Indiana University-Purdue University Indianapolis (IUPUI) / Hematopoiesis proceeds through three developmental phases, each with a unique and indispensable function. The individual roles of these phases in the pathogenesis of blood disorders is unknown. We have adapted murine lineage trace models to identify the relative contributions of embryonic, fetal, and adult hematopoietic phases to the origin of Juvenile Myelomonocytic Leukemia. We hypothesized that the fetal phase would have the most pronounced contribution to the development of JMML, a pediatric myeloproliferative disorder whose disease-initiating somatic mutations occur in utero. Progenitors expressing PTPN11E76K from all three waves were growth hypersensitive to GM-CSF due to hyperactive RAS-ERK signaling. However, fulminant myeloproliferation was only seen in fetal and adult cohorts. We observed equal disease severity in FLT3Cre; PTPN11E76K; ROSA26mTmG and CSF1R-MCM; PTPN11E76K; ROSA26YFP cohorts, which had high and low mutant allele frequencies, respectively. This led to the revelation that all progenitors in the BM niche of mutant animals have equal growth hypersensitivity and RAS-ERK hyperactivation due to non-cell autonomous effects of PTPN11E76K. We further identified that FLT3Cre has hematopoietic-restricted expression, and thereby circumvented morbidity from PTPN11E76K expression in endothelial and stromal cells. This led us to hypothesize that FLT3Cre; KrasG12D; ROSA26mTmG would be the first faithful model of JMML to express this disease-initiating mutation. Indeed, FLT3Cre; KrasG12D mice were born at expected Mendelian ratio and showed normal weight gain to 2 weeks of age. Thereafter, they acquired defining features of JMML including monocytosis, anaemia, thrombocytopenia, and hepatosplenomegaly. All FLT3Cre; KrasG12D mice succumb to a JMML-like disease, which was propagated following transplantation. This is in contrast with CSF1R-MCM; KrasG12D; ROSA26YFP mice, in which low mutant allele frequencies in either fetal or adult HSCs uniformly resulted in T-ALL. Our models reveal previously underappreciated features of JMML including an expansion of dendritic cells and a pronounced defect in T-lymphocyte development. We are the first to demonstrate non-cell autonomous effects of hematopoietic-restricted PTPN11E76K expression. Most importantly, we have shown that both the spatial and the temporal origin of JMML-initiating mutations will affect disease manifestations. Each of our findings suggest novel strategies to treat this intractable disease.

A 59-year-old man with lung cancer, peripheral blood leukocytosis and thrombocytosis without peripheral lymphadenopathy and hepatosplenomegaly was found to have pancreatic extramedullary hematopoiesis (EMH) in association with an 'atypical' myeloproliferative disorder. Studies for the Philadelphia chromosome and bcr-abl fusion product were negative. This is the first documented case in the literature of isolated EMH in the pancreas.

No description available.

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

Megakaryocytes (MK) are responsible for platelet biogenesis, which is thought to occur canonically in the adult bone marrow (BM) and in the fetal liver during development. However, emerging evidence highlights the lung as a previously underappreciated residence for MKs that may significantly contribute to circulating platelet mass. While a diversity of cells specific to the BM are known to promote the maturation and trafficking of MKs, little investigation into the impact of the lung niche on the development and function of MKs has been done. Here, we describe the application of single cell RNA sequencing (scRNA-Seq) coupled with histological, ploidy and flow cytometric analyses to profile primary MKs derived from syngeneic mouse lung and hematopoietic tissues. Transcriptional profiling demonstrated that lung MKs have a unique signature distinct from their hematopoietic counterparts with lung MKs displaying enrichment for maturation markers, potentially indicating a propensity for more efficient platelet production. Reciprocally, fetal lung MKs also showed the robust expression of cytokines and growth factors known to promote lung development. Lastly, lung MKs possess an enrichment profile skewed towards roles in immunity and inflammation. These findings highlight the existence of a lung-specific MK phenotype and support the notion that the lung plays an independent role in the development and functional maturation of MKs. In addition to MKs, the lung houses many resident hematopoietic cells, including hematopoietic stem and progenitor cells (HSPCs). The existence of lung HSPCs suggests that the differentiation and development of lung resident hematopoietic cells may occur in-situ. To investigate the potential role the lung has in instructing site specific hematopoiesis, we employed explant cultures of murine and human fetal lungs. This displayed adherent endothelial cells transitioning into floating hematopoietic cells, suggesting that the fetal lung is a source of hemogenic endothelial cells that have the functional capacity to undergo endothelial to hematopoietic transition (EHT) to produce HSPCs. Flow cytometric and functional assessment of fetal lung explants showed the production of HSPCs that expressed key EHT and pre-HSPC markers. Expression profiles revealed by scRNA-Seq and small molecule modulation demonstrated that fetal lung EHT is reliant on canonical EHT signaling pathways. These findings suggest that functional HECs are present in the fetal lung, thus establishing this location as a potential extramedullary site of de-novo hematopoiesis. Overall, these findings suggest that the lung may have a greater role in instructing tissue specific hematopoiesis and/or overall hematopoietic development.

