Since Yamanaka and Takahashi first described the isolation of induced pluripotent stem cells (iPSCs) in 2006, researchers have invested a vast amount of time and resources into trying to understand the process of reprogramming. However, the exact mechanisms underlying the induction of somatic cells to pluripotency is still incompletely understood. With this in mind, a screening approach was undertaken to identify shRNA that enhance the reprogramming process. A retrovirus based system was used to knock down candidate genes during reprogramming of mouse embryonic fibroblasts (MEF) containing doxycycline-inducible reprogramming factors and a Nanog-GFP reporter, which is activated when cells become iPSCs. The initial round of screening with over 150 shRNA vectors successfully identified several shRNAs that enhance reprogramming. One of these shRNA vectors exhibited both faster reprogramming kinetics as determined by activation of the Nanog-GFP reporter 2 to 3 days earlier and increased reprogramming efficiency giving rise to >5 fold more GFP+ colonies when compared with a control. Cell surface marker analysis with flow cytometry demonstrated that changes in CD44 and ICAM1 expression, which occur preceding Nanog-GFP expression, were also accelerated. Validation of this shRNA determined that the enhanced reprogramming phenotype is the result of an unknown off-target effect. Microarray and RNA-sequencing analysis was carried out to identify the off target gene with a view to investigate the functional importance of this knock down and its role in establishing the pluripotency transcriptional network during reprogramming.

BACKGROUND: The placenta is a multifunctional organ whose primary functions are to nourish and protect the fetus throughout gestation. The immune response of the placenta plays an important part in gestational outcome. Microbial infection during pregnancy can be detrimental to both maternal health and fetal development, increasing the risk for miscarriage, preterm birth, and congenital abnormalities. However, evaluating immunological response has been an on-going challenge for scientists and clinicians due to the complexity of the maternal-fetal interface. Research has been done to understand the mechanisms by which pathogens activate placental immune response, but our understanding is still lacking in many areas due to the dynamic changes that occur in immunology over the gestational timeline. The primary challenge faced by researchers is the availability of placental tissue, which is limited by donors and their finite viability in culture once harvested. Additionally, legal restrictions placed on fetal-tissue research have severely limited advancement in the field. Human induced pluripotent stem cells (hiPSCs) present a unique tool to study the differentiation of trophoblasts and maternal-placental immunology without the need of fetal tissue. OBJECTIVE: The goal of this project is to develop an in vitro model for studying placental immunology and pathogenesis using human induced pluripotent stem cell (hiPSC)-derived trophoblasts. Our aim is to report a robust protocol for producing hiPSC-derived trophoblasts and to characterize them against primary trophoblasts using both gene and protein expression detection techniques. Successful modeling of human trophoblasts would allow us the unique opportunity to investigate the cellular interface between the maternal and fetal systems without needing to isolate primary human trophoblasts. Once we produce and fully characterize several hiPSC cell clones from multiple normal individuals, we will demonstrate the use of these cells as a model for infections at the maternal-fetal interface by exposing them to viral pathogens known to target the placenta. METHODS: Earlier publications have reported the differentiation of embryonic stem cells into trophoblasts in culture by using bone morphogenetic protein-4 in conjunction with inhibitors of activin A and FGF2-signaling (BMP4/A83-01/PD173074; BAP-treatment). We applied this approach to hiPSC lines from two different lineage origins and characterized the outcome against known trophoblast markers. We also developed a novel approach to maintain proliferative trophoblast stem cells in culture long term. Two viral pathogens, a recombinant vesicular stomatitis virus strain engineered to express a green fluorescent protein (rVSV-GFP) and a strain of Zika virus (ZIKV-PRVABC59), were used to determine if it is possible to infect hiPSC-derived trophoblasts in culture. RESULTS: Using this approach, hiPSC readily differentiate into trophoblasts by day 8 of culture. These cells demonstrate formation of multinuclear syncytium, invasive capacities, and secretion of placental hormones. Further characterization using quantitative real-time PCR and immunofluorescent staining indicates that these cells express a number of trophoblast markers at levels comparable to those expressed by primary first-trimester trophoblasts. We were also able to maintain a putative CT population which retains the capacity to double and give rise to terminal cell types. HiPSC-derived trophoblasts infected with rVSV-GFP and ZIKV-PRVABC59 tested positive for viral infection by 72 hours post-infection (HPI), demonstrating the use of these cells as an in vitro model for studying placental pathogens at the maternal-fetal interface. / 2022-06-08T00:00:00Z

