Efficient generation of human NOTCH ligand-expressing haemogenic endothelial cells as infrastructure for in vitro haematopoiesis and lymphopoiesis.

Arterial endothelial cells (AECs) are the founder cells for intraembryonic haematopoiesis. Here, we report a method for the efficient generation of human haemogenic DLL4+ AECs from pluripotent stem cells (PSC). Time-series single-cell RNA-sequencing reveals the dynamic evolution of haematopoiesis and lymphopoiesis, generating cell types with counterparts present in early human embryos, including stages marked by the pre-haematopoietic stem cell genes MECOM/EVI1, MLLT3 and SPINK2. DLL4+ AECs robustly support lymphoid differentiation, without the requirement for exogenous NOTCH ligands. Using this system, we find IL7 acts as a morphogenic factor determining the fate choice between the T and innate lymphoid lineages and also plays a role in regulating the relative expression level of RAG1. Moreover, we document a developmental pathway by which human RAG1+ lymphoid precursors give rise to the natural killer cell lineage. Our study describes an efficient method for producing lymphoid progenitors, providing insights into their endothelial and haematopoietic ontogeny, and establishing a platform to investigate the development of the human blood system.

Cxcl10 and Cxcr3 regulate self-renewal and differentiation of hematopoietic stem cells.

BACKGROUND: The function of hematopoietic stem cells (HSC) is regulated by HSC internal signaling pathways and their microenvironment. Chemokines and chemokine ligands play important roles in the regulation of HSC function. Yet, their functions in HSC are not fully understood. METHODS: We established Cxcr3 and Cxcl10 knockout mouse models (Cxcr3-/- and Cxcl10-/-) to analyze the roles of Cxcr3 or Cxcl10 in regulating HSC function. The cell cycle distribution of LT-HSC was assessed via flow cytometry. Cxcr3-/- and Cxcl10-/- stem/progenitor cells showed reduced self-renewal capacity as measured in serial transplantation assays. To study the effects of Cxcr3 or Cxcl10 deficient bone marrow microenvironment, we transplanted CD45.1 donor cells into Cxcr3-/-or Cxcl10-/- recipient mice (CD45.2) and examined donor-contributed hematopoiesis. RESULTS: Deficiency of Cxcl10 and its receptor Cxcr3 led to decreased BM cellularity in mice, with a significantly increased proportion of LT-HSC. Cxcl10-/- stem/progenitor cells showed reduced self-renewal capacity in the secondary transplantation assay. Notably, Cxcl10-/- donor-derived cells preferentially differentiated into B lymphocytes, with skewed myeloid differentiation ability. Meanwhile, Cxcr3-deficient HSCs demonstrated a reconstitution disadvantage in secondary transplantation, but the lineage bias was not significant. Interestingly, the absence of Cxcl10 or Cxcr3 in bone marrow microenvironment did not affect HSC function. CONCLUSIONS: The Cxcl10 and Cxcr3 regulate the function of HSC, including self-renewal and differentiation, adding to the understanding of the roles of chemokines in the regulation of HSC function.

Lysosomal membrane protein TMEM106B modulates hematopoietic stem and progenitor cell proliferation and differentiation by regulating LAMP2A stability.

Hematopoietic stem and progenitor cells (HSPCs) are successfully employed for hematological transplantations, and impaired HSPC function causes hematological diseases and aging. HSPCs maintain the lifelong homeostasis of blood and immune cells through continuous self-renewal and maintenance of the multilineage differentiation potential. TMEM106B is a transmembrane protein localized on lysosomal membranes and associated with neurodegenerative and cardiovascular diseases; however, its roles in HSPCs and hematopoiesis are unknown. Here, we established tmem106bb-/- knockout (KO) zebrafish and showed that tmem106bb KO reduced the proliferation of HSPCs during definitive hematopoiesis. The differentiation potential of HSPCs to lymphoid lineage was reduced, whereas the myeloid and erythroid differentiation potentials of HPSCs were increased in tmem106bb-/- zebrafish. Similar results were obtained with morpholino knockdown of tmem106bb. Mechanistically, TMEM106B interacted with LAMP2A, the lysosomal associated membrane protein 2A, impaired LAMP2A-Cathepsin A interaction, and enhanced LAMP2A stability; tmem106bb KO or TMEM106B knockdown caused LAMP2A degradation and impairment of chaperone-mediated autophagy (CMA). Knockdown of lamp2a caused similar phenotypes to that in tmem106bb-/- zebrafish, and overexpression of lamp2a rescued the impaired phenotypes of HSPCs in tmem106bb-/- embryos. These results uncover a novel molecular mechanism for the maintenance of HSPC proliferation and differentiation through stabilizing LAMP2A via TMEM106B-LAMP2A interaction.

