京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19759号 / 農博第2155号 / 新制||農||1039(附属図書館) / 学位論文||H28||N4975(農学部図書室) / 32795 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 宮川 恒, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Technological advancement allowed for the advent of single-cell technologies capable of measuring a large number of cellular features simultaneously. These technologies have been subsequently used to shed light on the heterogeneity of cellular systems previously considered homogeneous, identifying the exclusive features of individual cells within cellular niches. Today, single-cell technologies represent an essential tool for studying the underlying immunological mechanisms correlating with disease. In this context, cytometry is one of the diverse high-throughput methods capable of examining more than 50 features per cell. However, utilising cytometry at its full potential requires the development of optimized assays. Additionally, the resulting high-dimensional data represent a challenge for existing computational techniques. This thesis attempts to address these challenges. The first part of the thesis is focused on developing a non-linear embedding algorithm for rapid analysis of cytometry datasets called EmbedSOM. The comparison of EmbedSOM with other state-of-the-art algorithms suggested the superiority of EmbedSOM with faster runtime. This is critical for the analysis of large datasets with millions of cells. Furthermore, EmbedSOM has additional functionality such as landmark guided...

Anti-CD19 CAR-T cell immunotherapy is a hopeful treatment option for patients with B cell lymphomas, however it copes with partly severe adverse effects like neurotoxicity. Single-cell resolved molecular data sets in combination with clinical parametrization allow for comprehensive characterization of cellular subpopulations, their transcriptomic states, and their relation to the adverse effects. We here present a re-analysis of single-cell RNA sequencing data of 24 patients comprising more than 130,000 cells with focus on cellular states and their association to immune cell related neurotoxicity. For this, we developed a single-cell data portraying workflow to disentangle the transcriptional state space with single-cell resolution and its analysis in terms of modularly-composed cellular programs. We demonstrated capabilities of single-cell data portraying to disentangle transcriptional states using intuitive visualization, functional mining, molecular cell stratification, and variability analyses. Our analysis revealed that the T cell composition of the patient’s infusion product as well as the spectrum of their transcriptional states of cells derived from patients with low ICANS grade do not markedly differ from those of cells from high ICANS patients, while the relative abundancies, particularly that of cycling cells, of LAG3-mediated exhaustion and of CAR positive cells, vary. Our study provides molecular details of the transcriptomic landscape with possible impact to overcome neurotoxicity.

The 15q13.3 microdeletion is a common genetic disorder associated with multiple neurodevelopmental disorders including autism spectrum disorder, epilepsy, and schizophrenia. Patients have diverse clinical presentations, often prompting genetic assays that identify the CNV in the clinic. This late-stage screening leaves a considerable gap in our understanding of the prenatal and prediagnostic developmental impairments in these individuals, providing a barrier to understanding the disease pathobiology. We provide the first investigation into embryonic brain development of individuals with the 15q13.3 microdeletion by generating multiple 3D neural organoid models from the largest clinical cohort in reported literature. We incorporated unguided and guided forebrain organoid models into our multi-transcriptomic phenotyping pipeline to uncover changes in cell type emergence and disruptions to circuit development, all of which had underlying changes to cell adhesion pathways. Specifically, we identified accelerated growth trajectories in 15q13.3del unguided neural organoids and used single cell RNA sequencing to identify changes in radial glia dynamics that affect neurogenesis. We measured changes in the pseudotemporal trajectory of matured unguided neural organoids, and later identified disruptions in synaptic signaling modules amongst the primary constituents to neural circuitry, excitatory and inhibitory neurons. We leveraged dorsal and ventral forebrain organoid models to better assess circuit dynamics, as they faithfully produce the excitatory and inhibitory neurons in the pallium and subpallium, respectively. We then used the entire 15q13.3del cohort and performed bulk RNA sequencing on each tissue type at two timepoints and discovered convergence on transcriptional dysregulation and disruptions to human-specific zinc finger proteins localized to chromosome 19. We also identified cell type-specific vulnerabilities to DNA damage and cell migration amongst the dorsal and ventral organoids, respectively, which was consistent with the excitatory and inhibitory neural subpopulations amongst the unguided neural organoids scRNA Seq, respectively. We then examined neuron migration in a 3D assembloid model by sparsely labeling dorsal-ventral forebrain organoids from multiple genotype-lineage combinations. Light sheet microscopy identified deficits in inhibitory neuron migration and morphology, but not migration distance, suggesting a complex disruption to cortical circuitry. This novel combination of cell type characterization, pathway identification, and circuitry phenotyping provides a novel perspective of how the 15q13.3 deletions impair prenatal development and can be applied to other NDD models to leverage understanding of early disease pathogenesis. / Dissertation / Doctor of Science (PhD) / The development of the human brain is a highly complex and tightly regulated process that requires the participation of multiple cell types throughout development. Disturbances to the emergence, differentiation, or placement of these cell types can cause disruptions and local miswiring of neural circuits, which is often associated with neurodevelopmental disorders (NDDs). The 15q13.3 microdeletion syndrome is a highly complex condition associated with multiple NDDs and has seldom been studied in a human context. To address this, we used stem cells derived from a 15q13.3 microdeletion syndrome cohort and their typically developing familial controls to generate unguided (“whole brain”) and region-specific organoids to investigate early fetal development across time. We used the largest 15q13.3 microdeletion cohort in reported literature to identify shared disruptions in early developmental milestones such as neurogenesis, neural migration, and neural patterning. We identified expansion of specific cell populations, including progenitors that later give rise to mature neurons. Abnormalities persisted in more mature cell populations, including the inhibitory neurons responsible for establishing critical microcircuitry in the human cortex. By generating guided organoids that enrich for excitatory and inhibitory neural populations, we were able to merge the models to form assembloids, where we captured early migratory and morphological deficits in inhibitory neuron populations, which is supported by the multi-transcriptomics experiments performed in both organoid models. This study provides a framework for examining fetal development in a neurodevelopmental disorder context. By using the 15q13.3 microdeletion background, we found novel disruptions in cell type emergence and circuit formation previously unreported in mouse or 2D neuron models, highlighting the utility of the phenotyping platform for disease modeling.