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22302号 / 医博第4543号 / 新制||医||1040(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 川口 義弥, 教授 妹尾 浩, 教授 濵﨑 洋子 / 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・論文博士 / 博士(医学) / 乙第13371号 / 論医博第2208号 / 新制||医||1047(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 川口 義弥, 教授 妹尾 浩, 教授 浅野 雅秀 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

According to the World Health Organization (WHO) depression is the leading cause of disability worldwide with more than 300 million patients affected. Current antidepressants have a delayed onset of action and moreover, only two-thirds of patients suffering from depressive disorder respond to antidepressant drug treatment. The N-methyl-D-aspartate (NMDA) receptor antagonist ketamine offers promising perspectives for the treatment of major depressive disorder. Although ketamine demonstrates rapid and long-lasting effects, even in treatment-resistant patients, to date, the underlying mode of action remains elusive. Thus, the aim of this thesis was to investigate the molecular mechanism of ketamine and its major metabolites at clinically relevant concentrations by establishing an in vitro model based on human induced pluripotent stem cells (iPSCs)-derived neural progenitor cells (NPCs). As the pathophysiology of depression correlates with decreased adult neurogenesis, I aimed to investigate the molecular effects of ketamine on neural progenitor cell proliferation using a human-based iPSC-model. The findings from this thesis substantially contribute to an enhanced understanding of the molecular mode of action of ketamine as a novel signaling pathway involved in ketamine-induced effects was identified. Ketamine induced proliferation of human iPSC-derived NPCs and bioinformatic analysis of RNA-Seq data revealed significant upregulation of insulin-like growth factor2 (IGF2) and p11, a member of the S100 EF-hand protein family, which are both implicated in the pathophysiology of depression, 24 hours after ketamine treatment. In line with this, ketamine dependent proliferation was significantly impaired after IGF2 knockdown. Moreover, ketamine was able to enhance cAMP signaling in NPCs and both, cell proliferation as well as IGF2 expression, were reduced after protein kinase A (PKA)-inhibition. Noteworthy, the Nestin-expressing NPCs do not express functional NMDA receptors, suggesting that the proproliferative effect of ketamine in NPCs is NMDA receptor-independent. Furthermore, 24 hours post administration of ketamine (15 mg/kg) in vivo confirmed phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in the subgranular zone (SGZ) of the hippocampus in C57BL/6 mice. In conclusion, ketamine promotes proliferation of NPCs presumably by involving cAMP-IGF2 signaling.

Permanent repair of the F9 gene is a significant goal to cure Hemophilia B disease. Advanced gene therapy using CRISPR/Cas9 system can increase circulation level of Factor IX proteins to a significant level without the need of demanding infusions of FIX concentrates. Induced pluripotent stem cells represent an ideal cell for gene therapy because patient-derived cells could be reprogrammed into iPSCs, genetically modified, selected, expanded and then induced to differentiate into fully functional hepatocytes in vitro. This study covered a portion of a 5-year project which ultimately aims at establishing therapeutic results in transgenic Hemophilia B mice by injecting genetically corrected iPSC-derived hepatocytes into the liver. The purpose of this thesis is to summarize what has been completed up to now: generation of the proper model of Hemophilia B human iPSCs using CRISPR/Cas9-mediated genome editing and differentiation of healthy and disease specific iPSCs into hepatocytes which will allow disease modelling to look for cell function, viability, homogeneity and drug screening. Further research will be done to effectively knock-in the F9 allele into liver safe harbor site of disease specific iPSCs, which will express FIX at a significant level to show therapeutic effects.

