Human Hematopoietic Cells Research Articles

Deciphering the Complexities of Adult Human Steady State and Stress-Induced Hematopoiesis: Progress and Challenges

Human hematopoietic stem cells (HSCs) have traditionally been viewed as self-renewing, multipotent cells with enormous potential in sustaining essential steady state blood and immune cell production throughout life. Indeed, around 86% (1011–1012) of new cells generated daily in a healthy young human adult are of hematopoietic origin. Therapeutically, human HSCs have contributed to over 1.5 million hematopoietic cell transplants (HCTs) globally, making this the most successful regenerative therapy to date. We will commence this review by briefly highlighting selected key achievements (from 1868 to the end of the 20th century) that have contributed to this accomplishment. Much of our knowledge of hematopoiesis is based on small animal models that, despite their enormous importance, do not always recapitulate human hematopoiesis. Given this, we will critically review the progress and challenges faced in identifying adult human HSCs and tracing their lineage differentiation trajectories, referring to murine studies as needed. Moving forward and given that human hematopoiesis is dynamic and can readily adjust to a variety of stressors, we will then discuss recent research advances contributing to understanding (i) which HSPCs maintain daily steady state human hematopoiesis, (ii) where these are located, and (iii) which mechanisms come into play when homeostatic hematopoiesis switches to stress-induced or emergency hematopoiesis.

Selenoprotein-Mediated Redox Regulation Shapes the Cell Fate of HSCs and Mature Lineages.

The maintenance of cellular redox balance is crucial for cell survival and homeostasis and is disrupted with aging. Selenoproteins, comprising essential antioxidant enzymes, raise intriguing questions about their involvement in hematopoietic aging and potential reversibility. Motivated by our observation of mRNA downregulation of key antioxidant selenoproteins in aged human hematopoietic stem cells (HSCs) and previous findings of increased lipid peroxidation in aged hematopoiesis, we employed tRNASec gene (Trsp) knockout (KO) mouse model to simulate disrupted selenoprotein synthesis. This revealed insights into the protective roles of selenoproteins in preserving HSC stemness and B-lineage maturation, despite negligible effects on myeloid cells. Notably, Trsp KO exhibited B lymphocytopenia and reduced HSCs' self-renewal capacity, recapitulating certain aspects of aged phenotypes, along with the upregulation of aging-related genes in both HSCs and pre-B cells. While Trsp KO activated an antioxidant response transcription factor NRF2, we delineated a lineage-dependent phenotype driven by lipid peroxidation, which was exacerbated with aging yet ameliorated by ferroptosis inhibitors such as vitamin E. Interestingly, the myeloid genes were ectopically expressed in pre-B cells of Trsp KO mice, and KO pro-B/pre-B cells displayed differentiation potential toward functional CD11b+ fraction in the transplant model, suggesting that disrupted selenoprotein synthesis induces the potential of B-to-myeloid switch. Given the similarities between the KO model and aged wild-type mice, including ferroptosis vulnerability, impaired HSC self-renewal and B-lineage maturation, and characteristic lineage switch, our findings underscore the critical role of selenoprotein-mediated redox regulation in maintaining balanced hematopoiesis and suggest the preventive potential of selenoproteins against aging-related alterations.

Hormonally mediated mechanical remodelling of human haematopoietic stem cells enhances their bone-marrow engraftment.

Hormonally mediated mechanical remodelling of human haematopoietic stem cells enhances their bone-marrow engraftment.

Genomic and phenotypic correlates of mosaic loss of chromosome Y in blood.

Mosaic loss of Y (mLOY) is the most common somatic chromosomal alteration detected in human blood. The presence of mLOY is associated with altered blood cell counts and increased risk of Alzheimer disease, solid tumors, and other age-related diseases. We sought to gain a better understanding of genetic drivers and associated phenotypes of mLOY through analyses of whole-genome sequencing (WGS) of a large set of genetically diverse males from the Trans-Omics for Precision Medicine (TOPMed) program. We show that haplotype-based calling methods can be used with WGS data to successfully identify mLOY events. This approach enabled us to identify differences in mLOY frequencies across populations defined by genetic similarity, revealing a higher frequency of mLOY in the European (EUR) ancestry group compared to other ancestries. We identify multiple loci associated with mLOY susceptibility and show that subsets of human hematopoietic stem cells are enriched for the activity of mLOY susceptibility variants. Finally, we found that certain alleles on chromosome Y are more likely to be lost than others in detectable mLOY clones.

