Multipotent Hematopoietic Stem Cells Research Articles

Targeting Leukemic Stem Cells in Chronic Myeloid Leukemia: Is It Worth the Effort?

Chronic myeloid leukemia (CML) is a classical example of stem cell cancer since it arises in a multipotent hematopoietic stem cell upon the acquisition of the t(9;22) chromosomal translocation, that converts it into a leukemic stem cell (LSC). The resulting BCR-ABL1 fusion gene encodes a deregulated tyrosine kinase that is recognized as the disease driver. Therapy with tyrosine kinase inhibitors (TKIs) eliminates progenitor and more differentiated cells but fails to eradicate quiescent LSCs. Thus, although many patients obtain excellent responses and a proportion of them can even attempt treatment discontinuation (treatment free remission [TFR]) after some years of therapy, LSCs persist, and represent a potentially dangerous reservoir feeding relapse and hampering TFR. Over the past two decades, intensive efforts have been devoted to the characterization of CML LSCs and to the dissection of the cell-intrinsic and -extrinsic mechanisms sustaining their persistence, in an attempt to find druggable targets enabling LSC eradication. Here we provide an overview and an update on these mechanisms, focusing in particular on the most recent acquisitions. Moreover, we provide a critical appraisal of the clinical relevance and feasibility of LSC targeting in CML.

Single-Cell Transcriptome Analysis of Embryonic Erythro-Myeloid Progenitor Cells Reveals Lineage Heterogeneity

The paradigm that multipotential hematopoietic stem cells (HSC) differentiate through a hierarchical series of increasingly specified progenitors has recently been challenged by single cell transcriptome analyses, which suggest greater heterogeneity in progenitor populations and hierarchy than previously thought. These studies are complicated by the wide variety of hematopoietic progenitors at different stages of maturation concurrently present in the adult bone marrow. In the early mammalian embryo, unique hematopoietic progenitors arise prior to, and independently of, HSC. These include erythro-myeloid progenitors (EMP) that arise from hemogenic endothelium in the yolk sac and migrate to the fetal liver to jump-start definitive hematopoiesis before HSC emerge. In vitro, EMP can generate definitive erythroid, megakaryocyte and myeloid cells, although not B-cells, and include at least some single cells with both erythroid and myeloid potential (McGrath et al. 2015 Cell Rep. 11:1892). We have previously shown that day 9.5 (E9.5) murine EMP have a unique and uniform (kit+CD41+CD16/32+) immunophenotype in the yolk sac and circulation, which is maintained as they colonize the E10.5 liver. We performed single-cell transcriptome analysis of this apparently poised, embryonic-specific multi-potential hematopoietic progenitor population, asking whether it contains an underlying heterogeneity or intrinsic lineage hierarchy. High quality data, as assessed by transcriptomic complexity, read depth, and alignment distributions, were generated for 133 single-cell EMP (scEMP) derived from E9.5 mouse embryos. Use both of supervised and of semi-supervised analytical approaches uncovered transcriptome heterogeneity that allowed clustering of scEMP by expression patterns. Based on expression of known lineage-directing transcription factors and cell type enrichment analysis, clusters of cells were identified that could be annotated as multipotential hematopoietic, megakaryocyte/erythroid, myeloid, and a more specific macrophage lineage potential. A novel binary reduction data integration and transformation methods was developed and utilized to further refine and characterize single-cell classifications by facilitating cross platform comparisons with single cell RNA-seq data obtained from EMP progeny in E11.5 liver and with published gene expression datasets derived from adult hematopoietic progenitors and fetal macrophage populations. Predictions of lineage potential based on our analytical approaches were tested by sorting cells based on immunophenotypic markers differentially expressed in specific clusters and analyzing their potential using single cell colony-forming assays. These functional studies support the concept that immunophenotypically homogeneous EMP contain subpopulations of multipotential progenitors differentiating into erythro-megakaryocyte, myeloid, and macrophage-specific lineage fates. DisclosuresNo relevant conflicts of interest to declare.

Robust Autonomous Generation of Definitive Hematopoietic Stem Cells from Pre-Circulation Murine Embryos

