Journal: Experimental hematology
Red cell production is primarily determined by the action of erythropoietin. Additional erythropoiesis-regulatory factors include molecules and cellular interactions occurring within the bone marrow (BM) microenvironment. Sotatercept (ACE-011) is an activin receptor ligand trap which binds several members of the TGF-β superfamily. Treatment with ACE-011 reverses bone loss and reduces the degree of osteoporosis. Surprisingly, this was accompanied by elevated hemoglobin and hematocrit levels. The mechanisms underlying the beneficial effects of ACE-011 on red cell production remain unknown. This study explores the means by which ACE-011 promotes erythropoiesis. We showed that ACE-011 does not directly affect erythroid differentiation of human CD34(+) cells in vitro. We next tested whether ACE-011 acts indirectly by affecting BM accessory cells. Conditioned media (CM) produced by BM stromal cells (SC) inhibited erythroid differentiation of CD34(+) cells while maintained their ability to proliferate. However, CM from SC treated with ACE-011 partially restored erythropoiesis coinciding with changes in the molecular and secretory profile of SC, including the expression and secretion of erythropoiesis-modulatory factors. We conclude that inhibitory factors produced by BM-SC in vitro might control erythropoiesis in vivo and that agents that reverse these microenvironmental signals may provide an approach to attenuate anemia in clinical conditions.
SHP-1, encoded by the PTPN6 gene, is a protein tyrosine phosphatase with two src-homology-2 (SH2) domains that is implicated as providing suppression of hematopoietic malignancies. A number of reports have shown protein-protein interactions between SHP-1 SH2 domains and tyrosine-phosphorylated proteins. However, despite its having three proline-rich, potential SH3-binding motifs, no reports of protein-protein interactions through src-homology-3 (SH3)-binding domains with SHP-1 have been described. Herein we show that the SH3 domain-containing CT10 regulator of kinase-like (CrkL) adaptor protein associates with SHP-1. We also provide results that suggest this association is due to CrkL binding to PxxP domains located at amino acid residues 158-161 within the SHP-1 C-terminal SH2 domain, and amino acid residues 363-366 within its phosphatase domain. This study is the first to identify and define an interaction between SHP-1 and an SH3 domain-containing protein. Our findings provide an alternative way that SHP-1 can be linked to potential substrates.
The genomic events responsible for the pathogenesis of relapsed adult B lymphoblastic leukemia (B-ALL) are not yet clear. We performed integrative analysis of whole genome, exome, custom capture, RNA-seq, and locus-specific genomic assays across nine time points from a patient with primary de novo B-ALL. Comprehensive genome and transcriptome characterization revealed a dramatic tumor evolution during progression, yielding a tumor with complex clonal architecture at second relapse. We observed and validated point mutations in EP300 and NF1, a highly expressed EP300-ZNF384 gene fusion, a microdeletion in IKZF1, a focal deletion affecting SETD2, and large deletions affecting RB1, PAX5, NF1, and ETV6. While the genome analysis revealed events of potential biological relevance, no clinically actionable treatment options were evident at the time of the second relapse. However, transcriptome analysis identified aberrant overexpression of the targetable protein kinase encoded by the FLT3 gene. Although the patient had refractory disease after salvage therapy for the second relapse, treatment with the FLT3 inhibitor sunitinib rapidly induced a near complete molecular response, permitting the patient to proceed to a matched unrelated donor stem cell transplant. The patient remains in complete remission more than 4 years later. Analysis of this patient’s relapse genome revealed an unexpected, actionable therapeutic target that led to a specific therapy associated with a rapid clinical response. For some patients with relapsed or refractory cancers, this approach may indicate novel therapeutic interventions that could alter patient outcomes.