Tfap2a gehört zur Familie der AP-2-Transkriptionsfaktoren. Heterozygote Mutationen von TFAP2A im Menschen führen zum Branchio-Okulo-Fazialen-Syndrom (BOFS) und sind mit Nierenanomalien assoziiert. Molekulare Mechanismen, die zu diesen BOFS-assoziierten Nierenanomalien führen, sind noch unbekannt. In diesem Projekt wurde die Expression von Mitgliedern der AP-2-Familie in neugeborenen und erwachsenen Wildtyp-Mäusen analysiert. Tfap2a wurde in der Ureterknospe und der distalen Region des S-förmigen Körpers in den Nieren neugeborener Mäuse exprimiert. Die Expression blieb in ausgereiften distalen Tubuli und Sammelrohren erhalten. Tfap2b, ein zweites Mitglied der AP-2-Familie, das in der Niere exprimiert wird und mit Zystenbildung assoziiert ist, wurde im aufsteigenden Ast der Henleschen Schleife sowie in den distalen Tubuli und dem in der Nierenrinde liegenden Sammelrohr exprimiert. Um die Rolle von Tfap2a in der Niere zu untersuchen, wurden Mäuse mit einer sammelrohrspezifischen Deletion von Tfap2a (Tfap2a-KO) erzeugt. Phänotypische und morphologische Analysen ergaben, dass Tfap2a-KO-Mäuse mäßig reduzierte Nierengewichte und eine fortschreitende Dilatation der äußeren medullären Sammelrohre aufwiesen. Einzelkern- und RNA-Sequenzierung der Nieren adulter Mäuse zeigte eine deregulierte Expression von Genen, die mit der Organisation von Aktinfilamenten, Zelladhäsion, Wnt-Signalen und anderen Signalwegen der Nierenentwicklung in Verbindung stehen. In einem isolierten Modell von kultivierten Sammelrohrzellen mit einer Deletion von Tfap2a waren ähnliche Signalwege dereguliert. Insgesamt deutet diese Studie darauf hin, dass Tfap2a für die Differenzierung des Sammelrohrepithels und die Regulierung des Durchmessers des Tubuluslumens erforderlich ist. Dies ermöglicht Einblicke in die molekularen Grundlagen der beim BOFS beobachteten Nierenfehlbildungen. / The transcriptional regulator Tfap2a is part of the AP-2 transcription factor family. Heterozygous mutations of TFAP2A in humans lead to branchio-oculo-facial syndrome (BOFS) and are associated with renal anomalies. Molecular mechanisms leading to BOFS-associated renal anomalies are still unknown. In this project, expression patterns of AP-2 family members were analyzed in newborn and adult wildtype mice. Tfap2a was expressed in the ureteric bud and distal region of the S-shaped body in kidneys of newborn mice. Expression was maintained in mature distal tubules and collecting ducts. Tfap2b, a second AP-2 family member expressed in the kidney and associated with cyst formation, was found in the ascending limb and showed overlapping expression with Tfap2a in distal tubules and the cortical collecting duct. To investigate the role of Tfap2a in the kidney, mice with a collecting duct-specific deletion of Tfap2a (Tfap2a-KO) were generated by crossing mice carrying a Cre-recombinase under the Hoxb7 promotor and mice with floxed Tfap2a alleles. Phenotypic and morphological analyses revealed that Tfap2a-KO mice displayed moderately reduced kidney weights and a progressive dilation of outer medullary collecting ducts. Single-nucleus and bulk RNA sequencing of kidneys of three months old Tfap2a-KO mice and littermate controls indicated deregulated expression of genes associated with actin filament organization, cell adhesion, Wnt signaling, and other kidney developmental pathways. Genes deregulated in Tfap2a-deficient mice included several genes previously implicated in the development of congenital anomalies of the kidney and urinary tract. In an isolated model of cultured collecting duct cells carrying a Tfap2a knockout similar pathways were deregulated. Taking together, this study indicates that Tfap2a is required for collecting duct epithelium differentiation and tubular lumen diameter regulation, providing insights into the molecular basis of renal defects observed in BOFS.