Regulation of the hematopoietic stem cell pool by C-Kit-associated trogocytosis.

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. Although fully functional, these HSCs 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 by trogocytosis, regulated by receptor tyrosine-protein kinase C-Kit (CD117), 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.

Sysmex XN-HPC: study of reference intervals and clinical decision limits in healthy allogeneic donors mobilised with G-CSF.

The Sysmex XN series haematopoietic progenitor cell (XN-HPC) is a novel tool for assessing stem cell yield before allogeneic haematopoietic stem cell transplantation. This study aimed to establish a reference interval (RI) for XN-HPC in peripheral blood allogeneic transplant donors following granulocyte colony-stimulating factor (G-CSF) stimulation and determine its clinical significance. All specimens were analysed using Sysmex XN-20. Samples were collected and analysed using non-parametric percentile methods to define the RIs. Quantile regression was used to explore the dependency of the RIs on sex and age. Samples were included in clinical decision limits for apheresis based on receiver operating characteristic curve analysis. The non-parametrically estimated RI for XN-HPC was 623.50 (90% confidence interval [CI90%] 510.00-657.00) to 4,144.28 (CI90% 3,761.00-4,547.00). The RIs for the XN-HPC were not age-dependent but were sex-dependent. The RI for males was 648.40 (CI90% 582.00-709.00)-4,502.60 (CI90% 4,046.00-5,219.00) and for females was 490.90 (CI90% 311.00-652.00)-3,096.90 (CI90% 2,749.00-3,782.00). Comparisons based on XN-HPC values between the poor and less-than-optimal groups, good and less-than-optimal groups, and good and non-good groups had areas under the curve of 0.794 (P < 0.001), 0.768 (P < 0.001), and 0.806 (P < 0.001), respectively, indicating a good predictive value for mobilisation effectiveness. XN-HPC data exceeding 3974 × 106/L suggested that a sufficient number of stem cells could be collected clinically. Values > 5318 < 106/L indicated 100% mobilisation effectiveness. We established an RI for XN-HPC in peripheral blood allogeneic transplant donors following G-CSF stimulation and determined clinical decision thresholds for mobilisation efficiency.

Canalizing kernel for cell fate determination.

The tendency for cell fate to be robust to most perturbations, yet sensitive to certain perturbations raises intriguing questions about the existence of a key path within the underlying molecular network that critically determines distinct cell fates. Reprogramming and trans-differentiation clearly show examples of cell fate change by regulating only a few or even a single molecular switch. However, it is still unknown how to identify such a switch, called a master regulator, and how cell fate is determined by its regulation. Here, we present CAESAR, a computational framework that can systematically identify master regulators and unravel the resulting canalizing kernel, a key substructure of interconnected feedbacks that is critical for cell fate determination. We demonstrate that CAESAR can successfully predict reprogramming factors for de-differentiation into mouse embryonic stem cells and trans-differentiation of hematopoietic stem cells, while unveiling the underlying essential mechanism through the canalizing kernel. CAESAR provides a system-level understanding of how complex molecular networks determine cell fates.

The heart is a resident tissue for hematopoietic stem and progenitor cells in zebrafish.

The contribution of endocardial cells (EdCs) to the hematopoietic lineages has been strongly debated. Here, we provide evidence that in zebrafish, the endocardium gives rise to and maintains a stable population of hematopoietic cells. Using single-cell sequencing, we identify an endocardial subpopulation expressing enriched levels of hematopoietic-promoting genes. High-resolution microscopy and photoconversion tracing experiments uncover hematopoietic cells, mainly hematopoietic stem and progenitor cells (HSPCs)/megakaryocyte-erythroid precursors (MEPs), derived from EdCs as well as the dorsal aorta stably attached to the endocardium. Emergence of HSPCs/MEPs in hearts cultured ex vivo without external hematopoietic sources, as well as longitudinal imaging of the beating heart using light sheet microscopy, support endocardial contribution to hematopoiesis. Maintenance of these hematopoietic cells depends on the adhesion factors Integrin α4 and Vcam1 but is at least partly independent of cardiac trabeculation or shear stress. Finally, blocking primitive erythropoiesis increases cardiac-residing hematopoietic cells, suggesting that the endocardium is a hematopoietic reservoir. Altogether, these studies uncover the endocardium as a resident tissue for HSPCs/MEPs and a de novo source of hematopoietic cells.

