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Cytogenetic evaluation of human bone marrow cells is one of the principal sources of diagnostic and prognostic information in the evaluation of the myeloid leukemias. In the majority of cases, these diseases are characterized by non-random chromosomal changes in the cells of the malignant clone. The chromosomal abnormalities are present only in the leukemic cells, which are distributed along with normal cells in the bone marrow and throughout the circulation. The objective of this thesis was to test the hypothesis that suitable criteria could be established for automated metaphase detection using human bone marrow preparations. This involved computerized, low resolution scanning of a specimen slide, and the measurement of object features which allowed metaphases to be adequately distinguished from nuclei and debris. Two approaches were investigated. The first used a line-scanning system, in which microscope slides were scanned line by line with a linear CCD detector, and focussing was performed automatically. Eighteen signal features were measured for each detected object. Three group discriminant function analysis was performed on objects from a large number of slides from both types of preparations, in order to distinguish metaphases from nuclei and debris. The second method evaluated the use of a frame scanning system. Objects were detected in a frame-by-frame scan of microscope slides, using a two dimensional CD camera. Feature measurements were performed for all objects within a specified area range, and three group discriminant function analysis was performed on data from a large number of slides. In both approaches, the performance of the discriminant functions was evaluated on independent samples collected from a number of patients, in order to determine the operational error rates of the systems. The sensitivity of the line scan system for metaphase detection was 86%, compared to 92% fror the frame scannning system, while the specificity was 84% for the line scan system, and 86% for the frame scanning system. The frame scan system was shown to be useful for determining the mitotic index of cells cultured for varying periods of time prior to fixation. Four patients with AML were examined, and the results of the analysis show that the mitotic indices could be determined in this way to an accuracy of approximately 5%. The mitotic indices differed as a function of time for different patients. / Science, Faculty of / Physics and Astronomy, Department of / Graduate

An experimental animal model was developed to study and define the relationship between the blood flow and the marrow cavity pressure of bone. The study was carried out in 24 rabbits and 32 dogs under nembutol anesthesia. A multichannel physiograph was used to record simultaneously the systemic blood pressure, bone blood flow, marrow cavity pressure of bone and time sequence. The systemic blood pressure was measured by cannulating a brachial or carotid artery. The bone blood flow was measured by cannulating the nutrient vein and/or the nutrient artery. The marrow cavity pressure was measured by inserting a steel cannula through a drill hole in the cortex into the marrow cavity. The bones studied were mainly the tibia and femur. During the normal control condition, the marrow cavity pressure was found to have a wide range from animal to animal. In the rabbits, the range was from 20 to 60 mm. Hg. (15 to 50 percent of the systemic blood pressure). In the dogs, it ranged from 40 to 120 mm. Hg. (20 to 90 percent of the systemic blood pressure). However, the systemic blood pressure, intramedullary pressure and nutrient venous outflow were remarkably constant in a control period, therefore, their changes under experimental conditions were readily recordable. Various factors affecting bone circulation were studied. If the femoral vein is occluded the marrow cavity pressure rises and the nutrient venous outflow increases indicating venous congestion of bone. Nutrient artery occlusion causes a sharp fall in marrow cavity pressure coupled with a marked decrease in the nutrient venous outflow. Adrenalin and noradrenalin intravenous infusions produce a fall in marrow cavity pressure coupled with a decreased bone blood flow, despite an elevation in the systemic blood pressure. Isoproterenol hydrochloride generally causes a fall in the systemic blood pressure, widening of the pulse pressure, a fall in the marrow cavity pressure and a decrease in the nutrient venous outflow. Electrical sympathetic stimulation produces a fall in the marrow cavity pressure and a decrease in bone blood flow. Lumbar sympathectomy causes a rise in the marrow cavity pressure and an increase in the bone blood flow. Skeletal muscle contraction produces bone venous congestion with elevation of the marrow cavity pressure and increased nutrient venous outflow. Muscular relaxation causes a momentary sharp fall in the marrow cavity pressure to sub control levels before returning to the normal pressure. It is concluded that the marrow cavity pressure is bone blood flow dependent and reflects well the changes in the hemodynamics of bone. The narrow pressure rises if the arterial blood supply to bone increases or the venous congestion occurs in bone. The marrow pressure falls if the arterial blood supply to bone decreases or the venous drainage of bone is facilitated. / Surgery, Department of / Medicine, Faculty of / Graduate

