No description available.

No description available.

No description available.

Acute myeloid leukemia (AML), a blood malignancy resulting in abnormal hematopoiesis, is associated with alterations in the bone marrow environment (BME). Current treatments for this heterogeneous disease, mainly targeting the leukemic cells, are largely unsuccessful for the majority of AML subtypes. By better understanding the mechanisms by which the BME contributes to leukemogenesis, it may be possible to introduce more effective treatments for AML. Mesenchymal stromal/stem cells (MSCs) are essential cellular components of the BME/hematopoietic niche and have been shown to support normal hematopoiesis. As a critical component, they may have several roles in altering the BME, thus providing an excellent model for studying the BME in-vitro. Several studies have characterized AML-derived MSCs (AML-MSCs). However, their exact role in altering BME remains unclear. Here, we investigated the MSCs' potential role in BME alteration by investigating the genetic profiles of previously characterized AMLMSCs (n=29) and healthy donor MSCs (HD-MSCs) (n=8). We identified that among 7565 common genes, 21 genes were significantly differentially expressed in AMLMSCs. The CD248 gene was identified among these significantly upregulated genes in AML/HD-MSCs (n=29). Focusing on AML-MSCs derived from high-risk patients (HR), CD248 protein was investigated and validated using HR AML-MSCs (n=11) and HD-MSCs (n=4). Interestingly, it was highly abundant in HR samples at the intracellular and cell-surface levels. CD248 is an MSC marker and has a biological significance potentially on their function. To better understand its potential role in MSC, CD248 was knocked down (KD) in HD-MSCs using siRNA (siCD248-MSCs). Functional capacity, the ability of HD-MSCs and siCD248-MSCs to differentiate into cell types that form the BME (adipocytes and osteocytes), and their ability to promote the growth of HL60 human leukemia cell line were assessed. Posttransfection functional assessments showed that siCD248-MSCs had a reduced adipogenic but not osteogenic potential via differentiation assays. Quantitative validation of the adipogenesis pathway by qRT-PCR confirmed the reduction. KD CD248 increased SIRT2 expression and potentially led to adipogenesis inhibition. However, co-culture experiments showed no effect of HD-MSCs or siCD248-MSCs on HL60 proliferation. Together these data showed that CD248 is a potential player in adipogenesis, essential to MSC’s functionality. Thus, it could serve as a prognostic marker and target for AML therapy.

No description available.

Myelodysplastic syndrome (MDS) is a group of clonal bone marrow disorders characterized by bone marrow dysplasia with myeloblasts <20%, typically seen in older patients. MDS has a significant risk of transformation to Acute Myeloid Leukemia (AML). We report a case of MDS transformed to AML, with sustained Complete Remission and incomplete count recovery (CRi) with treatment. A 78-year-old male with a 2-year history of leukopenia had a workup including bone marrow biopsy (BMBX) revealing intermediate- risk MDS with 13% blasts (Refractory Anemia Excess Blasts II), deletion 20 on cytogenetics and normal MDS FISH panel. He was categorized as revised IPSS score 4.5 on risk stratification. Patient initiated treatment with hypomethylating agent Azacytidine with subsequent improved BMBX with 7% blasts. He continued Azacytidine with dose reductions due to cytopenia only to develop 14% blasts on another follow up BMBX. He continued successful treatment for over 3 years before developing with 40-50% CD 34+/CD117+ blasts in the bone marrow consistent with transformation to AML. He commenced salvage treatment with Venetoclax and full dose Azacytidine as advanced age and performance status precluded transplant options. Repeat BMBX 4 weeks following Venetoclax showed hypocellular marrow, blasts percentage less than 2% indicating a CRi. Two other subsequent marrow exams have demonstrated sustained CRi twelve months after transformation with continued Venetoclax and Azacytidine administration. Around 30% of MDS patients eventually transform to secondary AML. Azacytidine therapy has significantly improved survival and time to AML transformation in intermediate-2 and high-risk MDS patients. Venetoclax, a BCL-2 inhibitor, in treating AML. Based on the results of the VIALE-A trial, the incidence of CR (complete remission) was higher around 36.7% with Azacytidine-Venetoclax (A-V) compared to 17.9% with Azacytidine. The composite CR (CR+ Cri) was higher in the A-V group, 66.4% compared to 28.3% with Azacytidine group. The median overall survival was 14.7 months in the A-V group compared to 9.6 months in the Azacytidine group. Our patient achieved a CRi with A-V treatment and has demonstrated a durable response beyond 16 months in secondary AML which has a bleak prognosis indicating the promise of this new combination treatment.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24522号 / 医博第4964号 / 新制||医||1065(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 小川 誠司, 教授 萩原 正敏, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Acute myeloid leukemia (AML) is a fatal cancer of the human hematopoietic system characterized by the rapid accumulation of non-functional, immature hematopoietic cells in the bone marrow (BM) and peripheral blood (PB) of affected patients. Limited sources of safe hematopoietic stem/progenitor cells (HSPCs) for transplantation and incomplete mechanistic understandings of disease initiation, progression and maintenance have impeded advances in therapy required for improvement of long-term AML patient survival rates. Toward addressing these unmet clinical needs, the ability to generate induced pluripotent stem cells (iPSCs) from human somatic cells may provide platforms from which to develop patient-specific (autologous) cell-based therapies and disease models. However, the ability to generate iPSCs from human AML patient somatic cells had not been investigated prior to this dissertation. Accordingly, I hypothesized that cellular reprogramming of human AML patient somatic cells to iPSCs is possible and will enable derivation of autologous sources of normal and dysfunctional hematopoietic progenitor cells (HPCs). I first postulated that reprogramming AML patient fibroblasts (AML Fibs) to pluripotency would provide a novel source of normal autologous HPCs. Our findings revealed that AML patient-specific iPSCs devoid of leukemia-associated aberrations found in the matched bone marrow (BM) could be generated from AML Fibs, and demonstrated that this cellular platform allowed for the derivation of healthy HPCs capable of normal differentiation to mature myeloid lineages in vitro. During the tenure of these experiments we also redefined conventional reprogramming methods by discovering that OCT4 transcription factor delivery combined with culture in pluripotent-supportive media was minimally sufficient to induce pluripotency in AML and normal Fibs. Toward capturing and modeling the molecular heterogeneity observed across human AML samples in vitro, we next asked whether reprogramming of AML patient leukemic cells would enable generation of iPSCs and derivative HPCs that recapitulated dysfunctional differentiation features of primary disease. Our results demonstrated that conventional reprogramming conditions were insufficient to induce pluripotency in leukemic cells, but that generation of AML iPSCs could be reproducibly achieved in one AML sample when reprogramming conditions were modified. These AML iPSCs and their derivative HPCs harboured and expressed the leukemia-associated aberration found in the BM leukemic cells and similarly possessed dysfunctional differentiation capacities. Together, this body of works provides the proof of principle that cellular reprogramming can be applied on a personalized basis to generate normal and dysfunctional HPCs from AML patient somatic cells. These foundational findings should motivate additional studies aimed at developing iPSC-based cell therapies and disease models toward improving AML patient quality of life and long-term survival rates. / Thesis / Doctor of Philosophy (PhD)

