京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19267号 / 医博第4031号 / 新制||医||1011(附属図書館) / 32269 / 京都大学大学院医学研究科医学専攻 / (主査)教授 小川 誠司, 教授 生田 宏一, 教授 松岡 雅雄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20663号 / 医博第4273号 / 新制||医||1024(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 藤渕 航, 教授 木村 剛, 教授 岩田 想 / 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20795号 / 医博第4295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 原田 浩, 教授 松原 和夫, 教授 坂井 義治 / 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(理学) / 甲第20940号 / 理博第4392号 / 新制||理||1631(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 三木 邦夫, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Embryonic stem (ES) cells have the ability to maintain pluripotency and self-renewal during in vitro maintenance, which is a key to their clinical applications. ES cells are a model in developmental biology studies due to their potential to differentiate in vitro. Understanding critical pathways of pluripotency, self-renewal, and differentiation during early embryonic development is important for the evaluation of the therapeutic potential of ES cells because of their ability for tumor transformation due to genetic and epigenetic instability acquired during in vitro culture maintenance. Single tandem repeats are sequences of DNA that have been implicated in the deregulation of gene expression in different human conditions. Understanding the origin of repetitive sequence instability and functions in the genome allow characterization of early genomic instability signals in ES cell pluripotency, differentiation, and tumor transformation pathways. The hypothesis of this study was that genetic stability, in repetitive sequences, located near embryonic developmental genes is responsible for pluripotency, self-renewal, differentiation, and chromatin assembly and could be a signal for adaptation, differentiation, or transformation of ES cells in vitro. Our result showed instability in specific repetitive sequences which increased during ES cell passages and embryoid body differentiation in vitro. ES cells displayed significant mean frequencies of genomic instability in repetitive regions that lead to ES cells pluripotency, self-renewal maintenance, or cell lineage specialization. The present study reports potentially biomarkers for identifying accumulation of genomic instability in specific genes that may contributes to adaptation of ES cells and could be the switch that initiates early ES cell lineage commitment in vitro. Determining genetic and epigenetic modifications, including single tandem repeat instability, gene expression changes, and chromatin modifications, is essential for elucidating possible molecular mechanisms of genomic instability and determining novel molecular characterization for diagnostic purposes to ensure ES cell stability and integrity that could potentially lead to use of ES cell derivatives that could then be a safe source needed for regenerative medicine applications

Stem cell biology is an exciting field that will lead to significant advancements in science and medicine. We hypothesize that inducing the expression of stem cell genes, using the embryonic stem cell gene nanog, will reprogram cells and dedifferentiate human mesenchymal stem cells into pluripotent stem cells capable of neural differentiation. The aims of initial studies are as follows: Aim 1: Demonstrate that forced expression of the embryonic stem cell gene nanog induces changes in human mesenchymal stem cells to an embryonic stem cell-like phenotype. Aim 2: Demonstrate that induced expression of nanog up-regulates the expression of multiple embryonic stem cell markers and expands the differentiation potential of the stem cells. Aim 3: Demonstrate that these nanog-expressing stem cells have the ability to differentiate along neural lineages in vitro and in vivo, while mock-transfected cells have an extremely limited capacity for transdifferentiation. Alternatively, we hypothesize that embryonic stem cell genes can become activated in malignant gliomas and differentially regulate the subpopulation of cancer stem cells. This study examines the role of embryonic stem cell genes in transformed cells, particularly cancer stem cells. These studies explore has the following objectives: Aim 1: Isolate different sub-populations of cells from tumors and characterize cells with stem cell-like properties. Aim 2: Characterize the expression of embryonic stem cell markers in the sub-population of cancer stem cells. Aim 3: Examine the effects of histone deacetylase inhibitors at inhibiting the growth and reducing the expression of stem cell markers. Our research has demonstrated the potential of the embryonic transcription factor, nanog, at inducing dedifferentiation of human bone marrow mesenchymal stem cells and allowing their recommitment to a neural lineage. Specifically, we used viral and non-viral vectors to induce expression of NANOG, which produced an embryonic stem cell-like morphology in transduced cells. We characterized these cells using real-time PCR and immunohistochemical staining and find an up-regulation of genes responsible for pluripotency and self-renewal. Embryonic stem cell markers including Sox2, Oct4 and TERT were up-regulated following delivery of nanog. The role of nanog in the expression of these markers was further demonstrated in our induced-differentiation method where we transfected embryonic stem cell-like cells, that have been transduced with nanog flanked by two loxP sites, with a vector containing Cre-recominase. We tested the ability of these nanog-transfected cells to undergo neural differentiation in vitro using a neural co-culture system or in vivo following intracranial transplantation. Our next study characterized patient-derived glioblastoma cancer stem cells. We found that cells isolated from serum-free stem cell cultures were enriched for stem cell markers and were more proliferative than the bulk population of cells grown in convention serum-supplemented media. These cancer stem cells expressed embryonic stem cell markers NANOG and OCT4 whereas non-tumor-derived neural stem cells do not. Moreover, the expression of stem cell markers was correlated with enhanced proliferation and could serve as a measure of drug effectiveness. We tested two different histone deacetylase inhibitors, trichostatin A and valproic acid, and found that both inhibited proliferation and significantly reduced expression of stem cell markers in our cancer stem cell lines. These data demonstrate the potential use of stem cell genes as therapeutic markers and supports the hypothesis that cancer stem cells are a major contributor to brain tumor malignancy.