How age affects human hematopoietic stem and progenitor cells and strategies to mitigate HSPC aging.

Hematopoietic stem cells (HSCs) are central to blood formation and play a pivotal role in hematopoietic and systemic aging. With aging, HSCs undergo significant functional changes, such as an increased stem cell pool, declined homing and reconstitution capacity, and skewed differentiation towards myeloid and megakaryocyte/platelet progenitors. These phenotypic alterations are likely due to the expansion of certain clones, known as clonal hematopoiesis (CH), which leads to disrupted hematopoietic homeostasis, including anemia, impaired immunity, higher risks of hematological malignancies, and even associations with cardiovascular disease, highlighting the broader impact of HSC aging on overall health. HSC aging is driven by a range of mechanisms involving both intrinsic and extrinsic factors, such as DNA damage accumulation, epigenetic remodeling, inflammaging and metabolic regulation. In this review, we summarize the update understanding of age-related changes in HSPCs and the mechanisms underlie the aging process in mammalian models, especially in human study. Additionally, we provide insights into potential therapeutic strategies to counteract aging process and enhance HSC regenerative capacity, which will support therapeutic interventions and promote healthy aging.

Enhanced engraftment of human haematopoietic stem cells via mechanical remodelling mediated by the corticotropin-releasing hormone

Enhanced engraftment of human haematopoietic stem cells via mechanical remodelling mediated by the corticotropin-releasing hormone

T Cell Development and Responses in Human Immune System Mice.

Human Immune System (HIS) mice constructed with mature human immune cells or with human hematopoietic stem cells and thymic tissue have provided an important tool for human immunological research. In this article, we first review the different types of HIS mice based on human tissues transplanted and sources of the tissues. We then focus on knowledge of human T cell development and responses obtained using HIS mouse models. These areas include the development of human T cell subsets, with a focus on $\upalpha$$\upbeta$ conventional T cells and regulatory T cells, and human T cell responses in the settings of infection, transplantation rejection and tolerance, autoimmune disease, cancer immunotherapy, and regulatory T cell therapy. We also discuss the limitations and potential future applications of HIS mouse models.

Molecular regulators of chemotaxis in human hematopoietic stem cells.

Hematopoietic stem cells (HSCs), essential for lifelong blood cell regeneration, are clinically utilized to treat various hematological disorders. These cells originate in the aorta-gonad-mesonephros region, expand in the fetal liver, and mature in the bone marrow. Chemotaxis, involving gradient sensing, polarization, and migration, directs HSCs and is crucial for their homing and mobilization. The molecular regulation of HSC chemotaxis involves chemokines, chemokine receptors, signaling pathways, and cytoskeletal proteins. Recent advances in understanding these regulatory mechanisms have deepened insights into HSC development and hematopoiesis, offering new avenues for therapeutic innovations. Strategies including glucocorticoid receptor activation, modulation of histone acetylation, stimulation of nitric oxide signaling, and interference with m6A RNA modification have shown potential in enhancing CXCR4 expression, thereby improving the chemotactic response and homing capabilities of human HSCs. This review synthesizes current knowledge on the molecular regulation of human HSC chemotaxis and its implications for health and disease.

Gene editing without exvivo culture evades genotoxicity in human hematopoietic stem cells.

Gene editing the BCL11A erythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for β-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here, we compare combined CRISPR-Cas9 editing of the BCL11A+58 and+55 enhancers with leading gene modification approaches under clinical investigation. Dual targeting of the BCL11A+58 and+55 enhancers with 3xNLS-SpCas9 and two single guide RNAs (sgRNAs) resulted in superior HbF induction, including in sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers. Unintended on-target outcomes of double-strand break (DSB) repair in hematopoietic stem and progenitor cells (HSPCs), such as long deletions and centromere-distal chromosome fragment loss, are a byproduct of cellular proliferation stimulated by exvivo culture. Editing quiescent HSPCs bypasses long deletion and micronuclei formation and preserves efficient on-target editing and engraftment function.