Heart disease is the leading cause of death in the world. Finding future therapeutics for heart disease requires the development of mature 3D engineered human cardiac tissues that can serve as reliable models for biological studies and drug development. The ability to measure and stimulate cardiac action potentials (APs) is key to the development of these 3D tissue models. Optical measurement and stimulation methods offer an enticing solution to longstanding tissue development testbed needs. However, existing optical measurement methods have relied on toxic voltage dyes and motion inhibiting drugs, and existing optical stimulation methods have mainly focused on optogenetic modification techniques. In this dissertation, I present two related optical systems built for 3D engineered human cardiac microtissues, organized into two chapters: 1) a dual-mode voltage and contraction imaging optical microscope for tracking action potential metrics with fluorescent genetically encoded voltage indicator Archon1, and 2) an all optical cardiac stimulation system for unmodified engineered human cardiac micro-tissues with red and blue laser pulse options. These systems offer cardiac researchers less invasive optical solutions for cardiac action potential visualization and pacing: the microscope system allows direct action potential measurements without the use of toxic dyes, and the stimulation system allows ex-vivo pacing without any modification to the sample, genetic or otherwise. I will discuss the system design motivations, challenges, results, and mechanisms of this work.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19595号 / 医博第4102号 / 新制||医||1014(附属図書館) / 32631 / 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 瀬原 淳子, 教授 前川 平 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p>Epilepsies are the results of abnormal brain hyperactivities caused by brain injury, drug intoxication, and genetic perturbations. In the group of genetic-related epilepsies, the ion channel mutations contribute 25% of total epilepsy cases. Many studies suggest some forms of severe epilepsies can start early in patients’ lives, with epilepsy starting during infancy and childhood. With the wide adoption of genomic sequencing in children having seizures, an increasing number of <em>SCN2A</em> genetic variants have been revealed as genetic causes of epilepsy. Voltage-gated sodium channel Nav1.2, encoded by gene SCN2A, is predominantly expressed in the pyramidal excitatory neurons and supports action potential (AP) firing. One recurrent SCN2A genetic variant is L1342P, which was identified in multiple patients with epileptic encephalopathy and intractable seizures. However, the mechanism underlying L1342P-mediated seizures and the pharmacogenetics of this variant in human neurons remain unknown. To probe the potential hypothesized biophysical property changes, we used a heterologous expression system expressing the Nav1.2-L1342P. We observed prominent but quite complex gating kinetics without significant changes in window current. To understand the core phenotypes of the L1342P variant in human neurons, we took advantage of a reference human-induced pluripotent stem cell (hiPSC) line from a male donor, in which L1342P was introduced by CRISPR/Cas9-mediated genome editing. Using patch-clamping and microelectrode array (MEA) recordings, we revealed that cortical neurons derived from hiPSCs carrying heterozygous L1342P variant have significantly increased intrinsic excitability, higher sodium current density, and enhanced bursting and synchronous network firing, suggesting hyperexcitability phenotypes. Interestingly, L1342P neuronal culture displayed a degree of resistance to the anticonvulsant medication phenytoin, which recapitulated aspects of clinical observation of patients carrying the L1342P variant. In contrast, phrixotoxin-3 (PTx3), a compound showing greater specificity on Nav1.2 over other sodium channel subtypes, can potently alleviate spontaneous and chemically induced hyperexcitability of neurons carrying the L1342P variant. Our results reveal a possible pathogenic underpinning of Nav1.2-L1342P mediated epileptic seizures and demonstrate the utility of genome-edited hiPSCs as an in vitro platform to advance personalized phenotyping and drug discovery.</p>

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24792号 / 医博第4984号 / 新制||医||1066(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 椛島 健治, 教授 辻川 明孝, 教授 山中 伸弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM