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.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20992号 / 医博第4338号 / 新制||医||1027(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 小柳 義夫, 教授 髙折 晃史, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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The enzyme gamma-glutamyl transferase (GGT) is a multifunctional enzyme that participates in a number of metabolic processes, including the conversion of leukotriene C4 (LTC4) to leukotriene D4 (LTD4). LTD4 is necessary for normal myeloid proliferation and differentiation. We have examined the ability of hematopoietic growth factors (HGF) to induce GGT enzyme activity and mRNA content in a HGF-responsive cell line (KG-1). Incubation of KG-1 with recombinant human cytokines interleukin-1β (IL- 1β), interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor (TNF), but not interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF) or monocyte colony-stimulating factor (M-CSF), results in significant increases in GGT enzyme activity. The increases in GGT activity are both dose- and time-dependent. In response to IL-1. Increases in enzyme activity are seen by 6 hours and activity is maximal by 24 hours. GGT mRNA increases also occur and peak by 3 to 6 hours. These results indicate that induction of increases in GGT mRNA levels and enzyme activity occur in myeloid cells in response to HGFs. This induction, together with the requirement for LTD4 for normal granulopoiesis, supports a role for GGT in the cellular events occurring in myeloid cells in response to HGFs.

Indiana University-Purdue University Indianapolis (IUPUI) / Adult hematopoietic stem cells (HSC) are routinely used to reconstitute hematopoiesis after myeloablation; however, transplantation efficacy and multilineage reconstitution can be limited by inadequate HSC number, or poor homing, engraftment or self-renewal. We have demonstrated that mouse and human HSC express prostaglandin E2 (PGE2) receptors, and that short-term ex vivo exposure of HSC to PGE2 enhances their homing, survival and proliferation, resulting in increased long-term repopulating cell and competitive repopulating unit (CRU) frequency. HSC pulsed with PGE2 are more competitive, as determined by head-to-head comparison in a competitive transplantation model. Enhanced HSC frequency and competitive advantage is stable and maintained upon multiple serial transplantations, with full multi-lineage reconstitution. PGE2 increases HSC CXCR4 mRNA and surface expression and enhances their migration to SDF-1α in vitro and homing to bone marrow in vivo and stimulates HSC entry into and progression through cell cycle. In addition, PGE2 enhances HSC survival, associated with an increase in Survivin mRNA and protein expression and reduction in intracellular active caspase-3. While PGE2 pulse of HSC promotes HSC self-renewal, blockade of PGE2 biosynthesis with non-steroidal anti-inflammatory drugs (NSAIDs) results in expansion of bone marrow hematopoietic progenitor cells (HPC). We co-administered NSAIDs along with the mobilizing agent granulocyte-colony stimulating factor (G-CSF) and evaluations of limiting dilution transplants, assays monitoring neutrophil and platelet recoveries, and secondary transplantations, clearly indicate that NSAIDs facilitate mobilization of a hematopoietic graft with superior functional activity compared to the graft mobilized by G-CSF alone. Enhanced mobilization has also been confirmed in baboons mobilized with G-CSF and a NSAID. Increases in mobilization are the result of a reduction of signaling through the PGE2 receptor EP4, which results in marrow expansion and reduction in the osteoblastic HSC niche. We also identify a new role for cannabinoids, an eicosanoid with opposing functions to PGE2, in hematopoietic mobilization. Additionally, we demonstrate increased survival in lethally irradiated mice treated with PGE2, NSAIDs, or the hypoxia mimetic cobalt chloride. Our results define novel mechanisms of action whereby eicosanoids regulate HSC and HPC function, and characterize novel translational strategies for hematopoietic therapies.

Cryopreservation is currently the only method allowing for the long-term preservation of hematopoietic stem and progenitor cell (HSPC) grafts until their use. However, cryoinjuries reduce cell viability and potency of HSPC. New cryoprotectant (CPA) solutions have recently emerged that have not yet been investigated that may improve the cryopreservation of HSPCs. The overarching hypothesis of the work described in this thesis, is that different CPAs have diverse impact on the key biochemical processes essential for HSPC homeostasis which influences post thaw cell viability and potency. To test this hypothesis, 4 CPAs were extensively characterized for their cryoprotective properties on cord blood (CB) HSPCs in comparison to DMSO control. CryoProtectPure (CPP) supported similar post thaw cell viability and engraftment as DMSO control, whereas pentaisomaltose (PIM) and cryonovo (CN) failed as CPAs for HSPCs. Subsequently, the impact of CPAs on key biological pathways was explored to identify potential biochemical pathways implicated in HSPC cryopreservation. The impact of CPAs on cell membrane integrity, oxidative phosphorylation, glycolysis, and autophagy was examined. CPP and DMSO had varying impact on glycolytic and mitochondrial respiratory activities of HSPCs post-thaw, whereas both CPAs as well as PIM and CN had negligible impact on cell membrane parameters prefreeze. Cryopreservation and thawing strongly induced autophagy in HSPCs. Importantly, early inhibition of autophagy with 3-Methyladenine (3-MA) reduced the recovery of functional CB HSPCs post thaw. Together, my findings provide new insights regarding the biological processes impacted by CPAs and cryopreservation of HSPCs and identify potential targets to improve cryopreservation of HSC grafts.

Alloreactivity between donor cells against disparate host tissue is a natural and normal physiologic phenomenon after engraftment. Consequently, GVHD is a universally expected side effect after allogeneic HSCT. An effective strategy to prevent severe or fatal acute GVHD is require if the transplant is to be successful. The HSCT field has witnessed significant progress in the prevention and treatment of acute GVHD. However, select interventions come at the cost of losing the alloimmune activity that prevents relapse, the GVL effect, as many of the mechanisms which cause GVHD are shared with those responsible for GVL. Current efforts are focused on therapeutic interventions that not only alleviate the burden of acute GVHD but does so in a way that maintains the GVL effect. This review will provide an up-to-date overview of our current understanding of the diagnosis, risk stratification, immunobiology of acute GVHD, summarize efforts to prevent and treat the disease, and provide a perspective on future directions.

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