No description available.

Qian, Wei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 184-199). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Thesis (Ph. D.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains viii, 168 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Indiana University-Purdue University Indianapolis (IUPUI) / The dawn of the genetic era has allowed for investigation of gene transfer therapy as a treatment for certain diseases. Fanconi anemia (FA) is a rare genetic disorder in which the majority of patients develops progressive bone marrow failure (BMF) and require bone marrow transplantation. A possible alternative treatment is autologous gene therapy; however, original clinical trials involving gene transfer for FA were unsuccessful. This has led to re-evaluation of the gene transfer protocols, the vectors and also a deeper investigation of the FA pathway itself. My work has focused on illuminating these areas to further advance gene transfer therapy for FA. Many gene transfer protocols require the hematopoietic stem and progenitor cells (HSC/HPC) to be collected and then transduced ex vivo. The most common collection method is mobilization of the HSC/HPC to the peripheral blood (PB) using granulocyte colony-stimulating factor (G-CSF) and collection via apheresis. In FA patients G-CSF fails to mobilize a sufficient number of HSC/HPC. This has led to research into agents such as AMD3100, a CXCR4 antagonist, which may replace or augment G-CSF mobilization. These data show in two FA murine models that AMD3100 synergizes with G-CSF resulting in a significant increase in mobilization as compared to G-CSF alone. Previous work in our lab has shown that prototype foamy virus (FV) is an efficient gene transfer vector. Here a modified FV vector is used to transduce mobilized FA cells. The data indicate that long-term repopulating cells mobilized with both G-CSF and AMD3100 can be efficiently transduced by our FV vector. Clinically, FA is characterized mainly by BMF, but also by myelodysplasia (MDS) and acute myeloid leukemia (AML). However, current FA murine models do not display these disease phenotypes. These data show that double-mutant Fancc-/-;Fancg-/- mice spontaneously develop BMF, MDS and complex random chromosomal abnormalities that the single-mutant mice do not. Importantly, this model closely recapitulates the phenotypes found in FA patients and may be useful as a preclinical platform to evaluate the molecular pathogenesis of spontaneous BMF and MDS in FA and novel gene transfer protocols for FA.

Indiana University-Purdue University Indianapolis (IUPUI) / Neurexin I alpha (NRXN1α) and Dystroglycan (DAG1) are membrane receptors which serve as mutual ligands in the neuronal system. Neurexophilins (NXPHs) bind NRXN1α. Both NRXN1α and DAG1 were expressed in primitive populations in human cord blood (huCB) and murine bone marrow (muBM), with high concentrations of NXPHs in huCB plasma. We evaluated effects of these molecules on huCB and muBM hematopoietic progenitor (HPC) and stem (HSC) cells. At both a single and population level in vitro, we found that NXPH1 is a potent inhibitor of HPC proliferation acting through NRXN1α, an effect antagonized by DAG1. Injection of recombinant NXPH1 in vivo resulted in myelo- and lymphosuppression, with absolute numbers and cycling status of functional and phenotypically defined HPCs dose- and time-dependently decreased, and absolute numbers and cycling status of phenotypically defined longer-term repopulation HSCs increased. Competitive transplants showed an initial decrease in engraftment of NXPH1-treated cells, with an intermediate stage increase in engraftment. The increase in HSCs is at least partially mediated by the mTOR pathway and is thought to be homeostatic in nature. These results demonstrate the presence and function of a regulated signaling axis in hematopoiesis centered on NRXN1α and its modulation by DAG1 and NXPH1.