The dynamics of hematopoiesis over the human lifespan.

Over a lifetime, hematopoietic stem cells (HSCs) adjust their lineage output to support age-aligned physiology. In model organisms, stereotypic waves of hematopoiesis have been observed corresponding to defined age-biased HSC hallmarks. However, how the properties of hematopoietic stem and progenitor cells change over the human lifespan remains unclear. To address this gap, we profiled individual transcriptome states of human hematopoietic stem and progenitor cells spanning gestation, maturation and aging. Here we define the gene expression networks dictating age-specific differentiation of HSCs and the dynamics of fate decisions and lineage priming throughout life. We additionally identifiy and functionally validate a fetal-specific HSC state with robust engraftment and multilineage capacity. Furthermore, we observe that classification of acute myeloid leukemia against defined transcriptional age states demonstrates that utilization of early life transcriptional programs associates with poor prognosis. Overall, we provide a disease-relevant framework for heterochronic orientation of stem cell ontogeny along the real time axis of the human lifespan.

Deciphering the evolving niche interactome of human hematopoietic stem cells from ontogeny to aging.

Hematopoietic stem cells (HSC) reside within specialized microenvironments that undergo dynamic changes throughout development and aging to support HSC function. However, the evolving cell-cell communication networks within these niches remain largely unexplored. This study integrates single-cell RNA sequencing datasets to systematically characterize the HSC niche interactome from ontogeny to aging. We reconstructed single-cell atlases of HSC niches at different developmental stages, revealing stage-specific cellular compositions and interactions targeting HSC. During HSC maturation, our analysis identified distinct patterns of ligand-receptor interactions and signaling pathways that govern HSC emergence, expansion, and maintenance. HSC aging was accompanied by a decrease in supportive niche interactions, followed by an adaptive increase in interaction strength in old adult bone marrow. This complex aging process involved the emergence of interactions associated with inflammation, altered stem cell function, and a decline in the efficacy of key signaling pathways. Our findings provide a comprehensive understanding of the dynamic remodeling of the HSC niche interactome throughout life, paving the way for targeted interventions to maintain HSC function and promote healthy aging. This study offers valuable insights into the intricate cell-cell communication networks that govern HSC behavior and fate, with implications for hematological disorders and regenerative medicine.

BRAF inhibitors enhance erythropoiesis and treat anemia through paradoxical activation of MAPK signaling

Erythropoiesis is a crucial process in hematopoiesis, yet it remains highly susceptible to disruption by various diseases, which significantly contribute to the global challenges of anemia and blood shortages. Current treatments like erythropoietin (EPO) or glucocorticoids often fall short, especially for hereditary anemias such as Diamond-Blackfan anemia (DBA). To uncover new erythropoiesis-stimulating agents, we devised a screening system using primary human hematopoietic stem and progenitor cells (HSPCs). We discovered that BRAF inhibitors (BRAFi), commonly used to treat BRAFV600E melanoma, can unexpectedly and effectively promote progenitor cell proliferation by temporarily delaying erythroid differentiation. Notably, these inhibitors exhibited pronounced efficacy even under cytokine-restricted conditions and in patient samples of DBA. Mechanistically, although these BRAFi inhibit the MAPK cascade in BRAFV600E mutant cells, they paradoxically act as amplifiers in wild-type BRAF cells, potently enhancing the cascade. Furthermore, we found that while the oncogenic BRAFV600E mutation disrupts hematopoiesis and erythropoiesis through AP-1 hyperactivation, BRAFi minimally impact HSPC self-renewal and differentiation. In vivo studies have shown that BRAFi can enhance human hematopoiesis and erythropoiesis in severe immunodeficient mouse models and alleviate anemia in the Rpl11 haploinsufficiency DBA model, as well as other relevant anemia models. This discovery underscores the role of the MAPK pathway in hematopoiesis and positions BRAFi as a promising therapeutic option for improving hematopoietic reconstitution and treating anemias, including DBA.

Monocytes generated by interleukin-6-treated human hematopoietic stem and progenitor cells secrete calprotectin that inhibits erythropoiesis.