Heterotypic interaction promotes asymmetric division of human hematopoietic progenitors.

Hematopoietic stem and progenitor cells (HSPCs) give rise to all cell types of the hematopoietic system through various processes, including asymmetric divisions. However, the contribution of stromal cells of the hematopoietic niches in the control of HSPC asymmetric divisions remains unknown. Using polyacrylamide microwells as minimalist niches, we show that specific heterotypic interactions with osteoblast and endothelial cells promote asymmetric divisions of human HSPCs. Upon interaction, HSPCs polarize in interphase with the centrosome, the Golgi apparatus, and lysosomes positioned close to the site of contact. Subsequently, during mitosis, HSPCs orient their spindle perpendicular to the plane of contact. This division mode gives rise to siblings with unequal amounts of lysosomes and of the differentiation marker CD34. Such asymmetric inheritance generates heterogeneity in the progeny, which is likely to contribute to the plasticity of the early steps of hematopoiesis.

Development of VLA4 and CXCR4 Antagonists for the Mobilization of Hematopoietic Stem and Progenitor Cells.

The treatment of patients diagnosed with hematologic malignancies typically includes hematopoietic stem cell transplantation (HSCT) as part of a therapeutic standard of care. The primary graft source of hematopoietic stem and progenitor cells (HSPCs) for HSCT is mobilized from the bone marrow into the peripheral blood of allogeneic donors or patients. More recently, these mobilized HSPCs have also been the source for gene editing strategies to treat diseases such as sickle-cell anemia. For a HSCT to be successful, it requires the infusion of a sufficient number of HSPCs that are capable of adequate homing to the bone marrow niche and the subsequent regeneration of stable trilineage hematopoiesis in a timely manner. Granulocyte-colony-stimulating factor (G-CSF) is currently the most frequently used agent for HSPC mobilization. However, it requires five or more daily infusions to produce an adequate number of HSPCs and the use of G-CSF alone often results in suboptimal stem cell yields in a significant number of patients. Furthermore, there are several undesirable side effects associated with G-CSF, and it is contraindicated for use in sickle-cell anemia patients, where it has been linked to serious vaso-occlusive and thrombotic events. The chemokine receptor CXCR4 and the cell surface integrin α4ß1 (very late antigen 4 (VLA4)) are both involved in the homing and retention of HSPCs within the bone marrow microenvironment. Preclinical and/or clinical studies have shown that targeted disruption of the interaction of the CXCR4 or VLA4 receptors with their endogenous ligands within the bone marrow niche results in the rapid and reversible mobilization of HSPCs into the peripheral circulation and is synergistic when combined with G-CSF. In this review, we discuss the roles CXCR4 and VLA4 play in bone marrow homing and retention and will summarize more recent development of small-molecule CXCR4 and VLA4 inhibitors that, when combined, can synergistically improve the magnitude, quality and convenience of HSPC mobilization for stem cell transplantation and ex vivo gene therapy after the administration of just a single dose. This optimized regimen has the potential to afford a superior alternative to G-CSF for HSPC mobilization.

Baf155 controls hematopoietic differentiation and regeneration through chromatin priming.

Chromatin priming promotes cell-type-specific gene expression, lineage differentiation, and development. The mechanism of chromatin priming has not been fully understood. Here, we report that mouse hematopoietic stem and progenitor cells (HSPCs) lacking the Baf155 subunit of the BAF (BRG1/BRM-associated factor) chromatin remodeling complex produce a significantly reduced number of mature blood cells, leading to a failure of hematopoietic regeneration upon transplantation and 5-fluorouracil (5-FU) injury. Baf155-deficient HSPCs generate particularly fewer neutrophils, B cells, and CD8+ T cells at homeostasis, supporting a more immune-suppressive tumor microenvironment and enhanced tumor growth. Single-nucleus multiomics analysis reveals that Baf155-deficient HSPCs fail to establish accessible chromatin in selected regions that are enriched for putative enhancers and binding motifs of hematopoietic lineage transcription factors. Our study provides a fundamental mechanistic understanding of the role of Baf155 in hematopoietic lineage chromatin priming and the functional consequences of Baf155 deficiency in regeneration and tumor immunity.