Introduction:The ontogeny of mammalian hematopoiesis is organized in at least three embryonic hematopoietic waves. The first wave is characterized by the generation of primitive erythrocytes emerging from the yolk sac (YS); a second one produces bipotent erythro-myeloid progenitors also in the YS. Finally, multipotent definitive Hematopoietic Stem and Progenitor Cells (HSPCs) with the ability to reconstitute all mature adult blood lineages and sustain life long hematopoiesis emerge from a subset of endothelial cells named hemogenic endothelium (HE). The embryonic anatomical site of origin of these HSPCs is still under debate in the field. An intra-embryonic origin is well accepted, which primarily encompasses the Aorta-Gonad-Mesonephros region (AGM) and umbilical and vitelline arteries. In contrast, a YS contribution to adult hematopoiesis remains controversial. The same developmental origin (e.g. endothelial cells with a mesodermal ancestor) makes it difficult to identify specific markers to discern between an AGM- vs. YS-origin using a lineage trace approach. This together with the highly migratory nature of blood cells and the inability of cells isolated from pre-circulatory embryos to robustly engraft in transplantation assays, even after ex vivo culture, has precluded scientists from properly answering these questions. For example, cultured YS and para-aortic splanchnopleura (PSP, the precursor of the AGM) dissected from embryos prior to the onset of circulation resulted in the generation of HSPCs with multilineage engraftment potential only from the embryonic PSP and not the YS (Cumano A. et al., Immunity 2002). However, blood chimerism was low (<5%) and engraftment was achieved only after transplantation into severely immuno-compromised mice (i.e. Rag2gc-/-). This low engraftment baseline makes difficult any comparison between intraembryonic tissues (IET) and Yolk Sac because a smaller frequency of YS-derived transplantable HSPCs would be difficult to detect, yielding a false negative result.Thus, here we sought to interrogate if we could generate robust engraftment from pre-circulation embryos that would allow to rigorously compare the ability to derive definitive HSC in the IET and YS.Results:We have established ex vivo culture conditions to allow for the derivation of robust multi-lineage engraftment activity from pre-circulation murine embryos. CD45.2+ embryos at the 2-3 somite pairs (s.p.) stage and 5-7 s.p. embryos were dissected from their YS, cultured as explants in our optimized conditions and transplanted into lethally irradiated CD45.1+/CD45.2+ recipients. Here, we observed multilineage reconstitution of the peripheral blood (PB) for >16 weeks post-transplant in 5/7 and 9/11 recipients, respectively. Moreover these cells were serially transplantable, confirming the generation of self-renewing HSCs. In primary recipients, PB chimerism was >50% in 8/18 total recipients. This robust PB chimerism allowed us to rigorously interrogate the corresponding CD45.2+ YS (cultured under the same conditions). In these transplants, we observed PB engraftment in 0/7 and 0/11 recipients of cells isolated from 2-3 s.p. and 5-7 s.p. cultured YSs, respectively. These data suggest that either YS do not autonomously generate HSPCs or that they can not support their maturation ex vivo . Moreover we found that the engraftment ability from the IET is confined to an emerging CD31+CD45+c-Kit+CD41- population. Importantly, this population also develops in the YS explants, but lacks engraftment ability. We have repeated these studies with cultured 19-22 s.p. embryos and YSs, a post-circulation time point and still observed a failure of YS-derived cells to engraft irradiated recipients (0/9) while embryo-derived cells (9/9) engraft robustly. These data suggest that YS likely cannot support the maturation of HSPCs that might have migrated from other sites. Only when we transplanted 32-36 s.p. or 46-47 s.p. YS-derived cells we observed robust PB engraftment under our culture conditions (3/7 and 4/6 recipients engrafted, respectively).Conclusions:We observed robust generation of HSCs with long-term and serially transplantable multilineage potential from pre-circulation murine embryos. Our study demonstrates that the YS is not able to support the maturation of HSPCs. Further studies would be required to determine if the YS is an actual source of HSPCs. DisclosuresNo relevant conflicts of interest to declare.

A Long-Term Safety and Efficacy Study of Givinostat in Patients with Polycythemia Vera: The First 4 Years of Treatment