Droplet-based microfluidics is a powerful tool permitting massive-scale single-cell analysis in pico-/nano-liter water-in-oil droplets. It has been integrated into various library preparation techniques to accomplish high-throughput scRNA-seq, scDNA-seq, scATAC-seq, scChIP-seq, as well as scMulti-omics-seq. These advanced technologies have been providing unique and novel insights into both normal differentiation and disease development at single-cell level. In this thesis, we develop four new droplet-based tools for single-cell omics profiling. First, the developed Drop-BS is the first droplet-based platform to construct single-cell bisulfite sequencing libraries for DNA methylome profiling and allows production of BS library of 2,000-10,000 single cells within 2 d. We applied the technology to separately profile mixed cell lines, mouse brain tissues, and human brain tissues to reveal cell type heterogeneity. Second, the new Drop-ChIP platform only requires two steps of droplet generation to achieve multiple steps of reactions in droplets such as single-cell lysis, chromatin fragmentation, ChIP, and barcoding. Third, we aim to establish a droplet-based platform to accomplish high-throughput full-length RNA-seq (Drop-full-seq), which both current tube-based and droplet-based methods cannot realize. Last, we constructed an in-house droplet-based tool to assist single-cell ATAC-seq library preparation (Drop-ATAC), which provided a low-cost and facile protocol to conduct scATAC-seq in laboratories without the expensive instrument. / Doctor of Philosophy / Microfluidics is a collection of techniques to manipulate fluids in the micrometer scale. One of microfluidic techniques is called "droplet-based microfluidics". It can manipulate (i.e., generate, merge, sort, split, etc) pico-/nano-liter of water-in-oil droplets. First, since the water phase is separated by the continuous oil phase, these droplets are discrete and individual reactors. Second, droplet-based microfluidics can achieve highly parallel manipulation of thousands to millions of droplets. These two advantages make droplet-based microfluidics an ideal tool to perform single-cell assays. Over the past 10 years, various droplet-based platforms have been developed to study single-cell transcriptome, genome, epigenome, as well as multi-ome. To expand droplet-based tools for single-cell analysis, we aim to develop four novel platforms in this thesis. First, Drop-BS, by integrating droplet generation and droplet fusion techniques, can achieve high-throughput single-cell bisulfite sequencing library preparation. It can generate 10,000 single-cell BS libraries within 2 days which is difficult to achieve for conventional library preparation in tubes/microwells. Second, we developed a novel and facile Drop-ChIP platform to prepare single-cell ChIP-seq library. It is easy to operate since it only requires two steps of droplet generation. It also generates higher quality of data compared to previous work. In addition, we are working on the development and characterization of the other two droplet-based tools to achieve full-length single-cell RNA-seq and single-cell ATAC-seq.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25491号 / 医博第5091号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 浅野 雅秀, 教授 篠原 隆司, 教授 近藤 玄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

This thesis describes the design, construction and optimisation of two compact setups to produce ⁸⁷Rb Bose-Einstein condensates and dual ⁷Li-⁸⁷Rb Magneto- Optical Traps (MOTs). The motivation for compact systems is to have simplified systems to cool the atoms. The first experimental setup is based on a single pyrex glass cell without the need for atom chips. Fast evaporation will be achieved in a hybrid trap comprising of a magnetic quadrupole trap and an optical dipole trap created by a Nd:YVO4 laser and with future plans of using a Spatial Light Modulator (SLM). To enhance an efficient and rapid evaporation, we have investigated Light-Induced Atomic Desorption (LIAD) to modulate the Rb partial pressure during the cooling and trapping stage. With this technique, a ⁸⁷Rb MOT of 7 x 10⁷ atoms was loaded by shining violet light from a LED source into the glass cell, whose walls are coated with rubidium atoms. The atoms were then cooled by optical molasses and then loaded into a magnetic trap where lifetime measurements demonstrated that LIAD improves on magnetically-trapped atoms loaded from constant background pressure by a factor of six. This is quite encouraging and opens the possibility to do a rapid evaporation. In a second experiment, we have designed a compact system based on a stainless steel chamber to trap either ⁷Li or ⁶Li atoms in a MOT loaded from alkali-metal dispensers without the need of conventional oven-Zeeman slower. This setup can also load ⁸⁷Rb atoms, allowing future projects to simultaneously produce degenerate quantum gases of bosonic ⁸⁷Rb and fermionic ⁶Li atoms.