Clonal analysis of fetal hematopoietic stem/progenitor cells reveals how post-transplantation capabilities are distributed.

It has been proposed that adult hematopoiesis is sustained by multipotent progenitors (MPPs) specified during embryogenesis. Adult-like hematopoietic stem cell (HSC) and MPP immunophenotypes are present in the fetus, but knowledge of their functional capacity is incomplete. We found that fetal MPP populations were functionally similar to adult cells, albeit with some differences in lymphoid output. Clonal assessment revealed that lineage biases arose from differences in patterns of single-/bi-lineage differentiation. Long-term (LT)- and short-term (ST)-HSC populations were distinguished from MPPs according to capacity for clonal multilineage differentiation. We discovered that a large cohort of long-term repopulating units (LT-RUs) resides within the ST-HSC population; a significant portion of these were labeled using Flt3-cre. This finding has two implications: (1) use of the CD150+ LT-HSC immunophenotype alone will significantly underestimate the size and diversity of the LT-RU pool and (2) LT-RUs in the ST-HSC population have the attributes required to persist into adulthood.

Hematopoietic stem cell heterogeneity and age-associated platelet bias are evolutionarily conserved.

Hematopoietic stem cells (HSCs) reconstitute multilineage human hematopoiesis after clinical bone marrow (BM) transplantation and are the cells of origin of some hematological malignancies. Although HSCs provide multilineage engraftment, individual murine HSCs are lineage biased and contribute unequally to blood cell lineages. Here, we performed high-throughput single-cell RNA sequencing in mice after xenograft with molecularly barcoded adult human BM HSCs. We demonstrated that human individual BM HSCs are also functionally and transcriptionally lineage biased. Specifically, we identified platelet-biased and multilineage human HSCs. Quantitative comparison of transcriptomes from single HSCs from young and aged BM showed that both the proportion of platelet-biased HSCs and their level of transcriptional platelet priming increase with age. Therefore, platelet-biased HSCs and their increased prevalence and transcriptional platelet priming during aging are conserved features of mammalian evolution.

Bioengineered niches that recreate physiological extracellular matrix organisation to support long-term haematopoietic stem cells.

Long-term reconstituting haematopoietic stem cells (LT-HSCs) are used to treat blood disorders via stem cell transplantation. The very low abundance of LT-HSCs and their rapid differentiation during in vitro culture hinders their clinical utility. Previous developments using stromal feeder layers, defined media cocktails, and bioengineering have enabled HSC expansion in culture, but of mostly short-term HSCs and progenitor populations at the expense of naive LT-HSCs. Here, we report the creation of a bioengineered LT-HSC maintenance niche that recreates physiological extracellular matrix organisation, using soft collagen type-I hydrogels to drive nestin expression in perivascular stromal cells (PerSCs). We demonstrate that nestin, which is expressed by HSC-supportive bone marrow stromal cells, is cytoprotective and, via regulation of metabolism, is important for HIF-1α expression in PerSCs. When CD34+ve HSCs were added to the bioengineered niches comprising nestin/HIF-1α expressing PerSCs, LT-HSC numbers were maintained with normal clonal and in vivo reconstitution potential, without media supplementation. We provide proof-of-concept that our bioengineered niches can support the survival of CRISPR edited HSCs. Successful editing of LT-HSCs ex vivo can have potential impact on the treatment of blood disorders.

Counting blood precursors.

A new mathematical model can estimate the number of precursor cells that contribute to regenerating blood cells in mice.

Ex Vivo Evaluation of the Function of Hematopoietic Stem Cells in Toxicology of Metals.