Background and purpose of the study: Chronic myeloproliferative neoplasms (cMPN) are clonal diseases resulting from the transformation of a multi-potent hematopoietic stem cell that leads to overactive hemopoiesis and bone marrow fibrosis [Tefferi et al., Am. J. Hematol. 2008; 83(6): 491-7].Several reports have documented that histone deacetylases inhibitors (HDACi) induce neoplastic cells to undergo growth arrest, differentiation and/or apoptotic cell death. Among these agents, Givinostat inhibits proliferation of cells bearing the JAK2V617F mutation and showed clinical significant activity with good tolerability in cMPN patients [Guerini et al., Leukemia 2008 Apr; 22(4): 740-7; Calzada et al., Exp. Hematol. 2012; 40 (8): 634-45; Rambaldi et al., Br. J. Haematol. 2010; 150 (4): 446-55; Finazzi et al., Br. J. Haematol. 2013;161(5):688-94]. In order to allow cMPN patients who have tolerated the drug and derived a clinical benefit to continue to receive Givinostat treatment, this multicenter, international, open label, long-term study testing the long-term safety, tolerability and efficacy of Givinostat was started (EudraCT N.: 2012-003499-37).Methods: Eligible patients for this study included JAK2V617F-positive cMPN patients of both genders, who had tolerated Givinostat treatment and achieved a clinical benefit at the end of the core protocols.Patients continued at their last tolerable dose and treatment schedule of Givinostat. Dose adjustments were permitted in order to optimize the response for each individual patient, avoiding specific drug-related toxicities.Patients remained on study therapy as long as they derive clinical benefit, evaluated at each quarterly visit according to the clinico-haematological European LeukemiaNet (ELN) criteria [Barosi et al., Blood 2009; 113 (20):4829-33], and European MF Network (EUMNET) criteria [Barosi et al., Blood 2005; 106:2849-53], for PV/ET and MF, respectively.Results: The study started on March 2013 and is currently recruiting patients in 15 European sites. A total of 48 cMPN patients were treated at the cut-off date of 26th July 2017. At this cut-off date, all treated myelofibrosis patients (n = 3) dropped out the study.Considering only Polycythemia Vera (PV) patients and the cut-off date of 26th July 2017, a total of 45 patients (median age: 65 years; 27 male/18 female) were treated, with a median time of treatment of 4 years (range, 6 months-9 years). The most used dose was 100 mg/die.Givinostat treatment was well tolerated for long-time treatment: no grade 4 adverse drug reaction (ADR) occurred; only one serious ADR (grade 3 anemia, resolved in 8 days) and a total of two grade 3 ADRs (asthenia and asymptomatic QTc prolongation) were reported.At the cut-off date of 26th July 2017, 33 out of 45 PV patients (73%) are still on treatment, whereas 12 (27%) stopped the drug for multiple reasons, including informed consent form withdrawal (n = 2), safety reasons (n = 6, mainly not drug related adverse events), disease progression (n = 3, after 4, 7 and 8 years of treatment, respectively) and loss of response (n = 1).In patients on treatment for a median of 4 years, CR and PR were observed in 11% and 89% patients, respectively: normal HCT - without phlebotomy -, PLT and WBC values were observed in 56%, 78% and 56% of PV patients, respectively. At that time, 56% of PV patients had a normal spleen size, evaluated by palpation and/or imaging. A net response in term of disease-related symptoms was also noted: after a median of 4 years of treatment, neither patient reported headache nor microvascular symptoms, and pruritus was absent in 89% of patients. At the cut-off date, four patients developed 5 thrombotic events (3 deep vein thrombosis of the legs, 1 transient ischemic attack, 1 myocardial infarction) with an overall incidence of thrombosis of 2.30% patients/years. After a median of 4 years of treatment, a decrease of JAK2V617F allele burden was also noted (22%).At the time of ASH Meeting 2017, the most updated data will be presented.Conclusions : This is the first report on the long-term treatment of PV patients with an HDACi (i.e. a median of 4 years of treatment, including 32% of patients treated for more than 7 years). In this study, the drug confirmed to be well tolerated. The efficacy is also remarkable: 73% of patients maintained at least a partial response for more than 4 years and the overall incidence of thrombosis was 2.30% patients/year. DisclosuresFinazzi:Italfarmaco S.p.A.: Consultancy. Iurlo:Bristol Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Vannucchi:Novartis: Honoraria, Speakers Bureau; Shire: Speakers Bureau. von Bubnoff:BMS: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. McMullin:Italfarmaco S.p.A.: Consultancy; Shire, Novartis: Honoraria, Speakers Bureau. Bettica:Italfarmaco S.p.A.: Employment. Manzoni:Italfarmaco S.p.A.: Employment. DI Tollo:Italfarmaco S.p.A.: Employment. Rambaldi:Novartis, Amgen, Celgene, Sanofi: Other: Travel, Accomodations, Expenses; Novartis, Roche/Genentech, Amgen, Italfarmaco: Consultancy.

Inflammation, Aging and Hematopoiesis: A Complex Relationship.

All vertebrate blood cells descend from multipotent hematopoietic stem cells (HSCs), whose activity and differentiation depend on a complex and incompletely understood relationship with inflammatory signals. Although homeostatic levels of inflammatory signaling play an intricate role in HSC maintenance, activation, proliferation, and differentiation, acute or chronic exposure to inflammation can have deleterious effects on HSC function and self-renewal capacity, and bias their differentiation program. Increased levels of inflammatory signaling are observed during aging, affecting HSCs either directly or indirectly via the bone marrow niche and contributing to their loss of self-renewal capacity, diminished overall functionality, and myeloid differentiation skewing. These changes can have significant pathological consequences. Here, we provide an overview of the current literature on the complex interplay between HSCs and inflammatory signaling, and how this relationship contributes to age-related phenotypes. Understanding the mechanisms and outcomes of this interaction during different life stages will have significant implications in the modulation and restoration of the hematopoietic system in human disease, recovery from cancer and chemotherapeutic treatments, stem cell transplantation, and aging.

Transplantation of CD34+ cells for myocardial ischemia.

CD34+ cells are multipotent hematopoietic stem cells also known as endothelial progenitor cells and are useful in regenerative medicine. Naturally, these cells are mobilized from the bone marrow into peripheral circulation in response to ischemic tissue injury. CD34+ cells are known for their high proliferative and differentiation capacities that play a crucial role in the repair process of myocardial damage. They have an important paracrine activity in secreting factors to stimulate vasculogenesis, reduce endothelial cells and cardiomyocytes apoptosis, remodel extracellular matrix and activate additional progenitor cells. Once they migrate to the target site, they enhance angiogenesis, neovascularization and tissue regeneration. Several trials have demonstrated the safety and efficacy of CD34+ cell therapy in different settings, such as peripheral limb ischemia, stroke and cardiovascular disease. Herein, we review the potential utility of CD34+ cell transplantation in acute myocardial infarction, refractory angina and ischemic heart failure.

Heterochromatin and Polycomb as regulators of haematopoiesis.

Haematopoiesis is the process by which multipotent haematopoietic stem cells are transformed into each and every type of terminally differentiated blood cell. Epigenetic silencing is critical for this process by regulating the transcription of cell-cycle genes critical for self-renewal and differentiation, as well as restricting alternative fate genes to allow lineage commitment and appropriate differentiation. There are two distinct forms of transcriptionally repressed chromatin: H3K9me3-marked heterochromatin and H3K27me3/H2AK119ub1-marked Polycomb (often referred to as facultative heterochromatin). This review will discuss the role of these distinct epigenetic silencing mechanisms in regulating normal haematopoiesis, how these contribute to age-related haematopoietic dysfunction, and the rationale for therapeutic targeting of these pathways in the treatment of haematological malignancies.

Multipotent Progenitors and Hematopoietic Stem Cells Arise Independently During the Endothelial to Hematopoietic Transition in the Early Mouse Embryo

During embryogenesis, waves of hematopoietic progenitors develop from hemogenic endothelium (HE) prior to the emergence of self-renewing hematopoietic stem cells (HSC). Although previous studies have shown that yolk sac-derived erythromyeloid progenitors and HSC emerge from distinct populations of HE, it remains unknown whether the earliest lymphoid-competent progenitors, multipotent progenitors, and HSC originate from common HE. Here we demonstrate by clonal assays and single cell transcriptomics that rare HE with functional HSC potential in the early murine embryo are distinct from more abundant HE with multilineage hematopoietic potential that fail to generate HSC. Specifically, HSC-competent HE are characterized by expression of CXCR4 surface marker and by higher expression of genes tied to arterial programs regulating HSC dormancy and self-renewal. Together, these findings suggest a revised model of developmental hematopoiesis in which the initial populations of multipotent progenitors and HSC arise independently from HE with distinct phenotypic and transcriptional properties.

Polyvinyl alcohol increased growth, migration, invasion, and sphere size in the PK-8 pancreatic ductal adenocarcinoma cell line

Polyvinyl alcohol (PVA) is a water-soluble synthetic polymer used in eye drops, embolization particles, and artificial cartilage. It has also been shown to cause expansion of functional multipotent self-renewing hematopoietic stem cells under serum-free conditions. In this study, we examined the effects of PVA on human pancreatic ductal adenocarcinoma (PDAC) cell lines using 2-dimensional (2D) and 3D-cultures with serum-free medium. In the 2D-culture, PVA-treatment induced an aggregated colony-like appearance in PDAC cells. It increased the growth of PK-8 cells in a dose-dependent manner as well as significantly increasing migration and invasion abilities. qRT-PCR showed an increase in α2 integrin and a decrease in matrix metalloprotease levels in PVA-treated PK-8 cells. Through qRT-PCR analysis, β1 integrin expression at the mRNA level was found to be decreased; however, it was unaltered at the protein level when assessed using FACS analysis.PVA further induced mesenchymal to epithelial transition-like alterations, including increased E-cadherin and decreased Vimentin and N-cadherin expression. Four cancer stem cell (CSC) markers were higher in PVA-treated PK-8 cells compared to controls. In 3D-culture, PVA-treated PK-8 cells showed a rod-like appearance with larger sphere size and higher growth ability. qRT-PCR showed that CSC markers did not increase and 2 of 4 drug transporters had decreased in PVA-treated PK-8 cells. These findings suggest that PVA increases the growth, migration, invasion, and sphere size of PK-8 cells, but does not increase the proportion of pancreatic CSCs under 3D-culture conditions with serum-free medium.

Cas9-AAV6 gene correction of beta-globin in autologous HSCs improves sickle cell disease erythropoiesis in mice

CRISPR/Cas9-mediated beta-globin (HBB) gene correction of sickle cell disease (SCD) patient-derived hematopoietic stem cells (HSCs) in combination with autologous transplantation represents a recent paradigm in gene therapy. Although several Cas9-based HBB-correction approaches have been proposed, functional correction of in vivo erythropoiesis has not been investigated previously. Here, we use a humanized globin-cluster SCD mouse model to study Cas9-AAV6-mediated HBB-correction in functional HSCs within the context of autologous transplantation. We discover that long-term multipotent HSCs can be gene corrected ex vivo and stable hemoglobin-A production can be achieved in vivo from HBB-corrected HSCs following autologous transplantation. We observe a direct correlation between increased HBB-corrected myeloid chimerism and normalized in vivo red blood cell (RBC) features, but even low levels of chimerism resulted in robust hemoglobin-A levels. Moreover, this study offers a platform for gene editing of mouse HSCs for both basic and translational research.

Experimental Modeling of Eosinophil-Associated Diseases.