The cellular diversity of the hematopoietic system has been extensively studied and a plethora of cell-surface markers have been used to discriminate and prospectively purify different blood cell types. However, even within phenotypically-identical fractions of hematopoietic stem and progenitor cells (HSPCs) or lineage-restricted progenitors, significant functional heterogeneity is observed when single cells are analyzed. To address these challenges, researchers are now utilizing techniques to follow single cells and their progeny in order to improve our understanding for the underlying functional heterogeneity. On November 19(th) 2015 Drs. David Kent and Leïla Perié, two emerging young group leaders, presented their recent efforts to dissect the functional properties of individual cells in a webinar series organized by the International Society for Experimental Hematology (ISEH). Here, we provide a summary of the presented methods for cell labeling and clonal tracking and discuss how these different techniques have been employed to study hematopoiesis.
Improving the understanding of the intricacies of hematopoietic specification of induced or embryonic pluripotent stem cells is beneficial for many areas of research and translational medicine. Currently, it is not clear whether during hPSC hematopoietic differentiation in vitro the maturation of definitive progenitors proceeds through a primitive progenitor (hemangioblast) intermediate or develops independently. The objective of this study is to investigate the early stages of hematopoietic specification of pluripotent stem cells in vitro. By implementing an adherent culture, serum-free differentiation system, which utilizes a small molecule CHIR99021 to induce hPSC toward various hematopoietic lineages, we established that, compared to OP9 co-culture hematopoietic induction system, the application of CHIR99021 alters the early steps of hematopoiesis such as hemangioblast (HB), angiogenic hematopoietic progenitors (AHP) and hemogenic endothelium (HE). Importantly, it is associated with the loss of hemangioblast progenitor, loss of CD43+ (primitive hematopoietic marker) expression, and predominant development of BFU erythroid colonies in semisolid media. These data support the hypothesis that the divergence of primitive and definitive programs during hPSC differentiation precedes the hemangioblast stage. Furthermore, we showed that the inhibition of primitive hematopoiesis is associated with increase in hematopoietic potential, which is a fruitful finding for a growing need of lymphoid and myeloid cells in translational applications.
Major haemoglobinopathies place tremendous strain on global resources. Intrauterine haemopoietic cell (IUHCT) and gene (IUGT) therapies can potentially reduce perinatal morbidities with greater efficacy than postnatal therapy alone. We performed both procedures in the thalassaemic HbbTh3/+ murine model. Intraperitoneal delivery of coisogenic cells at E13-14 produced dose-dependent chimerism. High-dose adult bone marrow (BM) cells maintained 0.2-3.1% chimerism over ~24 weeks and treated heterozygotes demonstrated higher chimerism than wild-type pups (1.6 vs. 0.7%). Fetal liver cells produced higher chimerism compared to adult BM when transplanted at the same doses, maintaining 1.8-2.4% chimerism over ~32 weeks. We boosted transplanted mice postnatally with adult BM cells following busulfan conditioning. Engraftment was maintained at >1% only in recipients which were chimeric prior to boosting. IUHCT-treated non-chimeras and non-IUHCT mice showed micro- or no chimerism. Additional fludarabine treatment produced higher chimerism than busulfan alone. Engraftment was more effective following higher starting chimerism prior to boosting and in heterozygotes. Chimeric heterozygotes expressed 2.2-15.1% donor cells with eventual decline at 24 weeks (vs. <1% in non-chimeras) and demonstrated improved haematological indices and smaller spleens compared to untreated heterozygotes. Intravenous delivery of GLOBE lentiviral-vector expressing HBB (human β-globin) resulted in vector concentration of 0.001-0.6 copies/cell. Most haematological indices were higher in treated than untreated heterozygotes including haemoglobin and mean corpuscular volume, though still lower than in wild-types. Thus both direct IUGT and IUHCT strategies can be used to achieve haematological improvement but require further dose optimisation. IUHCT will be useful combined with postnatal transplantation to further enhance engraftment.
HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets) is a severe variant of preeclampsia whose pathogenesis remains unclear. Recent evidence and clinical similarities suggest a link to atypical hemolytic uremic syndrome (aHUS), a disease of excessive activation of the alternative complement pathway effectively treated with a complement inhibitor, eculizumab. Therefore, we utilized a functional complement assay, the modified Ham test, to test sera of women with classic or atypical HELLP syndrome, preeclampsia with severe features, normal pregnancies and healthy non-pregnant women. Sera were also evaluated using levels of the terminal product of complement activation (C5b-9). We tested the in vitro ability of eculizumab to inhibit complement activation in HELLP serum. Increased complement activation was found in participants with classic or atypical HELLP compared to normal pregnancy and non-pregnant controls. Mixing HELLP serum with eculizumab containing serum resulted in a significant decrease in cell killing compared to HELLP serum alone. In conclusion, HELLP syndrome is associated with increased complement activation demonstrated by the modified Ham test. This assay may aid in the diagnosis of HELLP syndrome and confirm its pathophysiology relates to aHUS.
The Human Cell Atlas (HCA) is expected to facilitate the creation of reference cell profiles, marker genes and gene regulatory networks that will provide a deeper understanding of healthy and disease cell types from clinical biospecimens. The hematopoietic system includes dozens of distinct transcriptionally-coherent cell types, including intermediate transitional populations, which have not been previously described at a molecular level. Using the first data release from the HCA bone marrow tissue project, we resolved common, rare and potential transitional cell populations from over 100,000 hematopoietic cells, spanning 35 transcriptionally coherent groups across eight healthy donors using emerging new computational approaches. These data highlight novel mixed-lineage progenitor populations and putative trajectories governing granulocytic, monocytic, lymphoid, erythroid, megakaryocytic and eosinophil specification. Our analyses suggest significant variation in cell-type composition and gene expression among donors, including biological processes impacted by donor age. To enable broad exploration of these findings, we provide an interactive website to probe intra- and extra-cell population differences within and between donors, and reference markers for cellular classification and cellular trajectories through associated progenitor states.
Hematopoietic stem cells (HSCs) maintain a quiescent state in the bone marrow to preserve their self-renewal capacity, but also undergo cell divisions as required. Organelles such as the mitochondria sustain cumulative damage during these cell divisions, and this damage may eventually compromise the cells' self-renewal capacity. HSC divisions result in either self-renewal or differentiation, with the balance between the two directly impacting hematopoietic homeostasis; but the heterogeneity of available HSC-enriched fractions, together with the technical challenges of observing HSC behavior, has long hindered the analysis of individual HSCs, and prevented the elucidation of this process. However, recent advances in genetic models, metabolomics analyses and single-cell approaches have revealed the contributions made to HSC self-renewal by metabolic cues, mitochondrial biogenesis, and autophagy/mitophagy, which have highlighted mitochondrial quality as a key control factor in the equilibrium of HSCs. A deeper understanding of precisely how specific modes of metabolism control HSC fate at the single cell level is therefore not only of great biological interest, but will have clear clinical implications for the development of therapies for hematological disease.
Arginine methylation is an abundant covalent modification that regulates diverse cellular processes including transcription, translation, DNA repair, and RNA processing. The enzymes that catalyze these marks are known as the protein arginine methyltransferases (PRMTs), and they can generate asymmetric dimethyl arginine (Type I arginine methyltransferases), symmetric dimethylarginine (Type II arginine methyltransferases), or monomethyarginine (Type III arginine methyltransferases). The PRMTs are capable of modifying diverse substrates, from histone components to specific nuclear and cytoplasmic proteins. Additionally, the PRMTs can orchestrate chromatin remodeling by blocking the docking of other epigenetic modifying enzymes or by recruiting them to specific gene loci. In the hematopoietic system, PRMTs can regulate cell behavior, including the critical balance between stem cell self-renewal and differentiation in at least two critical ways, via: 1) The covalent modification of transcription factors, and 2) The regulation of histone modifications at promoters critical to cell fate determination. Given these important functions, it is not surprising that these processes are altered in hematopoietic malignancies, such as acute myeloid leukemia (AML), where they promote increased self-renewal and impair hematopoietic stem and progenitor cell (HSPC) differentiation.