Elevated circulating levels of calprotectin (CAL), the S100A8/A9 heterodimer, are biomarkers of severe systemic inflammation. Here, we investigate the effects of CAL on early human hematopoiesis. CAL demonstrates limited impact on gene expression in stem and progenitor cells, in contrast with interleukin-6 (IL6), which promotes the expression of the S100A8 and S100A9 genes in hematopoietic progenitors and the generation of monocytes that release CAL. The main target of CAL is an erythroid-megakaryocyte progenitor (EMP) subset. CAL prevents both erythropoietin-driven differentiation of healthy progenitors and JAK2-V617F-driven erythropoiesis. In the context of JAK2-V617F, CAL also promotes the expression of S100A8 and S100A9 genes in monocytes. The signature of CAL effects is detected in the bone marrow progenitors of patients with myeloid malignancy or severe infection. These results position CAL as a mediator of IL6 effects on triggering anemia during inflammation, an effect that is amplified in the context of JAK2-V617F-driven hematopoiesis.

ALDH Enzymes and Hematological Diseases: A Scoping Review of Literature.

Aldehyde dehydrogenases (ALDHs) constitute a group of enzymes that catalyze the oxidation of aldehydes to carboxylic acids. The human ALDH superfamily, including 19 different isoenzymes (ALDH1A1, ALDH1A2, ALDH1A3, AHDH1B1, ALDH1L1, ALDH1L2, ALDH2, ALDH3A1, ALDH3A2, ALDH3B1, ALDH3B2, ALDH4A1, ALDH5A1, ALDH6A1, ALDH7A1, ALDH8A1, ALDH9A1, ALDHA16A1, ALDH18A1), displays different key physiological and toxicological functions, with specific tissue expression and substrate specificity. Several studies have established that ALDH are interesting markers for the identification and quantification of human hematopoietic stem cells and cancer stem cells, notably leukemic stem cells. ALDH2 is the best-documented enzyme, in this family, as having an impact on hematology, particularly myeloid malignancies. ALDH2 mainly catalyzes the detoxification of toxic aldehydes (acetaldehyde, formaldehyde). For example, ALDH2 detoxifies formaldehyde, which is produced during the differentiation of hematopoietic progenitors. The trigger of alcohol dehydrogenase 5 (also known as formaldehyde dehydrogenase or S-nitrosoglutathione reductase, ADH5/FDH/GSNOR)/ALDH2 allows to eliminate formaldehyde and ensures normal hematopoiesis. Moreover, the ALDH2*2 variant allele is the most frequent ALDH2 variant, found in 35-45% of individuals of East Asian origin. It is associated with altered acetaldehyde metabolism and is involved in several hematological diseases (aplastic anemia, bone marrow failure, myelodysplastic syndrome). This review presents current knowledge of different members of the ALDH family and their involvement in normal and malignant hematopoiesis. Focus was brought to the ALDH2 isoenzyme in congenital (Fanconi anemia, Aplastic anemia, mental retardation, and dwarfism (AMeD) syndrome, and idiopathic aplastic anemia) and acquired (acute myeloid leukemia and myelodysplastic syndrome) hematological diseases. It also describes the possibilities of using ALDH as both a biomarker and therapeutic target, to identify and eradicate leukemic stem cells in malignant diseases.

Resolving the chromatin impact of mosaic variants with targeted Fiber-seq.

Accurately quantifying the functional consequences of noncoding mosaic variants requires the pairing of DNA sequences with both accessible and closed chromatin architectures along individual DNA molecules-a pairing that cannot be achieved using traditional fragmentation-based chromatin assays. We demonstrate that targeted single-molecule chromatin fiber sequencing (Fiber-seq) achieves this, permitting single-molecule, long-read genomic, and epigenomic profiling across targeted >100 kb loci with ∼10-fold enrichment over untargeted sequencing. Targeted Fiber-seq reveals that pathogenic expansions of the DMPK CTG repeat that underlie Myotonic Dystrophy 1 are characterized by somatic instability and disruption of multiple nearby regulatory elements, both of which are repeat length-dependent. Furthermore, we reveal that therapeutic adenine base editing of the segmentally duplicated γ-globin (HBG1/HBG2) promoters in primary human hematopoietic cells induced toward an erythroblast lineage increases the accessibility of the HBG1 promoter as well as neighboring regulatory elements. Overall, we find that these non-protein coding mosaic variants can have complex impacts on chromatin architectures, including extending beyond the regulatory element harboring the variant.

