Experimental hematology最新文献

Hematopoietic stem and progenitor cells (HSPCs) reside in niches that provide regulatory signals for their function. HSPC clones have been examined by cellular barcoding but the clonality of niche endothelial (ECs) and stromal cells (SCs) is unknown. We hypothesized that leukemia alters niche clones to support leukemogenesis. We developed a zebrafish model of acute erythroid leukemia (AEL) by overexpression of CMYC under the blood specific promotor draculin (drl). We used the GESTALT technique to uniquely barcode single cells using CRISPR-CAS9 during embryonic development. We injected GESTALT embryos with drl:CMYC to induce AEL, barcode HSPCs and their niche. Barcode and scRNA-Seq of ECs revealed a decrease in EC clones (fc=-3.5,p< 0.05) and an AEL-induced angiogenic venous EC population. AEL marrows had less SC clones (fc=-2.1,p< 0.01) and scRNA-Seq of SCs revealed an increased fraction of lepr+ SCs (66 vs 24%). We hypothesized that AEL cells secrete a signal to remodel niche clones. We mined our transcriptome data for ligands upregulated in AEL cells and receptors expressed on ECs and/or SCs. We identified apelin upregulated in AEL cells (p< 0.0001) and receptors aplnra/b specifically expressed on niche ECs. We tested if apelin alone could remodel the niche by overexpressing apelin in HSPCs and found fewer (p=0.004) and larger (p< 0.02) EC clones. HSPC barcode analysis revealed expanded myeloid clones (p< 0.0001) characterized by increased macrophage and erythroid differentiation. Immunohistochemistry on human sections revealed that acute myeloid leukemia (AML) marrows express higher levels APLN and APLNR compared to controls demonstrating the relevance of apelin signaling in human disease. Our data reveals that apelin signaling mediates AEL-induced clonal and transcriptional remodeling of niche ECs to promote disease progression.

Hematopoietic Stem Cells (HSC) are known for their regenerative potential which allowed their use in bone marrow transplantation to treat hematological disorders. However, aging results in HSC functional decline. Some consequences of HSC aging include inflammation leading to clonal hematopoiesis and myelodysplastic syndrome. The central goal of this project is to understand the mechanisms leading to HSC aging. Mitochondria are critical for HSC differentiation and homeostasis. We show that in aged HSC, mitochondria have increased sphericity, polarized network, lower mitochondrial membrane potential (MPP), but increased mass. We also show a decrease in number of lysosomes and in mitophagy events in aged HSC. A lipid trafficking assay showed an atypical pattern of lipid incorporation by mitochondria in aged HSC suggesting that mitochondrial lipids become abnormal upon aging. Cardiolipin (CL), a signature mitochondrial membrane lipid is essential to maintain mitochondrial membrane structure for optimum organelle-to-organelle interactions. We found reduced CL content in aged HSC, along with decreased protein expression of tafazzin, encoded by the gene TAZ, which is crucial for remodeling CL, compared to young. Using a doxycycline inducible, sh-RNA mediated TAZ KD mouse model, reduced Taz expression caused decreased HSC regenerative potential in competitive serial transplant assay. Furthermore, TAZ KD HSC exhibited fewer lysosomes localized near mitochondria, suggesting CL is crucial for channeling lysosomes towards mitochondria and initiating mitophagy. Incubation with a cardiolipin booster, Alcar, rescued the MPP and morphology in aged HSC. This work suggests that reduced levels of CL results in accumulation of abnormal mitochondria in aged HSC further contributing to decline in HSC functions with age.

Hematopoietic stem cells (HSCs) possess the ability to sustain the continuous production of all blood cell types throughout an organism's lifespan. Although primarily located in the bone marrow of adults, HSCs originate during embryonic development. Visualization of the birth of HSCs, their developmental trajectory, and the specific interactions with their successive niches have significantly contributed to our understanding of the biology and mechanics governing HSC formation and expansion. Intravital techniques applied to live embryos or non-fixed samples have remarkably provided invaluable insights into the cellular and anatomical origins of HSCs. These imaging technologies have also shed light on the dynamic interactions between HSCs and neighboring cell types within the surrounding microenvironment or niche, such as endothelial cells or macrophages. This review delves into the advancements made in understanding the origin, production, and cellular interactions of HSCs, particularly during the embryonic development of mice and zebrafish, focusing on studies employing (live) imaging analysis.