The mammalian central nervous system (CNS) has a high degree of complexity and cell type diversity that enables sophisticated processing of sensory information, circuit formation, and behaviour. While much is known about the patterning and specification of the major neuronal classes in the CNS, through processes such as morphogen gradient signaling and transcription factor combinatorial coding, much less is known about how subtypes within each cell class are specified. Bipolar cells are one of the main classes of interneurons in the vertebrate retina and consist of fifteen different subtypes based on their physiological function, morphology, and unique gene expression. The cellular mechanisms behind the specification of these subtypes are not fully known. In this thesis, I examine these mechanisms by investigating the role of extrinsic and intrinsic factors on the specification and differentiation of bipolar cell subtypes. We hypothesize that the specification of bipolar cell subtypes occurs in a multi-step manner and is dependent on non-cell autonomous (extrinsic) signals. To test this hypothesis, I conducted a series of experiments on the early postnatal mouse retina, which is the period when bipolar cells are generated. First, I examined whether bipolar cell marker onset was temporally ordered as would be predicted in a multi-step model. Postnatal day 3 (P3) mice were injected with EdU (5-ethynyl-2’-deoxyuridine), a thymidine analog that labels proliferating cells and then dissociated and fixed the retinal cells 24-120 hours after injection. My results show that Vsx2-5.3-PRE-Cre, a marker of pan-bipolar cells specification, is first detected 36 hrs after cell cycle exit, whereas specialized bipolar subtype-specific markers are expressed 48-60 hrs post-EdU injection. This observation is consistent with the idea that bipolar cells develop in a stepwise manner, first as an unspecified, pan-bipolar cell intermediate and then into one of the 15 subtypes. To further investigate this possibility, I developed a novel dissociated retinal culture assay that enabled me to accurately track retinal progenitor cells and postmitotic precursor cells and determine the requirement of cell autonomous and non-cell autonomous mechanisms during bipolar cell subtype specification. This assay involves culturing dissociated retinal cells from P3 EdU-injected mice at high density (abundant cell contact) or low density (scarce cell contact) at various timepoints, thereby allowing me to probe the role of these mechanisms in RPCs, early postmitotic cells, and late postmitotic cells. My findings revealed the first 24-48 hrs post cell cycle exit to be a critical, cell contact-dependent period for the specification of bipolar cell subtypes. This assay also allowed us to test the effect of blocking or activating the Notch and the Sonic Hedgehog (Shh) signal transduction pathways by using pharmacological compounds and recombinant ligands. Co-activation of Notch and Shh pathways increased the specification of Vsx1+ subtypes suggesting they play a role in their specification. Altogether, our results suggest that bipolar cell subtype specification follows a multi-step model, through an undifferentiated bipolar cell intermediate, and that cell contact plays a role in the specification mechanisms of bipolar cell subtype development. This is a novel finding that provides insight into the mechanisms underlying retinal neuronal subtype development and possibly in other neuronal cell types throughout the CNS. / Graduate / 10000-01-01