FoxO1 regulates human haematopoietic stem cells self-renewal and engraftment.

Hematopoietic stem cell transplantation (HSCT) is one of the most effective ways to treat hematological malignant diseases, but the traditional culture of hematopoietic stem cells (HSCs) in vitro will soon lose their ability to self-renewal or differentiate into multilineage blood cells. To determine whether Forkhead boxO1 (FoxO1) is implicated in the development of HSCs, lentiviral vectors expressing knockdown (KD) or overexpression (OE) of FoxO1 were utilized in fetal liver-derived hematopoietic stem and progenitor cells (FL-HSPCs). The impacts on the proliferation and hematopoietic differentiation of FL-HSPCs were subsequently evaluated via flow cytometry (FCM). Furthermore, the effect of FoxO1-OE on the self-renewal of cord blood-derived hematopoietic stem and progenitor cells (CB-HSPCs) was investigated. Additionally, the transplantation ability of hematopoietic stem cells derived from these CB-HSPCs in mice after secondary transplantation was also assessed by FCM. After knocking down FoxO1 in FL-HSPCs, the apoptosis rate was significantly increased, and the expression of hCD45 was significantly decreased. Conversely, overexpression of FoxO1 reversed this phenomenon, effectively promoting the expansion and differentiation of FL-HSPCs in vitro. Similarly, it was found that FoxO1-OE could effectively enhance the expansion of CB-HSPCs. Furthermore, upon transplantation of CB-HSPCs overexpressing FoxO1 into NSG mice, multilineage human hematopoietic reconstruction was promoted. Notably, the results of secondary transplantation revealed that only the FoxO1-OE group exhibited multilineage reconstitution. In conclusion, our study confirmed that FoxO1-OE could enhance the self-renewal and engraftment of CB-HSPCs.

A proof-of-concept study to investigate the radiolabelling of human mesenchymal and hematopoietic stem cells with [89Zr]Zr-Df-Bz-NCS

BackgroundThe transplantation of hematopoietic stem and progenitor cells (HSPCs) or mesenchymal stromal/stem cells (MSCs) for the treatment of a wide variety of diseases has been studied extensively. A challenge with cell-based therapies is that migration to and retention at the target site is often difficult to monitor and quantify. Zirconium-89 (89Zr) is a positron-emitting radionuclide with a half-life of 3.3 days, which allows long-term cell tracking. Para-isothiocyanatobenzyl-desferrioxamine B (Df-Bz-NCS) is the chelating agent of choice for 89Zr-cell surface labelling. We utilised a shortened labelling method, thereby avoiding a 30–60-min incubation step for [89Zr]Zr-Df-Bz-NCS chelation, to radiolabel HSPCs and MSCs with zirconium-89.ResultsThree 89Zr-MSC labelling attempts were performed. High labelling efficiencies (81.30 and 87.30%) and relatively good labelling yields (59.59 and 67.00%) were achieved with the use of a relatively larger number of MSCs (4.425 and 3.855 million, respectively). There was no significant decrease in MSC viability after 89Zr-labeling (p = 0.31). This labelling method was also translatable to prepare 89Zr-HSPC; preliminary data from one preparation indicated high 89Zr-HSPC labelling efficiency (88.20%) and labelling yield (71.06%), as well as good HSPC viability after labelling (68.65%).ConclusionsSuccessful 89Zr-MSC and 89Zr-HSPC labelling was achieved, which underlines the prospects for in vivo cell tracking studies with positron emission tomography. In vitro investigations with larger sample sizes and preclinical studies are recommended.

Models to study myelodysplastic syndrome and acute myeloid leukaemia.

