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For over 50 years, bone marrow transplants have used CD34 to select stem cells. Recent research suggests that the most primitive hematopoietic stem cells (HSCs), long-term HSCs (LT-HSCs), are found in the CD34-negative portion of murine and human bone marrow cells. LT-HSCs are rare and cannot be isolated directly, making them difficult to study. During a bone marrow transplant, these stem cells must find their way to the bone marrow niche and engraft to become blood cells. Several cell adhesion molecules on the stem cell engage with their ligands on the endothelial cells lining the bone marrow vasculature to control this migration. Human LT-HSCs cells do not migrate and engraft well when infused in vivo, which may be due to a lack of adhesion molecules. Thus, the goal of this study was to determine whether this population of HSCs lacked adhesion systems (proteins and carbohydrate modifications) and, if so, to improve their migration and engraft ability by modifying key mechanistic steps in the adhesion cascade. Therefore, we investigated how distinct hematopoietic stem cell populations migrate to the bone marrow using adhesion mechanisms. This study represents the first direct analysis of adhesion molecules expression in LT-HSC and will potentially shed light on methods to optimally use these very valuable cells in the clinical bone marrow and cord blood transplants worldwide.

No description available.

On average, 65 Canadian men will be diagnosed with prostate cancer (PC) every day, making it the most common male cancer in Canada. Despite the prevalence, the etiology of PC is unknown. Evidence nonetheless supports the role of prostate cancer stem cells (PCSCs) in PC initiation and metastasis. In spite of almost a decade worth of research on PCSCs our knowledge on their biology remains fragmented. By taking advantage of the availability of DU145 cell-derived PCSCs in our laboratory, this thesis research focuses on investigating the unique properties of PCSCs and their function in promoting PC tumorigenesis. We identified two PCSC-specific proteins, ALDH3A1 and CNTN1. In mouse models of xenograft tumors, ALDH3A1 was expressed at higher levels in PCSC-derived tumors than in DU145 non-PCSC-produced tumors and in lung metastases than local tumors. In clinical settings, elevation of ALDH3A1expression was observed from normal prostate tissues to carcinomas and from local PCs to the paired lymph node metastases. Additionally, ALDH3A1 was clearly detected in bone metastases. Similar to ALDH3A1, CNTN1 expression associates with PC progression and biochemical recurrence following radical prostatectomy. The clear presence of CNTN1 in lymph node and bone metastases was also demonstrated. Furthermore, CNTN1 expression promoted PC metastasis to the lungs and tumor initiation in NOD/SCID mice. Mechanistically, CNTN1 increased AKT activation and reduced E-cadherin expression. Collectively, our research revealed important roles of both PCSC proteins in promoting PC tumorigenesis and progression. PC develops chemotherapy resistance in which PCSCs play a major role. In supporting this knowledge, we demonstrated that PCSCs are innately more resistant to the chemotherapeutic drugs, etoposide and docetaxel and that this resistance was in part attributable to their enhanced DNA damage response. Taken together, the findings of this thesis advances our knowledge on two specific PCSC markers and their association with prostate cancer progression and metastasis. As well as to the mechanism whereby PCSCs promote resistance to chemotherapeutic drugs. / Thesis / Doctor of Philosophy (PhD)

Indiana University-Purdue University Indianapolis (IUPUI) / Inner ear development requires the complex interaction of numerous cell types arising from multiple embryologic origins. Current knowledge of inner ear organogenesis is limited primarily to animal models. Although most mechanisms of cellular development show conservation between vertebrate species, there are uniquely human aspects of inner ear development which remain unknown. Our group recently described a model of in vitro human inner ear organogenesis using pluripotent stem cells in a 3D organoid culture system. This method promotes the formation of an entire sensorineural circuit, including hair cells, inner ear neurons, and Schwann cells. Our past work has characterized certain aspects of this culture system, however we have yet to fully define all the cell types which contribute to inner ear organoid assembly. Here, our goal was to reconstruct a time-based map of in vitro development during inner ear organoid induction to understand the developmental elements captured in this system. We analyzed inner ear organoid development using single-cell RNA sequencing at ten time points during the first 36 days of induction. We reconstructed the on-target progression of undifferentiated pluripotent stem cells to surface ectoderm, pre-placodal, and otic epithelial cells, including supporting cells, hair cells, and neurons, following treatment with FGF, BMP, and WNT signaling modulators. Our data revealed endogenous signaling pathwayrelated gene expression that may influence the course of on-target differentiation. In addition, we classified a diverse array of off-target ectodermal cell types encompassing the neuroectoderm, neural crest, and mesenchymal lineages. Our work establishes the Inner ear Organoid Developmental Atlas (IODA), which can provide insights needed for understanding human biology and refining the guided differentiation of in vitro inner ear tissue. / 2024-08-02

京都大学 / マギル大学 / 新制・課程博士 / 博士(ゲノム医学) / 甲第24204号 / 医博JD第2号 / 新制||医||JD1(附属図書館) / 京都大学大学院医学研究科京都大学マギル大学ゲノム医学国際連携専攻 / (主査)准教授 Wilson Michael (トロント大学), 教授 竹内 理, 准教授 Bailey Swneke (マギル大学), 教授 伊藤 貴浩, 教授 Shoubridge Eric (マギル大学) / 学位規則第4条第1項該当 / Doctor of Philosophy in Human Genetics / Kyoto University / McGill University / DFAM