Recent progress in magnetic resonance imaging (MRI) has opened the way for micron-scale resolution, and thus for imaging biological cells. In this thesis work, we performed magnetic resonance microscopy (MRM) on the nervous system of Aplysia californica, a model particularly suited due to its simplicity and to its very large neuronal cell bodies, in the aim of studying cellular-scale processes with various MR contrasts. Experiments were performed on a 17.2 Tesla horizontal magnet, at resolutions down to 25 µm isotropic. Initial work consisted in conceiving and building radiofrequency microcoils adapted to the size of single neurons and ganglia. The first major part of the project consisted in using the manganese ion (Mn2+) as neural tract tracer in the buccal ganglia of Aplysia. Manganese is an MR contrast agent that enters neurons via voltage-gated calcium channels. We performed the mapping of axonal projections from motor neurons into the peripheral nerves of the buccal ganglia. We also confirmed the existence of active Mn2+ transport inside the neural network upon activation with the neurotransmitter dopamine. In the second major part of the project, we tested the potential of two diffusion MRI sequences for microscopy. On the one hand, we explored a very original mechanism for diffusion weighting, DESIRE (Diffusion Enhancement of SIgnal and REsolution), particularly suited for small samples. The two-dimensional DESIRE sequence was implemented and successfully tested on phantoms. The measured enhancement was consistent with theoretical predictions. Using this sequence to produce diffusion weighted images with an unprecedented contrast in biological tissue remains a challenge. On the other hand, a more "standard" sequence was implemented to measure the apparent diffusion coefficient (ADC) in nervous tissue with MRM. This sequence was a three-dimensional DP-FISP (Diffusion Prepared Fast Imaging with Steady-state free Precession), which met criteria for high resolution in a short acquisition time, with minimal artifacts. Using this sequence, we studied the changes in water ADC at different scales in the nervous system, triggered by cellular challenges. The challenges were hypotonic shock or exposure to ouabain. ADC measurements were performed on single isolated neuronal bodies and on ganglia tissue, before and after challenge. Both types of stress produced an ADC increase inside the cell and an ADC decrease at tissue level. The results favor the hypothesis that the increase in membrane surface area associated with cell swelling is responsible for the decrease of water ADC in tissue, typically measured in ischemia or other conditions associated with cell swelling.

Droplet microfluidics is an emerging technology for analysis of single cells and biomolecules at high throughput. The controlled encapsulation of particles along with the surrounding microenvironment in discrete droplets, which acts as miniaturized reaction vessels, allows millions of particles to be screened in parallel. By utilizing the unit operations developed to generate, manipulate and analyze droplets, this technology platform has been used to miniaturize a wide range of complex biological assays including, but not limited to, directed evolution, rare cell detection, single cell transcriptomics, rare mutation detection and drug screening. The aim of this thesis is to develop droplet microfluidics based methods for analysis of single cells and nucleic acids. In Paper I, a method for time-series analysis of mammalian cells, using automated fluorescence microscopy and image analysis technique is presented. The cell-containing droplets were trapped on-chip and imaged continuously to assess the viability of hundreds of isolated individual cells over time. This method can be used for studying the dynamic behavior of cells. In Paper II, the influence of droplet size on cell division and viability of mammalian cell factories during cultivation in droplets is presented. The ability to achieve continuous cell division in droplets will enable development of mammalian cell factory screening assays in droplets. In Paper III, a workflow for detecting the outcome of droplet PCR assay using fluorescently color-coded beads is presented. This workflow was used to detect the presence of DNA biomarkers associated with poultry pathogens in a sample. The use of color-coded detection beads will help to improve the scalability of the detection panel, to detect multiple targets in a sample. In Paper IV, a novel unit operation for label-free enrichment of particles in droplets using acoustophoresis is presented. This technique will be useful for developing droplet-based assays that require label-free enrichment of cells/particles and removal of droplet content. In general, droplet microfluidics has proven to be a versatile tool for biological analysis. In the years to come, droplet microfluidics could potentially be used to improve clinical diagnostics and bio-based production processes. / <p>QC 20160926</p>