Eosinophils are an important subtype of leukocytes derived from bone marrow multipotent hematopoietic stem cells and represent about 1% of leukocytes in circulating blood. In homeostatic conditions, eosinophils reside in the intestine to maintain the balance of immune responses by communicating with gut microbes without causing inflammation. However, under the stressed or diseased condition, eosinophils degranulate, releasing their granule-derived cytotoxic proteins that are involved in inflammatory responses. Various eosinophil-associated inflammatory diseases are eosinophilic esophagitis (EoE), eosinophilic gastroenteritis (EG), and eosinophilic colitis (EC), together called EGID, asthma, hypereosinophilic syndrome, and eosinophilic pneumonia (EP). Eosinophildegranulation results in thereleaseof their four toxic proteins [major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), and eosinophil-derived neurotoxin (EDN)]which promote disease pathogenesis. Pancreatitis is the inflammatory disease of the pancreas that arises due to blockage of the pancreatic duct, trypsinogen mutation, alcohol consumption, and repeated occurrence of pancreatitis leading to chronic pancreatitis (CP); subsequently some CP patients may also develop pancreatic cancer. The presence of eosinophils is now shown in various case reports with acute, recurrent acute, and chronic pancreatitis and pancreatic cancer indicating the role of eosinophils in the pathogenesis of various pancreatic inflammatory disorders. However, the details of eosinophil accumulation during pancreatic diseases are not well explored and need further attention. Overall, the chapter provides the current understanding of reported eosinophils associated with inflammatory diseases like EGID diseases, asthma, and pancreatic disorders, i.e., acute, chronicpancreatitis, and pancreatic cancer. This knowledge will be helpful for future studies to develop novel treatment options for the eosinophils associated diseases. Therefore, more efforts are needed to perform preclinical and clinical studies in this field for the successful development of eosinophil-targeting treatments for a variety of eosinophil-associated diseases.

Distinct Transcriptional Signatures Distinguish the Emergence of Multipotent Progenitors and Hematopoietic Stem Cells from Endothelial Precursors in the Murine Embryo

During embryonic development, blood cells emerge from hemogenic endothelium (HE), producing waves of hematopoietic progenitors prior to the emergence of rare hematopoietic stem cells (HSCs), which have the unique ability to self-renew and generate all cell types of the adult hematopoietic system. HSCs have significant potential for use in cellular therapies and disease modeling. However, efforts to generate HSCs in vitro from pluripotent stem cells (PSCs) have been limited by an incomplete understanding of the unique phenotypic markers and transcriptional programs that distinguish HE with HSC potential. Previous studies have demonstrated that yolk sac-derived erythromyeloid progenitors and HSCs originate from distinct populations of HE. However, it is not known whether the earliest lymphoid-competent progenitors, multipotent progenitors, and HSCs originate from HE with common phenotypic and transcriptional properties. To investigate this, we combined index sorting of single hemogenic precursors with stromal co-culture that enables simultaneous detection of HSC and multilineage hematopoietic potential, to functionally validate surface markers that may distinguish hemogenic precursors with different hematopoietic fates. We previously found that the co-expression of two markers, CD61 and EPCR, identifies a subset of VE-Cadherin+ endothelial cells from the mouse P-Sp/AGM region (para-aortic splanchnopleura/aorta-gonad-mesonephros, where the first HSCs are generated from HE between E9 and E11 in development) enriched phenotypically for arterial endothelial surface markers (e.g. Dll4, CD44) and functionally for hemogenic precursors with HSC potential. However, this population remains heterogeneous, containing clonal hemogenic precursors with the potential for HSC as well as multilineage progenitor-restricted fates. Here, we report that expression of arterial marker CXCR4 further enriched for functional HSC potential in hemogenic precursors in the P-Sp/AGM between E9 and E10, when the first clonal HSC precursors are detected at rare frequency. In contrast, we detected more abundant clonal HE with multilineage hematopoietic potential (producing lymphoid, erythroid, and myeloid progeny in vitro but lacking HSC potential) at the same stage, which are distinguished by comparatively lower CXCR4 expression. To investigate transcriptional differences between HE populations differentially expressing CXCR4, we performed single-cell RNA sequencing of E9 P-Sp-derived VE-Cadherin+CD61+EPCR+ cells. Using an unbiased gene module analysis based on graph autocorrelation in the Monocle 3 platform to identify genes that co-vary over pseudotime, we found that Cxcr4 is uniquely expressed in a subset of cells simultaneously enriched for arterial-specific genes (including Dll4, Efnb2, Hey2, Sox17, Cd44) and genes with established roles in HSC maintenance and self-renewal (including Mecom, Cdkn1c, H19, Txnip, Kmt2a). Conversely, expression of these genes is decreased in cells undergoing the endothelial to hematopoietic transition at this stage based on pseudotemporal ordering, concomitant with increasing expression of hematopoietic-specifying transcription factors Runx1 and Gfi1, and other genes associated with definitive hematopoiesis (egs. Myb, Kit, Hlf, Gata2, Mpl, Lyl1). We also examined the aggregate expression of established HSC-specific signature genes from published data sets across pseudotime, and found that they exhibit similar expression dynamics to that of Cxcr4 and Dll4, reaching peak expression prior to the initiation of Runx1 and Gfi1 expression. Altogether, our studies support a model in which the initial populations of multipotent progenitors and HSCs emerge independently from HE in the P-Sp/AGM. Furthermore, our findings suggest that HE with HSC competence is uniquely defined by co-expression of arterial endothelial genes and genes encoding HSC self-renewal factors, providing insight into the earliest transcriptional programs that must be coordinated to drive HSC fate from endothelial precursors. Future studies will focus on identifying the signal pathways whose integration promotes expression of these HSC-defining transcriptional programs in endothelial cells, with the goal of advancing methods for HSC generation in vitro. Disclosures Bernstein: Lyell Immunopharma: Current equity holder in publicly-traded company, Other: Laboratory Support; Deverra Therapeutics: Current equity holder in publicly-traded company.