Acute myeloid leukemia (AML) often remains refractory to current chemotherapy and transplantation approaches despite many advances in our understanding of mechanisms in leukemogenesis. The bone marrow “niche” or microenvironment, however, may be permissive to leukemia development and studying interactions between the microenvironment and leukemia cells may provide new insight for therapeutic advances. Mesenchymal stem cells (MSCs) are central to the development and maintenance of the bone marrow niche and have been shown to have important functional alterations derived from patients with different hematological disorders. The extent to which MSCs derived from AML patients are altered remains unclear. The aim of this study was to detect changes occurring in MSCs obtained from human bone marrow in patients with AML by comparing their function and gene expression pattern with normal age-matched controls. MSCs expanded from patients diagnosed with acute leukemia were observed to have heterogeneous morphological characteristics compared to the healthy controls. Immunohistochemistry and flow data confirmed the typical cell surface immunophenotype of CD90+ CD105+ CD73+ CD34- CD45-, although MSCs from two patients with AML revealed reduced surface expression of CD105 and CD90 antigens respectively. Differentiation assays demonstrated the potential of MSCs from AML patients and healthy donors to differentiate into bone, fat and cartilage. However, the ability of MSCs from AML samples to support hematopoietic function of CD34+ progenitors was found to be impaired while the key hematopoietic genes were found to be differentially expressed on AML-MSCs compared to nMSCs. These studies indicate that there exist differences in the biologic profile of MSCs from AML patients compared to MSCs derived from healthy donors. The results described in the thesis provide a formulation for additional studies that may allow us to identify new targets for improved treatment of AML.

Osteoporosis is a systemic skeletal disease that affects millions of people worldwide. There are many possible etiologies for osteoporosis, including inherent variables like genetics and sex, and lifestyle variables like diet and exercise. Characterized by low bone mass and increased fracture risk, the disease places a burden on both the patients and the healthcare industry. Therefore, it is vital that research determine the mechanisms by which the risk factors affect BMD so that better diagnosis and treatment options may be developed. The purpose of this study was to examine the relationship between various osteoporosis risk factors and biochemical markers of osteogenic cell activity derived from bone-marrow MSCs. It was hypothesized postmenopausal white women, having the greatest risk for osteoporosis, would have elevated hydroxyproline and decreased ALP, indicative of greater bone resorption. Acetabular reamings were collected from 26 patients (15 males and 11 females) undergoing total hip arthroplasty at Boston Medical Center. MSCs from the reamings were plated and underwent osteoinduction into osteoblasts. The cells were then harvested and assayed for various indicators of cell growth and bone cell activity, such as DNA, ALP, and hydroxyproline. Our hypothesis was generally supported in that postmenopausal white women did have less ALP, an indicator of bone deposition, than premenopausal women and postmenopausal African American women. Additional findings and directions for future studies are further discussed in this paper.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22319号 / 医博第4560号 / 新制||医||1041(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 濵﨑 洋子, 教授 稲垣 暢也, 教授 清水 章 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