Wolbachia are widespread obligate intracellular bacteria that are maternally transmitted and modulate reproduction of their invertebrate host. Mosquitoes transinfected with Wolbachia have reduced capacity for transmitting vector borne diseases and can replace native populations in the field because of a reproductive advantage. The cellular mechanisms of how reproduction is altered by Wolbachia are poorly understood. In this work Wolbachia-induced reproductive changes in the model organism Drosophila were used to pinpoint underlying cellular processes affected by the bacteria. Specifically, egg production (or fecundity) of Wolbachia-infected Drosophila mauritiana was compared to non-infected flies that had been generated by antibiotic treatment of infected flies. Immediately before the fecundity experiment backcrossing of both fly lines ensured an equivalent nuclear genetic background. Initially egg production in Wolbachia-infected flies was increased by 4-fold but in less than 30 generations this changed to a 0.84 fold decrease with a slight advantage for the non-infected line. Additional backcrossing experiments determined that selection on the host nuclear genome is one of the factors underlying this reversion of fecundity gains. Other non-Mendelian factors, such as the microbiota, may also play a role in this rapid change. Wolbachia alterations in egg production were always linked to Wolbachia induced changes in programmed cell death (PCD) in the germarium during oogenesis and germline stem cell (GSC) division. Germline stem cells are maintained and regulated through their interaction with the germline stem cell niche (GSCN). Interestingly, these cells are both frequently infected with Wolbachia and possess a high bacterial titer. A developmental time course revealed the mechanism of how Wolbachia accumulate in the niche cells. The data suggest that the bacteria actually coordinate their replication with the differentiation of the niche cells. Future work on understanding the cellular and molecular basis of Wolbachia - host interaction will not only give insight into novel mechanisms of host manipulation by a pathogen, but will also expand our current understanding of stem cell niche morphogenesis and modulation of stem cell proliferation. / 2018-06-05T00:00:00Z

Acetylsalicylic acid (ASA), aspirin, is a renowned NSAID that its role in the process of bone metabolism has recently come to light. However, the influence of ASA on the odontogenesis of human dental pulp cells (HDPCs) remains elusive. In search of materials that would synergize the healing potential of the dental pulp, this study aimed to investigate the role of ASA on the odontogenesis of HDPCs in vitro and the influence of ASA on TGF-ß1 liberation from dentin. HDPCs were cultured in a culture medium with different concentrations of ASA: 25, 50, 75, 100, 200 μg/mL and 0 μg/mL as a control. The mitochondria activity of HDPCs was assessed using an MTT assay. Crystal violet staining and triton were used to evaluate cell proliferation rates. ALP activity was measured with the fluorometric assay. Expressions of DSP and RUNX2 were determined with ELISA. DSP and RUNX2 mRNA levels were measured with RT‐qPCR. Alizarin red staining was conducted to evaluate the mineralized nodule formation. Dentin slices were submerged in PBS (negative control), 17% EDTA (positive control), and ASA before collecting the solution for TGF-ß1quantification by ELISA. The data were analyzed by t tests and ANOVA followed by the Tukey post hoc tests. P values < 0.05 were considered statistically significant. The results showed that 25-50 μg/mL ASA promoted mitochondria activity of HDPCs at 72h (P<0.05) and yielded significantly higher proliferation rates of HDPCs than the control at 14d and 21d (P<0.001). All concentrations of ASA promoted odontogenic differentiation of HDPCs by enhancing the mineralization and the levels of DSP, RUNX2, and their mRNA expression in a dose-dependent manner (P<0.05). Also, ASA yielded significantly higher TGF-ß1 liberation after conditioning dentin for 5min (P<0.001) and 10min (P<0.05). In conclusion, the data suggest that ASA promotes the odontogenic potential of HDPCs and TGF-ß1 liberation from dentin in vitro and might be incorporated into the novel pulp capping materials for dental tissue regeneration.

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