Cytoreductive treatments, such as 5-fluorouracil (5-FU), are often used as conditioning regimens in bone marrow (BM) transplantation and cancer therapy, eliminating highly proliferative hematopoietic progenitor cells and partially damaging the BM microenvironment. While the responses of the hematopoietic compartment to irradiation and chemotherapy have been studied in detail, the impact of these treatments on specific stromal components is less understood.

Here, we employ customized 3D microscopy and image-based analytical pipelines to investigate the dynamics and kinetics of injury and repair following treatment with 5-FU on sinusoidal endothelial and arterial cells (SECs and AECs), and CXCL12-abundant reticular cells (CARc) within the regenerated BM, as well as mapping the spatial distribution of HSCs. Finally, we integrate scRNA-seq data to reveal compositional changes in stromal networks and pathways involved in tissue regeneration.

We report that i) contrary to previous reports, CARc mostly survive 5-FU treatments and their numbers remain largely unaltered as determined by 3D-QM ii) myeloablation causes severe structural damage to CARc and vascular networks and fragmentation of CARc mesh iii) despite this, SECs and CARc demonstrate significant regenerative potential, restoring structural integrity and quantitative morphometric parameters iv) the regeneration of BM stroma coincides with HSC recovery and re-entry into quiescence v) while stromal networks regain their structure, the transcriptomic landscapes of both EC and MSC subsets remain strongly perturbed even after 16 weeks post 5-FU. These findings show that stromal networks possess self-organizing capabilities for rapid structural repair, but 5-FU treatment leads to long-term molecular changes in stromal cells, potentially affecting their functional regulation of hematopoiesis and HSC maintenance.

Acute myeloid leukemia (AML) is an acquired hematological malignancy resulting in the expansion of undifferentiated leukemic blasts at the expense of healthy hematopoiesis. The sympathetic nervous system (SNS) plays a key role in regulating leukemogenesis, but the precise mechanism remains unclear. We have found that in a mouse model of MLL-AF9-driven AML, ROS levels in the leukemic niche are elevated, particularly in myeloid-lineage cells. Treatment with antioxidants or genetically targeting NADPH Oxidase (NOX)-derived ROS prolonged survival and reduced leukemic burden. Inhibiting ROS in AML resulted in higher levels of CD8 cytotoxic T cell activation, suggesting that niche-derived ROS may suppress T cell activity. We hypothesize that this occurs due to a loss of sympathetic nerves. Indeed, chemical sympathectomy increased myeloid-derived ROS and reduced CD8 T cell activation in healthy and leukemic mice, and leukemic mice devoid of β2 adrenergic signaling had fewer total CD8 T cells and higher leukemic burden. The precise cell types suppressing CD8 T cells via ROS are likely to be myeloid lineage cells. Macrophages, neutrophils, and myeloid-derived suppressor cells express high levels of NOX, generate the highest levels of ROS during leukemia, and are implicated in the suppression of lymphocyte activation in other malignancies. The loss of sympathetic nerves in the bone marrow and CD8 T cell dysfunction, both which occur in patients, may be linked. Indeed, our data point to a role for the loss of SNS activity during leukemia as a driver of NOX-derived ROS production by myeloid cells and suppression of CD8 T cell responses. Promoting these beneficial neuro-immune interactions could help boost anti-AML immunity and improve survival in AML patients.

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Clonal Hematopoiesis of Indeterminate Potential (CHIP), a phenomenon in which a single hematopoietic stem cell (HSC) disproportionately contributes to the peripheral blood, is associated with an increased likelihood of developing many age-associated diseases. However, the connection to neurodegenerative diseases, such as Alzheimer's Disease (AD) is less clear. Prior studies have established that inflammation plays a central role in AD pathogenesis and mounting evidence suggests systemic inflammatory signals can be transmitted to the CNS, where they may play a direct role in microglia activation and plaque clearance. Here we investigated the role of the two most commonly mutated CHIP-associated genes, Dnmt3a and Tet2, in the pathogenesis of Alzheimer's Disease. We transplanted 5xFAD transgenic mice predisposed to develop familial AD with Dnmt3a-/-, Tet2-/- or wildtype (WT) bone marrow (BM). Mice were then challenged weekly with LPS to mimic systemic chronic inflammation seen in aging. 5xFAD mice transplanted with Dnmt3a-/- BM showed signs of exacerbated AD, including increased cognitive impairment and decreased microglia activation compared to those transplanted with WT BM. Mice transplanted with Dnmt3a-/- BM also had fewer peripheral immune cells infiltrating the brain. In contrast, 5xFAD mice transplanted with Tet2-/- BM showed improved cognitive status, increased amyloid plaque clearance and increased microglia activation, while having a higher percentage of activated infiltrating myeloid cells within the brain compared to WT controls. Our data suggest that Dnmt3a and Tet2 mutations impact peripheral immune cell infiltration leading to changes in microglia activation and AD pathogenesis. Overall, our study of CHIP and AD marks the first report in which Dnmt3a and Tet2, which have opposite roles in DNA methylation, induce opposing effects on disease progression.

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Background

Mean platelet volume (MPV) is an efficient, easily available, and cost-effective marker, which can be used in monitoring glycaemic status in Type 2 Diabetes Mellitus patients. Platelet volume indices (PVI) such as MPV are the indicators of increased platelet activity and can be considered as potential biomarkers for diabetic complications.

Methods & Results

Sixty four patients were included to study the link, between blood sugar levels and MPV in conjunction with glycated hemoglobin (HbA1c), in both men and women. Men were notably older with an age of 58.4 years compared to women who had an average age of 50.6 years. Men had a higher MPV of 9.2 fl compared to women 8.4 fl indicating differences in platelet characteristics based on gender within the diabetic population. Additionally, men had HbA1c levels at 8.4% compared to women at 7.9%, which suggests variations in blood sugar control between the genders. Similarly, men showed blood sugar levels at 194.7 mg/dL compared to women's 180 mg/dL. These results highlight the importance of considering gender factors in managing diabetes and stress the need for approaches to enhance patient care and outcomes.

Conclusion

Understanding the relationship between diabetes and thrombosis is crucial for assessing risks and devising management plans. Our research emphasizes the importance of Mean Platelet Volume (MPV) as a marker for evaluating the likelihood of blood clots in individuals, particularly concerning blood sugar control. By acknowledging the significance of MPV in foreseeing clotting events, healthcare providers can improve their ability to pinpoint individuals at risk and tailor treatments accordingly, ultimately enhancing outcomes in diabetes care. Further exploration of MPVs usefulness as a tool in blood clotting issues is necessary to fully exploit its clinical benefits.

Primary myelofibrosis (PMF) has hypercoagulability and venous thromboembolism (SVT). JAK2 V617F mutated patients with lower-risk IPSS have more thrombosis and myeloproliferation vs high-risk IPSS. JAK2 mutated splanchnic endothelium releases von Willebrand factor (VWF), P-selectin, Factor VIII (FVIII), and plasminogen activator inhibitor-1 (PAI-1). We studied VWF, soluble P-selectin (s-P-selectin), P-selectin expression, FVIII, and PAI-1 in 20 patients with PMF according to WHO-criteria. 11/20 were JAK2, 8/20 CALR, and 1/20 MPL mutated. 6/20 were low-, 6/20 intermediate-1-, 8/20 intermediate-2-risk IPSS. The mutations were conducted by ARMS-PCR gel electrophoresis. Hemoglobin and platelets, and VWF, sP-selectin, and PAI-1 were measured by automated analyzer and ELISA, respectively. P-selectin and FVIII were measured by flow cytometry and chromogenic assay, respectively. Patients were 67 years old (23-84 years). Nobody had thrombotic risk factors. 3/20 JAK2 mutated had portal vein thrombosis, 2 with intermediate-2 risk IPSS and 1 with low-risk IPSS, 2/20 JAK2 mutated had mesenteric vein thrombosis and splenic vein thrombosis with intermediate-1 risk IPSS and low-risk IPSS. SVT was diagnosed with computed tomographic scan and abdominal magnetic resonance imaging. JAK2 burden was higher in thrombosis vs without thrombosis (35% vs 10%) as well as hemoglobin (12 g/dl vs 10 g/dl) while platelets were comparable (300x109/L vs 370x109/L9). VWF and s-P-selectin were higher in thrombosis (55±5 ng/mL vs 72±5 ng/mL) vs without thrombosis (30±2 ng/mL vs 35±5 ng/mL) as well as P-selectin (40±5% vs 9±2%). FVIII was higher in thrombosis (330±30%) vs without thrombosis (170±5%). PAI-1 was elevated in thrombosis vs without thrombosis (88±5 ng/ml vs 62±2 ng/ml). These results suggest that PMF-SVT may underly a JAK2 mutated prothrombotic endothelium. Further studies are needed.