Neural stem cells (NSCs) have been recently identified in the inferior colliculus (IC). These cells are of particular interest, as no casual therapeutic options for impaired neural structures exist. This research project aims to evaluate the neurogenic potential in the rat IC from early postnatal days until adulthood. The IC of rats from postnatal day 6 up to 48 was examined by neurosphere assays and histological sections. In free-floating IC cell cultures, neurospheres formed from animals from early postnatal to adulthood. The amount of generated neurospheres decreased in older ages and increased with the number of cell line passages. Cells in the neurospheres and the histological sections stained positively with NSC markers (Doublecortin, Sox-2, Musashi-1, Nestin, and Atoh1). Dissociated single cells from the neurospheres differentiated and were stained positively for the neural lineage markers β-III-tubulin, glial fibrillary acidic protein, and myelin basic protein. In addition, NSC markers (Doublecortin, Sox-2, CDK5R1, and Ascl-1) were investigated by qRT-PCR. In conclusion, a neurogenic potential in the rat IC was detected and evaluated from early postnatal days until adulthood. The identification of NSCs in the rat IC and their age-specific characteristics contribute to a better understanding of the development and the plasticity of the auditory pathway and might be activated for therapeutic use. / Neuronale Stammzellen wurden kürzlich im unteren Colliculus inferior (CI) identifiziert. Diese Zellen sind von besonderem Interesse, da es keine therapeutischen Optionen für geschädigte neuronale Strukturen gibt. Ziel dieses Forschungsprojekts ist es, das neurogene Potenzial im CI der Ratte von den ersten postnatalen Tagen bis zum Erwachsenenalter zu untersuchen. Der CI von Ratten vom 6. bis zum 48. postnatalen Tag wurde mit Neurosphären-Assays und histologischen Schnitten untersucht. In frei schwimmenden CI-Zellkulturen bildeten sich Neurosphären bei Tieren vom frühen postnatalen Alter bis zum Erwachsenenalter. Die Menge der gebildeten Neurosphären nahm im höheren Alter ab und stieg mit der Anzahl der Zelllinienpassagen. Die Zellen in den Neurosphären und die histologischen Schnitte zeigten eine positive Färbung mit neuronalen Stammzell-Markern (Doublecortin, Sox-2, Musashi-1, Nestin und Atoh1). Dissoziierte Einzelzellen aus den Neurosphären differenzierten und wurden positiv für die neuralen Abstammungsmarker β-III-Tubulin, GFAP und MBP angefärbt. Darüber hinaus wurden neuronalen Stammzell-Marker (Doublecortin, Sox-2, CDK5R1 und Ascl-1) mittels qRT-PCR untersucht. Zusammenfassend lässt sich sagen, dass ein neurogenes Potenzial im CI der Ratte von den frühen postnatalen Tagen bis zum Erwachsenenalter nachgewiesen und bewertet wurde. Die Identifizierung von neuronalen Stammzellen im CI der Ratte und ihre altersspezifischen Merkmale tragen zu einem besseren Verständnis der Entwicklung und der Plastizität der Hörbahn bei und könnten für eine therapeutische Nutzung aktiviert werden.

No description available.

Initial investigation of Moxtek wire grid polarizers composed of Al and coated with SiO2 - SiX - SiO2 (where SiX is used to indicate a Si rich layer whose complete composition is not to be disclosed for proprietary reasons) showed a growth of 3x in the inner (closest to Al) SiO2 layer after baking. Upon removing the X and varying rib composition and layering composition and geometries in 12 sets of before and after samples, no obvious growth was observed. Even baking the original unbaked sample yielded no growth. Our data suggest that the initial conclusion of buried oxide growth was flawed and that the observed changes in optical properties upon baking are either very sensitive to layer thicknesses (smaller than we can confidently observe) or due to some other mechanism. Here we present our sample preparation and analysis using the Focused Ion Beam (FIB), Scanning Transmission Electron Microscopy (STEM), and Energy Dispersive Xray Spectroscopy (EDXS).

Indiana University-Purdue University Indianapolis (IUPUI) / Hematopoietic stem and progenitor cells (HSCs/HPCs) transplantation is a curative treatment for a variety of hematologic and non-hematologic diseases. Successful HSC transplantation requires infusing patients with a sufficient number of long-term engrafting HSCs. As a result, research efforts have focused on optimizing the collection process. Previous work established that harvesting mouse bone marrow HSCs under low oxygen tension similar to that reported for the bone marrow niche in situ (physioxia), results in enhanced HSC recovery and function. However, collecting bone marrow cells under physioxia is not a clinically viable approach. Here, I demonstrated that the collection and processing of peripheral blood mobilized with G-CSF alone or G-CSF and Plerixafor under physioxia resulted in a greater number of phenotypically defined long-term engrafting HSCs. Using high-resolution single cell sequencing to explore the molecular programs governing HSCs under physioxia, I identified increased expression of genes involved in HSC self-renewal and maintenance. In contrast, HSCs under ambient air upregulated genes implicated in HSC differentiation, apoptosis, and inflammatory pathways. Furthermore, wild-type HSCs under physioxia revealed a significant reduction in gene expression and activity of the epigenetic modifier Tet2. Consequently, I evaluated the phenotyping, engraftment potential and gene expression of preleukemic Tet2-/- bone marrow cells under physioxia and ambient air. Unlike wild-type HSCs, Tet2-/- HSCs/HPCs were unresponsive to changes in oxygen tension. Notably, we observed similar phenotypes, functions, and self-renewal and quiescence gene expression in wild-type HSCs under physioxia and Tet2- /- HSCs under physioxia or ambient air. These findings imply that the preserved stemness and enhanced engraftment of HSCs under physioxia may in part be a result of Tet2 downregulation. Understanding the mechanisms regulating wild-type and preleukemic HSCs under physioxia will have therapeutic implications for optimizing HSC transplantation and mitigating the growth advantage of preleukemic stem cells. / 2022-12-15