Metabolomic Characterization of Red Blood Cell Differentiation

Background: Erythropoiesis is the highly coordinated multi-step process by which multipotent hematopoietic stem cells differentiate into mature enucleated red blood cells (RBCs). As erythroid cells become more terminally differentiated, they undergo changes in morphology and gene expression, start synthesizing hemoglobin, commit to an irreversible loss of proliferation, and eventually expulse their nuclei and other cytoplasmic organelles. Thus, RBCs must rely on their proteome and metabolome for proper function. The RBC proteome is estimated to contain 2,800 proteins, including a variety of receptors and transporters that allow RBCs to uptake xenobiotics or endogenous metabolites as they circulate for ∼120 days. Furthermore, they are metabolically active with glycolysis, nucleotide catabolism, and glutathione metabolism as the major pathways supporting cell survival and function. However, it is unclear how the metabolome is altered during erythropoiesis, what role metabolites play in normal erythropoiesis, and if dysregulation of metabolites contributes to diseases of ineffective erythropoiesis, such as sickle cell anemia and thalassemia. Methods: Four models of erythropoiesis were used in this study. 1) Mice were treated with phenylhydrazine (Phz) to induce acute hemolysis followed by erythropoietic recovery, leading to an increase in circulating reticulocytes. 2) Mice were lethally irradiated and transplanted with wild-type or sickle cell bone marrow, leading to anemic profiles in sickle cell chimeras. 3) The mouse erythroleukemic (MEL) cell line was treated with DMSO to induce differentiation. 4) The human erythroleukemic (K562) cell line was treated with sodium butyrate to induce differentiation. For the in vivo mouse models, blood was collected from control and treated animals, and complete blood count (CBC) analysis was performed. For the in vitro cell culture models, the mRNA levels of β-globin were measured by Q-RT-PCR in control and differentiated cells, and the degree of hemoglobinization was determined visually and via staining for heme. In addition, metabolites were extracted from the collected RBCs and erythroleukemic cell lines, and a Snapshot LC/MS metabolomic platform was used to identify commonly altered metabolites. Results: We first validated our four models of erythropoiesis. Treatment with Phz decreased the number of total RBCs while increasing the RBC distribution width, indicating an increased number of reticulocytes (more immature RBCs) in circulation. Similar results were seen in the sickle cell chimeras. Treatment of MEL and K562 cells with DMSO and sodium butyrate, respectively, resulted in increased expression of β-globin, increased levels of heme, and increased red color. Then, using our Snapshot metabolomic platform, we identified global changes in RBC metabolism during erythropoiesis. Analyses of the commonly altered metabolites in the in vitro and in vivo models revealed an increase in amino acid, mitochondrial, and urea cycle metabolism during erythropoiesis. L-aspartate levels were particularly upregulated, especially in DMSO-treated MEL cells. We are now investigating the role of aspartate in the regulation of erythropoiesis. Conclusions: We defined how the metabolome was altered in multiple in vitro and in vivo models of erythropoiesis and identified global changes in RBC metabolism between the different models. Specifically, we found that L-aspartate was upregulated during RBC differentiation in all four models. Aspartate is an amino acid that plays a role in many processes in cells, including nucleotide biosynthesis, redox homeostasis, and amino acid biosynthesis. We hypothesize that aspartate metabolism is critical for RBC differentiation and that its dysregulation exacerbates disease of ineffective erythropoiesis, such as sickle cell anemia and β-thalassemia. We are currently testing its role in inducing hemoglobinization and in regulating the commitment of erythroid progenitor cells to an irreversible loss of proliferation. Overall, we believe that understanding the precise mechanisms by which cellular metabolism plays a role in proper RBC differentiation may lead to better therapies for diseases of ineffective erythropoiesis, such as sickle cell anemia and thalassemia. Disclosures No relevant conflicts of interest to declare.

Role of thrombopoiesis in leishmaniasis

The blood vascular system of mammals is unique in nature; inhabited with a pool of tiny small cell fragments called platelets; attributed with the most important patrolling tasks to check integrity of the entire endothelial landscape. Their production is tightly coupled with hematopoietic system where everything starts from self renewable multipotent hematopoietic stem cells (HSCs) which eventually undergo dual step (megakaryopoiesis-thrombopoiesis) thrombocytes production. Several cytokines tune the fate of every progenitor cells during hematopoiesis through temporal activation of specific transcription factors. Though platelets generated through steady state hematopoiesis are involved in the regulation of vascular homeostasis, these cells can sense pathogens through its innate immune sensors and can mount crucial responses against the invading pathogen. For this, the primary aim of many infections including Leishmania is to induce thrombocytopenia within infected host. But the underlying mechanism of this induced thrombocytopenia in Leishmania infection has not been evaluated. Elucidation of these mechanisms will be fruitful to design new chemotherapeutic strategies.

