efficient in generating iPS cell lines, and cell lines with silent transgenes are most efficient in differentiating to different cell types . Second, we are interested in generating hematopoietic stem cells (HSCs) from fibroblasts directly, without going through the pluripotent state, to increase efficiency and to avoid complications associated with a stem cell intermediate. However, a robust hematopoietic reporter system remains elusive. There are multiple hematopoietic reporter candidates, but we demonstrate that the CD45 gene was the most promising. CD45 is expressed early during hematopoiesis on the surface of HSCs; and as HSCs differentiate CD45 levels increase. Furthermore, the CD45 reporter is only active in hematopoietic cells. We were able to confirm the utility of the CD45 reporter using an in vitro and an in vivo murine model. In conclusion, The goal of this research was to expand the knowledge of stem cell reprogramming, specifically the reprogramming of iPS cells. Furthermore, it is our desire that the CD45 reporter system will undergo further validation and find utility in clinical and cell therapy environments.

Genetic associations have been discovered for many human complex traits, and yet for most associated loci the causal variants and molecular mechanisms remain unknown. Studies mapping quantitative trait loci (QTLs) for molecular phenotypes, such as gene expression, RNA splicing, and chromatin accessibility, provide rich data that can link variant effects in specific cell types with complex traits. These genetic effects can also now be modeled in vitro by differentiating human induced pluripotent stem cells (iPSCs) into specific cell types, including inaccessible cell types such as those of the brain. In this thesis, I explore a range of approaches for using QTLs to identify causal variants and to link these with molecular functions and complex traits. In Chapter 2, I describe QTL mapping in 123 sensory neuronal cell lines differentiated from human iPSCs. I observed that gene expression was highly variable across iPSC-derived neuronal cultures in specific gene categories, and that a portion of this variability was explained by commonly used iPSC culture conditions, which influenced differentiation efficiency. A number of QTLs overlapped with common disease associations; however, using simulations I showed that identifying causal regulatory variants with a recall-by- genotype approach in iPSC-derived neurons is likely to require large sample sizes, even for variants with moderately large effect sizes. In Chapter 3, I developed a computational model that uses publicly available gene expression QTL data, along with molecular annotations, to generate cell type-specific probability of regulatory function (PRF) scores for each variant. I found that predictive power was improved when the model was modified to use the quantitative value of annotations. PRF scores outperformed other genome-wide scores, including CADD and GWAVA, in identifying likely causal eQTL variants. In Chapter 4, I used PRF scores to identify relevant cell types and to fine map potential causal variants using summary association statistics in six complex traits. By examining individual loci in detail, I showed how the enrichments contributing to a high PRF score are transparent, which can help to distinguish plausible causal variant predictions from model misspecification.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18850号 / 医博第3961号 / 新制||医||1007(附属図書館) / 31801 / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 吉村 長久, 教授 中畑 龍俊 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19561号 / 医博第4068号 / 新制||医||1013(附属図書館) / 32597 / 京都大学大学院医学研究科医学専攻 / (主査)教授 吉村 長久, 教授 戸口田 淳也, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第24885号 / 医科博第152号 / 新制||医科||10(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 河本 宏, 教授 濵﨑 洋子, 教授 上野 英樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM