Experimental hematology最新文献

DNMT3A encodes an enzyme that carries out de novo DNA methylation, which is essential for acquiring cellular identity and specialized functions during cellular differentiation. DNMT3A is the most frequently mutated gene in age-related clonal hematopoiesis (CH). DNMT3A-mutated hematopoietic stem and progenitor cells retain their ability to differentiate, giving rise to DNMT3A-mutated differentiated immune cells in circulation and tissues. Previously, we demonstrated that DNMT3A haploinsufficiency causes dichotomous DNA methylation defects at enhancers in human macrophages and alters the expression of a subset of genes involved in immune response and inflammation. Intriguingly, DNA methylation at the promoters of differentially expressed genes remained unchanged in the mutant macrophages, and the molecular link between DNA methylation defects at enhancers and altered gene expression was unclear. Through a deep characterization of chromatin features and mapping of enhancer-promoter interactions, we demonstrate that the genes whose expression is altered in DNMT3A-mutated macrophages are enriched with repressive histone marks that have been shown to cross-talk extensively with DNA methylation. Similar results were obtained from independent model systems including murine models of inducible DNMT3A mutation. These findings provide insights into the mechanism of immune dysfunction associated with CH and acquired DNMT3A mutations.

Pediatric acute leukemia is clinically and biologically differentiated from adult acute leukemia. The latter is a disease associated with aging while in childhood leukemia is associated with growth and development. In fact, pediatric leukemia is considered and studied as a developmental disease. In this session, I will present the advances that our laboratory has made in recent years in the biology and clinical picture of pediatric B-cell acute lymphoblastic leukemia (B-ALL), especially that associated with rearrangements in the KTM2A (MLL) gene. I will discuss aspects of the cell of origin and the leukemia-initiating cell as well as other cellular, molecular and genetic aspects that underlie the etiology and pathogenesis of these pediatric/childhood leukemias. Finally, I will share recently discovered therapeutic aspects to circumvent glucocorticoid resistance.

Hematopoietic stem cell (HSC) gene therapy is a promising treatment option for various genetic blood diseases/disorders. To enable efficient, precise, and safe modification of HSCs for gene therapies ex vivo and in vivo, robustly expressed and specific antigens are necessary to isolate and target HSCs. Here, we investigated the expression and cell-surface abundance of various commonly used HSC antigens such as CD34, CD90, CD117, and others using quantitative flow cytometry and bulk/single-cell RNAseq of human and NHP HSCs. We further studied the impact of mobilization on the transcription and cell surface presentation of antigens to inform the best route for the in vivo administration of HSC-targeted agents. Our analysis found the glycoprotein CD90 to be a robustly and stably expressed antigen on the surface of human and NHP HSCs regardless of donor or collection methodology, whereas expression of CD117 was downregulated on mobilized HSCs. As such, we developed second-generation targeted viral particles (VP) to the CD90 antigen, capable of highly specific transduction and editing of human HSCs in vivo using a murine xenograft model. CD90-targeted VPs targeted HSCs with over 100-fold more efficiency than any other hematopoietic subset in vivo. Additionally, modified HSCs were capable of unbiased repopulation of the hematopoietic hierarchy six weeks after VP administration and secondary transplantation. These results support previous studies identifying the HSC containing CD34+CD45+CD90+CD45RA- (CD34+CD90+HSCs) subset as responsible for long-term hematopoietic reconstitution. Thus, targeting CD90 on human HSCs provides a novel platform to modify quiescent HSCs for in vivo gene therapy without perturbing normal hematopoietic output.

Organismal aging is thought to be mediated by the interaction of multiple genetic and environmental variables acting cumulatively over long periods of time, confounding mechanistic insights into this process. In this study, we measured a wide range of hematologic variables (cell counts, histology, flow cytometry, HSC transplantation, scRNA and scATAC-seq) from hematopoietic tissues across a large cohort (>100 individuals) of young (8 weeks), middle aged (18 months) and old (>24 months) female C57BL/6J mice housed in the same controlled environment. Surprisingly, the aged phenotypes across the cohort were highly variable, with some 24-month-old mice displaying parameters in line with 8-week-old controls while others demonstrating extreme aged outcomes, despite the minimal variance in genotype and environment. This suggests a dominant stochastic basis to hematopoietic aging that is rarely considered in the literature. Importantly, canonical age-associated phenotypes that are thought to have a causal relationship (HSC functional potential, HSC expansion, myeloid bias, anemia) poorly correlated across the cohort, challenging the concept that HSC dysfunction drives the evolution of aged hematopoiesis. scRNAseq of bone marrow HSCs, progenitors, mature hematopoietic and niche cells identified a new population of inflammatory adipocytes precursors exclusive to, but heterogenous across aged individuals. Interaction analysis suggests that these cells receive inflammatory signals from neutrophils (Il1b and Tnf), and downregulate ligands (Kitl, Vcam1, and Angpt2) that typically signal to HSCs, potentially mediating HSC decline during aging. Taken together, these findings challenge the notion of a uniform hematological aging process stemming from compromised HSCs, but rather indicate a stochastic process, which extends to a heterogenous niche composition.

Thrombopoietin (TPO), acting via its receptor (MPL), is a master regulator of hematopoiesis and an exemplar pleiotropic cytokine – supporting HSC maintenance and driving megakaryocyte differentiation. The importance of TPO signalling is exemplified by human diseases; activating mutations in MPL or associated proteins JAK2 and CALR, lead to sustained MPL activation and myeloid malignancy, whereas loss-of-function mutations cause thrombocytopenia, HSC depletion and bone marrow failure. Recently, we have re-defined the molecular mechanisms of MPL activation, demonstrating that the receptor is monomeric at the membrane and is dimerized by TPO. In the same study, we showed that JAK2V617F, the primary driver mutation in MPN development, was able to promote TPO-independent MPL dimerization, and now have data showing we can block MPL oncogenic activity by targeting the MPL extracellular domain.

Our understanding of the TPO-2xMPL complex was further improved when we solved the structure of the complex using cryo-electron microscopy. This has, for the first time, uncovered detailed information on molecular interactions between TPO and MPL, allowing us to manipulate these interactions to alter receptor activity and uncouple TPO pleiotropic activity. By engineering TPO variants, which can fine-tune signalling output, we now have the potential switch between the role of TPO in HSC maintenance and platelet production.

Macrophages are fascinating immune cells involved in a variety of processes in both health and disease. Although they were first discovered and characterized by their functions as professional phagocytes and antigen-presenting cells, it is now clear that macrophages have multiple roles within embryonic development, tissue homeostasis, regulation of inflammation, and host response to pathogens and tissue insults. Interestingly, macrophages, or macrophage-like cells, exist in a variety of organisms, from echinoderms to humans, and are present also in species that lack an adaptive immune system or hematopoietic stem cells (HSCs). In mammals, macrophages can be generated from bone marrow precursors through a monocyte intermediate, but it is now known that they are also generated during earlier hematopoietic waves in the embryo. Seeding a variety of tissues at different times, macrophages contribute to embryonic organogenesis and tissue homeostasis. Interestingly, in species where embryonic macrophages are generated before HSC specification, they seem to be an important component of the HSC generative microenvironment. There are many excellent reviews reporting the current knowledge on the ontogeny and functions of macrophages in adult tissues. Here, we aim to summarize the current knowledge on the development and functions of embryonic macrophages across the most used animal models, with a special focus on developmental hematopoiesis.

Adult blood cells are produced in the bone marrow by hematopoietic stem cells (HSCs), the origin of which can be traced back to fetal developmental stages. Indeed, during mouse development, at days 10–11 of gestation, the aorta–gonad–mesonephros (AGM) region is a primary site of HSC production, with characteristic cell clusters related to stem cell genesis observed in the dorsal aorta. Similar clusters linked with hematopoiesis are also observed in the other sites such as the yolk sac and placenta. In this review, I outline the formation and function of these clusters, focusing on the well-characterized intra-aortic hematopoietic clusters (IAHCs).

Embryonic and fetal hematopoietic stem and progenitor cells differ in some key properties from cells that are part of the adult hematopoietic system. These include higher proliferation and self-renewal capacity, different globin gene usage, and differing lineage biases. Although these evolved to cover specific requirements of embryonic development, they can have serious consequences for the pathogenesis of hematologic malignancies that initiate prebirth in fetal blood cells and may result in a particularly aggressive disease that does not respond well to treatments that have been designed for adult leukemias. This indicates that these infant/pediatric leukemias should be considered developmental diseases, where a thorough understanding of their unique biology is essential for designing more effective therapies. In this review, we will highlight some of these unique fetal properties and detail the underlying molecular drivers of these phenotypes. We specifically focus on those that are pertinent to disease pathogenesis and that may therefore reveal disease vulnerabilities. We have also included an extensive description of the origins, phenotypes, and key molecular drivers of the main infant and pediatric leukemias that have a known prenatal origin. Importantly, successes in recent years in generating faithful models of these malignancies in which cellular origins, key drivers, and potential vulnerabilities can be investigated have resulted in uncovering potential, new therapeutic avenues.

Hematopoietic development goes through a number of embryonic sites that host hematopoietic progenitor and stem cells with function required at specific developmental stages. Among embryonic sites, the fetal liver (FL) hosts definitive hematopoietic stem cells (HSCs) capable of engrafting adult hematopoietic system and supports their rapid expansion. Hence, this site provides an excellent model to understand the cellular and molecular components of the machinery involved in HSC-proliferative events, leading to their overall expansion. It has been unequivocally established that extrinsic regulators orchestrate events that maintain HSC function. Although most studies on extrinsic regulation of HSC function are targeted at adult bone marrow (BM) hematopoiesis, little is known about how FL HSC function is regulated by their microniche. This review provides the current state of our understanding on molecular and cellular niche factors that support FL hematopoiesis.

Hematopoietic stem cells (HSCs) are routinely mobilized from the bone marrow (BM) to the blood circulation for clinical transplantation. However, the precise mechanisms by which individual stem cells exit the marrow are not understood. This study identified cell-extrinsic and molecular determinants of a mobilizable pool of blood-forming stem cells. We found that a subset of HSCs displays macrophage-associated markers on their cell surface. While fully functional, these HSC are selectively niche-retained as opposed to stem cells lacking macrophage markers which exit the BM upon forced mobilization. Macrophage markers on HSCs could be acquired through direct transfer via trogocytosis, regulated by cKIT, from BM-resident macrophages in mouse and human settings. Our study provides proof-of-concept that adult stem cells utilize trogocytosis to rapidly establish and activate function-modulating molecular mechanisms.