A new lymphoid-primed progenitor marked by Dach1 downregulation identified with single cell multi-omics.

A classical view of blood cell development is that multipotent hematopoietic stem and progenitor cells (HSPCs) become lineage-restricted at defined stages. Lin-c-Kit+Sca-1+Flt3+ cells, termed lymphoid-primed multipotent progenitors (LMPPs), have lost megakaryocyte and erythroid potential but are heterogeneous in their fate. Here, through single-cell RNA sequencing, we identify the expression of Dach1 and associated genes in this fraction as being coexpressed with myeloid/stem genes but inversely correlated with lymphoid genes. Through generation of Dach1-GFP reporter mice, we identify a transcriptionally and functionally unique Dach1-GFP- subpopulation within LMPPs with lymphoid potential with low to negligible classic myeloid potential. We term these 'lymphoid-primed progenitors' (LPPs). These findings define an early definitive branch point of lymphoid development in hematopoiesis and a means for prospective isolation of LPPs.

Isolation of a Highly Purified HSC-enriched CD34+CD90+CD45RA- Cell Subset for Allogeneic Transplantation in the Nonhuman Primate Large-animal Model.

Background.Allogeneic hematopoietic stem cell transplantation (allo-HCT) is a common treatment for patients suffering from different hematological disorders. Allo-HCT in combination with hematopoietic stem cell (HSC) gene therapy is considered a promising treatment option for millions of patients with HIV+ and acute myeloid leukemia. Most currently available HSC gene therapy approaches target CD34-enriched cell fractions, a heterogeneous mix of mostly progenitor cells and only very few HSCs with long-term multilineage engraftment potential. As a consequence, gene therapy approaches are currently limited in their HSC targeting efficiency, very expensive consuming huge quantities of modifying reagents, and can lead to unwanted side effects in nontarget cells. We have previously shown that purified CD34+CD90+CD45RA− cells are enriched for multipotent HSCs with long-term multilineage engraftment potential, which can reconstitute the entire hematopoietic system in an autologous nonhuman primate transplant model. Here, we tested the feasibility of transplantation with purified CD34+CD90+CD45RA− cells in the allogeneic setting in a nonhuman primate model.Methods.To evaluate the feasibility of this approach, CD34+CD90+CD45RA− cells from 2 fully major histocompatibility complex-matched, full sibling rhesus macaques were sort-purified, quality controlled, and transplanted. Engraftment and donor chimerism were evaluated in the peripheral blood and bone marrow of both animals.Results.Despite limited survival due to infectious complications, we show that the large-scale sort-purification and transplantation of CD34+CD90+CD45RA− cells is technically feasible and leads to rapid engraftment of cells in bone marrow in the allogeneic setting and absence of cotransferred T cells.Conclusions.We show that purification of an HSC-enriched CD34+ subset can serve as a potential stem cell source for allo-HCTs. Most importantly, the combination of allo-HCT and HSC gene therapy has the potential to treat a wide array of hematologic and nonhematologic disorders.

3042 – CAS9-AAV6 GENE CORRECTION OF AUTOLOGOUS HSCS IMPROVES SICKLE CELL DISEASE ERYTHROPOIESIS IN MICE

With the ability to reconstitute the entire hematopoietic system, hematopoietic stem cells (HSCs) are an attractive target for a range of disease therapies. CRISPR/Cas9-mediated beta-globin (HBB) gene correction of Sickle Cell Disease (SCD) patient-derived HSCs in combination with autologous transplantation represents an exciting new paradigm in gene therapy. Although several Cas9-based HBB-correction approaches have been proposed, functional correction of in vivo erythropoiesis has not been investigated to date. Here, we have applied a humanized globin-cluster SCD mouse model to study Cas9-AAV6-mediated HBB-correction in functional HSCs within the context of autologous transplantation using a novel ex vivo HSC culture system (Wilkinson et al., Nature 2019). We have discovered that long-term multipotent HSCs can be gene corrected ex vivo and stable hemoglobin-A production can be achieved in vivo from HBB-corrected HSCs following autologous transplantation back into SCD mice. There was a direct correlation between increased HBB-corrected myeloid chimerism and normalized in vivo RBC features, but even low levels of chimerism resulted in significant HgbA levels. Our results provide a useful framework for evaluating Cas9-based HSC gene therapies and support the clinical investigation of Cas9-AAV6 technologies.

Single-cell lineage tracing unveils a role for TCF15 in haematopoiesis.