In patients with aplastic anaemia that respond to immunosuppressive therapy, quantitative, morphological and functional haematologic derangement have been reported. To explain these findings, abnormalities in the marrow stroma or the stem cell have been postulated. To define the relative contribution of each of the latter, the integrity of the bone marrow from sixteen patients that responded to anti-lymphocyte globulin and high dose methyl prednisolone was compared to normal individuals. Bone marrow mononuclear cells were divided into two fractions. From the first, stroma was cultured in aMEM containing 12.5% of both horse and foetal calf serum and 10-5 M hydrocortisone at 37° C in 5% CO2 in 90% humidity. The medium was changed weekly. Upon confluence, these stromal layers were studied morphologically and with cytospin preparations stained with Sudan black, 0 red oil, alkaline and acid phosphatases. The remainder was monocyte and lymphocyte depleted, CD 34+ progenitors were selected with paramagnetic beads and the population morphologically and immunophenotypically defined. To determine the functional status, control or patient CD 34+ progenitors, were suspended for two hours on normal or aplastic stroma for adherence to take place. The non-adhesive fraction was decanted by standardised washing and cultured for fourteen days in the presence of PHA-conditioned medium in the CFU-gm assay. Strama-adherent progenitors were covered with 0.3% agar and cultured for five days. Aggregates with more than twenty cells were scored (CFU-bl). The remaining CD 34+ cells were cultured in the mixed colony assay with combinations of recombinant cytokines belonging to the G protein super-family and the tyrosine kinase group in dose response studies. Light density cells from patients with treated aplasia contained significantly fewer CD 34+ cells than those present in the control suspensions (mean 0.65%, SD 0.35% vs 1.62%, SD 1.4%; p= 0.002). Normal and aplastic stroma became confluent at three and four weeks. There was no difference on the morphology or the cytochemical stains between the two groups. Functionally, aplastic bone marrow stroma supported CFU-bl formation no differently from normal layers. However, CD 34+ precursors from the patients cultured on control stroma resulted in significantly fewer CFU-bl (p= 0.0002,) and CFU-gm (p= 0.0009). This work provides original evidence supporting the reduced clonogenicity of the corresponding populations of CFU-bl from patients with aplasia is unrelated to attachment to the stroma, but intrinsic to the CD 34+ cells. Moreover, this study shows for the first time that exposure of these progenitors to growth factors belonging to the G protein and tyrosine kinase receptor families have defective responses, correctable only at supra physiological concentrations, while effects on combinations containing c-kit ligand, appear preserved. Following immunosuppressive therapy, the bone marrow is repopulated by a hypoproliferative progenitor cell population which responds suboptimally to physiological cytokine stimulation. This suggests that abnormal interactions between receptors and their ligands or alterations in the signal transduction for cell division by the cytokines belonging to the G superfamily lead to suboptimal growth.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23372号 / 医博第4741号 / 新制||医||1051(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 滝田 順子, 教授 髙折 晃史, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / The entire hematopoietic system rests upon a group of very rare cells called hematopoietic stem cells (HSCs). Due to this extraordinarily crucial role, after birth HSCs are localized to the deep bone marrow niche, a hypoxic environment inside the bone where HSCs are under well-orchestrated regulation by both cellular and humoral factors. Among the cellular components regulating hematopoiesis are Leptin Receptor (LEPR)-expressing mesenchymal/stromal cells and adipocytes; both have been demonstrated to have significant influence on the maintenance of HSCs under homeostasis and in stress-related conditions. It has been reported in early work by others that HSCs and hematopoietic progenitor cells (HPCs) express LEPR. However, whether LEPR+ HSCs/HPCs are functionally different from other HSCs/HPCs was unknown. In this study, I demonstrated for the first time that murine LEPR+ Lineage-Sca-1+cKit+ (LSK, a heterogenous population consisting of HSCs/HPCs) cells even though constituting a small portion of total LSK cells are significantly enriched for both phenotypic and functional self-renewing long-term (LT) HSCs as shown in primary and secondary transplants in lethally irradiated recipients. LEPR+LSK cells are also more enriched for colony-forming progenitor cells assessed by colony-forming unit (CFU) assays. In addition, LEPR+ HSCs (defined as LSKCD150+CD48-) exhibited robust repopulating potential as compared to LEPR-HSCs in long-term competitive transplantation assays. To elucidate the molecular pathways that may govern functional properties of LEPR+HSCs, bulk RNA-seq on freshly sorted cells was done. Gene set enrichment analyses (GSEA) revealed Interferon Type I and Interferon γ (IFNγ) Pathways were significantly enriched in LEPR+HSCs while mitochondrial membrane protein gene set was significantly enriched in LEPR-HSCs. Interestingly, proinflammatory signaling including IFNγ pathway has been suggested to be critical for the emergence of embryonic HSCs from the hemogenic endothelium. Altogether, our work demonstrated that LEPR+HSCs represent a small subset of highly engrafting adult BM HSCs. These results may have potential therapeutic implications in the field of hematopoietic transplantation as LEPR is highly conserved between mice and humans.

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