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are hematological malignancies characterized by complex genetic alterations, leading to poor clinical outcomes. Despite advances in treatment, there is an urgent need for novel therapeutic approaches. This review outlines recent progress in humanized models of MDS and AML and highlight their role in advancing our understanding of these diseases. Patient derived xenografts (PDXs) were among the first humanized models for studying MDS and AML, allowing researchers to analyze patient-specific cancer properties in vivo. However, they face challenges related to sample availability and consistent engraftment in mice. New methods, including specialized mouse strains and human tissue scaffolds, have been developed to address these issues. Induced pluripotent stem cells (iPSCs) offer the advantage of indefinite expansion and genetic modification, making them valuable for in vitro research, though protocols to enhance their engraftment in vivo are still being refined. Genetically engineered human primary hematopoietic stem and progenitor cells (HSPCs) provide reliable in vivo models with good engraftment in mice, and recent advancements in culture systems and gene-editing techniques are helping to overcome challenges related to ex vivo expansion and genetic modification. PDXs, iPSCs, and genetically engineered HSPCs are crucial models for the study of MDS and AML. This review discusses strengths, limitations, and recent advancements of these humanized models, which provide insights into human-specific disease biology and therapeutic development.

A strategy to design protein-based antagonists against type I cytokine receptors.

Excessive cytokine signaling resulting from dysregulation of a cytokine or its receptor can be a main driver of cancer, autoimmune, or hematopoietic disorders. Here, we leverage protein design to create tailored cytokine receptor blockers with idealized properties. Specifically, we aimed to tackle the granulocyte-colony stimulating factor receptor (G-CSFR), a mediator of different types of leukemia and autoinflammatory diseases. By modifying designed G-CSFR binders, we engineered hyper-stable proteins that function as nanomolar signaling antagonists. X-ray crystallography showed atomic-level agreement with the experimental structure of an exemplary design. Furthermore, the most potent design blocks G-CSFR in acute myeloid leukemia cells and primary human hematopoietic stem cells. Thus, the resulting designs can be used for inhibiting or homing to G-CSFR-expressing cells. Our results also demonstrate that similarly designed cytokine mimics can be used to derive antagonists to tackle other type I cytokine receptors.

Mechanism of bone-marrow mesenchymal stem cell-derived exosomes mediating microRNA-139-5p to regulate β-catenin in the modulation of proliferation and apoptosis of acute myeloid leukemia cells

ABSTRACT Objective: Acute myeloid leukemia (AML) stands out as a malignancy of the stem cell precursors of the myeloid lineage. Bone-marrow mesenchymal stem cell-derived exosomes (BMSC-exos) affect AML progression. We explored the effects and mechanism of BMSC-exos on AML cell proliferation and apoptosis. Methods: Human AML cells (MOLM-16, MV-4-11) and normal human hematopoietic cells (GM12878) cultured in vitro were treated with exos extracted from BMSCs that transfected with microRNA (miR)-139-5p-mimics, ovβ-catenin, or miR-139-5p-inhibitor. BMSC morphology was observed by a microscopy, and its adipogenic and osteogenic differentiation abilities were assessed by oil red O staining and alizarin red S staining. BMSC-exos were extracted by ultracentrifugation, and their morphology was observed by a transmission electron microscopy. BMSC-exos were identified by nanoparticle tracking analysis and Western blot. The binding sites between miR-139-5p and β-catenin were predicted by TargetScan database, and then validated by dual-luciferase reporter assay. mRNA levels of miR-139-5p and β-catenin, cell proliferation, and apoptosis were evaluated by RT-qPCR, CCK-8, and flow cytometry. The expressions of CD63, CD81, TSG101, and GRP94 and the proteins of β-catenin, Bax, and Bcl-2 were determined by Western blot. Results: miR-139-5p was poorly expressed in AML cell lines. miR-139-5p overexpression reduced AML cell viability/proliferation/Bcl-2 level, and raised apoptosis/Bax level. BMSC-exos repressed AML cell proliferation and promoted apoptosis via miR-139-5p. miR-139-5p targeted to inhibit β-catenin expression. Subsequently, up-regulated β-catenin partially counteracted the effects of miR-139-5p in BMSC-exos on AML cell proliferation and apoptosis. Conclusion: BMSC-exos targeted to repress β-catenin expression by miR-139-5p, limited AML cell proliferation and facilitated apoptosis.