SummaryBone marrow transplantation therapy relies on the life-long regenerative capacity of haematopoietic stem cells (HSCs)1,2. HSCs present a complex variety of regenerative behaviours at the clonal level, but the mechanisms underlying this diversity are still undetermined3–11. Recent advances in single cell RNA sequencing (scRNAseq) have revealed transcriptional differences amongst HSCs, providing a possible explanation for their functional heterogeneity12–17. However, the destructive nature of sequencing assays prevents simultaneous observation of stem cell state and function. To solve this challenge, we implemented expressible lentiviral barcoding, which enabled simultaneous analysis of lineages and transcriptomes from single adult HSCs and their clonal trajectories during long-term bone marrow reconstitution. Differential gene expression analysis between clones with distinct behaviour unveiled an intrinsic molecular signature that characterizes functional long-term repopulating HSCs. Probing this signature through in vivo CRISPR screening, we found the transcription factor Tcf15 to be required, and sufficient, to drive HSC quiescence and long-term self-renewal. In situ, Tcf15 expression labels the most primitive subset of true multipotent HSCs. In conclusion, our work elucidates clone-intrinsic molecular programs associated with functional stem cell heterogeneity, and identifies a mechanism for the maintenance of the self-renewing haematopoietic stem cell state.

The Source and Dynamics of Adult Hematopoiesis: Insights from Lineage Tracing.

The generation of all blood cell lineages (hematopoiesis) is sustained throughout the entire life span of adult mammals. Studies using cell transplantation identified the self-renewing, multipotent hematopoietic stem cells (HSCs) as the source of hematopoiesis in adoptive hosts and delineated a hierarchy of HSC-derived progenitors that ultimately yield mature blood cells. However, much less is known about adult hematopoiesis as it occurs in native hosts, i.e., without transplantation. Here we review recent advances in our understanding of native hematopoiesis, focusing in particular on the application of genetic lineage tracing in mice. The emerging evidence has established HSCs as the major source of native hematopoiesis, helped to define the kinetics of HSC differentiation, and begun exploring native hematopoiesis in stress conditions such as aging and inflammation. Major outstanding questions about native hematopoiesis still remain, such as its clonal composition, the nature of lineage commitment, and the dynamics of the process in humans.

Коррекция морфофункционального состояния печени при остром гепатите с помощью стволовых клеток

Цель работы - изучение влияния сочетанной трансплантации мультипотентных мезенхимальных стромальных и гемопоэтических стволовых клеток на регенерацию печени в физиологических условиях и в условиях токсического гепатита. Методика. Эксперименты выполнены на 84 белых лабораторных мышах-самцах, возраст 7-8 мес. Токсический гепатит вызывали внутрибрюшинным введением четыреххлористого углерода (CCl4 в дозе 50 мкг/кг. Животным опытной группы в хвостовую вену вводили мультипотентные мезенхимальные стромальные (ММСК, 4 млн клеток/кг) и гемопоэтические стволовые клетки (ГСК, 330 тыс. клеток/кг). Для инъекции полученные клетки суспендировали в 0,2 мл 0,9 % раствора NaCl. Животным контрольной группы внутривенно вводили 0,2 мл 0,9 % раствора NaCl. Внутривенные введения осуществляли через 1 ч после введения четыреххлористого углерода однократно. Для трансплантации использовали ММСК третьего пассажа, а ГСК не подвергались культивированию. Изучали влияние сочетанной трансплантации ММСК и ГСК на биохимические показатели периферической крови и морфометрические показатели печени в физиологических условиях и после введения CCl4 на 1-е, 3-и, 7-е сут. Результаты. Показано, что проведение сочетанной трансплантации мультипотентных мезенхимальных стромальных и гемопоэтических стволовых клеток при токсическом гепатите приводило к снижению активности ферментов цитолиза, активации белоксинтезирующей функции печени. Также отмечалось увеличение количества гепатоцитов, повышение митотической активности, что указывает на активацию внутриклеточной регенерации. Обнаруженное увеличение числа двуядерных гепатоцитов, размеров ядра, ядерно-цитоплазматического соотношения говорит об усилении процесса внутриклеточной регенерации в печени. The aim of the work was studying the effect of combined transplantation of multipotent mesenchymal stromal and hematopoietic stem cells on regeneration of the liver under the physiological conditions and in toxic hepatitis. Methods. Experiments were performed on 84 white laboratory male mice aged 7-8 months. Toxic hepatitis was induced by administration of carbon tetrachloride (CCl4) at a dose of 50 µg/kg, i.p. Mice were divided into experimental and control groups. The experimental group received caudal vein injections of multipotent mesenchymal stromal (MMSC) and hematopoietic stem cells (HSC) derived from the placenta chorion of female mice at respective doses of 4M cells/kg and 330K cells/kg suspended in 0.2 ml of 0.9% NaCl. Control animals were given 0.2 ml of 0.9 % NaCl, i.v. Intravenous injections were performed once 1 hour following the administration of carbon tetrachloride. MMSCs of the third passage were used for transplantation, while transplanted HSCs were not cultured. Effects of the combined MMSC and HSC transplantation on blood biochemistry and liver morphometry were studied under the physiological conditions and at 1, 3, and 7 days after administration of CCl4. Results. In toxic hepatitis, the combined transplantation of multipotent mesenchymal stromal and hematopoietic stem cells resulted in decreased activity of cytolytic enzymes, activation of hepatic protein synthesis, increased number of hepatocytes, and increased mitotic activity indicative of activation of intracellular regeneration. Increases in the number of binuclear hepatocytes, nucleus size, and the nuclear-cytoplasmic ratio suggested an enhancement of intracellular regeneration in the liver.