Acute myeloid leukemia (AML) is a deadly cancer of the blood and bone marrow defined by the accumulation of immature and non-functional myeloid progenitor cells. While AML is associated with a high success of chemotherapy-induced remission, it is accompanied by high relapse rates with poor response to subsequent therapies. Therefore, relapsed AML patients only have a 10% probability of long-term survival. An effective postinduction therapy is allogeneic hematopoietic stem cell transplantation (HSCT). However, complications associated with HSCT can be more severe than the AML disease itself. To date, no robust methodology is available to prospectively identify and distinguish AML patients that are more likely to benefit from HSCT. Our group has shown that AML patients with high leukemic progenitor cell content (LPC+) have a significantly lower overall survival (OS) when compared to patients with lower LPC content (LPC-). The objective of this study was to determine whether the LPC assay can be used as a functional predictor of post-HSCT survival. We hypothesized that LPC content correlates to post-HSCT survival times. We performed LPC assays on over 100 primary AML patient samples, showing that HSCT significantly improved OS in both LPC+ and LPC- patients, but LPC+ patients benefited more strongly than LPC- patients. This provides an initial basis to suggest that HSCT can offset the negative prognostic impact associated with high LPC content. To understand the biology of LPCs, we employed the Infinium HumanMethylation450 BeadChip assay to determine whether there are any methylation patterns that distinguish LPC+ and LPC- patients. However, we were not able to discover any uniquely methylated regions that separate the two groups, suggesting for further studies with an increased patient cohort, or extending the analyses to the transcript level. Given the rarity of curative approaches to cancers, a prognostic measure that could determine whether any single patient will benefit from HSCT will have an immediate impact. / Thesis / Master of Science (MSc)

Tissue engineering is a promising approach that combines the interactions of biomaterials, cells, and growth factors to stimulate tissue growth and regeneration. As such, selecting a suitable biomaterial is vital to the success of the procedure. Ideally, the material should show similarity to the extracellular matrix in the structure and relative stiffness, and biofunctionality beside others to provide a comfortable environment for the cells. Additionally, the biomaterial properties should allow for the effective diffusion of relevant growth factors and nutrients throughout the material to enable cell growth. Because peptides are composed of amino acids found naturally within the human body, they are considered non-toxic and biocompatible. Ultrashort peptides are peptides with three to seven amino acids that can be self-assembled into helical fibers forming scaffolds of supramolecular structures. These peptide hydrogels formed a highly porous network of nanofibers which can quickly solidify into nanofibrous hydrogels that resemble the extracellular matrix (ECM) and provide a 3D environment for cells with suitable mechanical properties. Furthermore, we can easily tune the stiffness of these peptide hydrogels by just increasing peptide concentration, thus providing a wide range of peptide hydrogels with different stiffness for 3D cell culture applications. Herein we describe the use of ultrashort peptide hydrogels for the maintenance and the differentiation of human mesenchymal stem cells into the osteogenic lineage. Furthermore, we develop a three dimensional (3D) biomimicry acute myeloid leukemia (AML) disease model using biomaterial from a tetramer ultrashort self-assembling peptide. In addition, we evaluate the potential application of peptide hydrogels as a hemostatic agent. The results presented in this study suggest that our biomimetic ultrashort tetrapeptide hydrogels are an excellent candidate for tissue engineering and biomedical applications.