A variety of metals, e.g., lead (Pb), cadmium (Cd), and lithium (Li), are in the environment and are toxic to humans. Hematopoietic stem cells (HSCs) reside at the apex of hematopoiesis and are capable of generating all kinds of blood cells and self-renew to maintain the HSC pool. HSCs are sensitive to environmental stimuli. Metals may influence the function of HSCs by directly acting on HSCs or indirectly by affecting the surrounding microenvironment for HSCs in the bone marrow (BM) or niche, including cellular and extracellular components. Investigating the impact of direct and/or indirect actions of metals on HSCs contributes to the understanding of immunological and hematopoietic toxicology of metals. Treatment of HSCs with metals ex vivo, and the ensuing HSC transplantation assays, are useful for evaluating the impacts of the direct actions of metals on the function of HSCs. Investigating the mechanisms involved, given the rarity of HSCs, methods that require large numbers of cells are not suitable for signal screening; however, flow cytometry is a useful tool for signal screening HSCs. After targeting signaling pathways, interventions ex vivo and HSCs transplantation are required to confirm the roles of the signaling pathways in regulating the function of HSCs exposed to metals. Here, we describe protocols to evaluate the mechanisms of direct and indirect action of metals on HSCs. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Identify the impact of a metal on the competence of HSCs Basic Protocol 2: Identify the impact of a metal on the lineage bias of HSC differentiation Basic Protocol 3: Screen the potential signaling molecules in HSCs during metal exposure Alternate Protocol 1: Ex vivo treatment with a metal on purified HSCs Alternate Protocol 2: Ex vivo intervention of the signaling pathway regulating the function of HSCs during metal exposure.

Hematopoietic Stem Cells and Their Niche in Bone Marrow.

Extensive research has explored the functional correlation between stem cells and progenitor cells, particularly in blood. Hematopoietic stem cells (HSCs) can self-renew and regenerate tissues within the bone marrow, while stromal cells regulate tissue function. Recent studies have validated the role of mammalian stem cells within specific environments, providing initial empirical proof of this functional phenomenon. The interaction between bone and blood has always been vital to the function of the human body. It was initially proposed that during evolution, mammalian stem cells formed a complex relationship with the surrounding microenvironment, known as the niche. Researchers are currently debating the significance of molecular-level data to identify individual stromal cell types due to incomplete stromal cell mapping. Obtaining these data can help determine the specific activities of HSCs in bone marrow. This review summarizes key topics from previous studies on HSCs and their environment, discussing current and developing concepts related to HSCs and their niche in the bone marrow.

Donor and recipient hematopoietic stem and progenitor cells mobilization in liver transplantation patients.

BACKGROUND: Hematopoietic stem and progenitor cells (HSPCs) mobilize from bone marrow to peripheral blood in response to stress. The impact of alloresponse-induced stress on HSPCs mobilization in human liver transplantation (LTx) recipients remains under-investigated. METHODS: Peripheral blood mononuclear cell (PBMC) samples were longitudinally collected from pre- to post-LTx for one year from 36 recipients with acute rejection (AR), 74 recipients without rejection (NR), and 5 recipients with graft-versus-host disease (GVHD). 28 PBMC samples from age-matched healthy donors were collected as healthy control (HC). Multi-color flow cytometry (MCFC) was used to immunophenotype HSPCs and their subpopulations. Donor recipient-distinguishable major histocompatibility complex (MHC) antibodies determined cell origin. RESULTS: Before LTx, patients who developed AR after transplant contained more HSPCs in PBMC samples than HC, while the NR group patients contained fewer HSPCs than HC. After LTx, the HSPC ratio in the AR group sharply decreased and became less than HC within six months, and dropped to a comparable NR level afterward. During the one-year follow-up period, myeloid progenitors (MPs) biased differentiation was observed in all LTx recipients who were under tacrolimus-based immunosuppressive treatment. During both AR and GVHD episodes, the recipient-derived and donor-derived HSPCs mobilized into the recipient's blood-circulation and migrated to the target tissue, respectively. The HSPCs percentage in blood reduced after the disease was cured. CONCLUSIONS: A preoperative high HSPC ratio in blood characterizes recipients who developed AR after LTx. Recipients exhibited a decline in blood-circulating HSPCs after transplant, the cells mobilized into the blood and migrated to target tissue during alloresponse.

Umbilical cord blood derived cell expansion: a potential neuroprotective therapy.

Umbilical cord blood (UCB) is a rich source of beneficial stem and progenitor cells with known angiogenic, neuroregenerative and immune-modulatory properties. Preclinical studies have highlighted the benefit of UCB for a broad range of conditions including haematological conditions, metabolic disorders and neurological conditions, however clinical translation of UCB therapies is lacking. One barrier for clinical translation is inadequate cell numbers in some samples meaning that often a therapeutic dose cannot be achieved. This is particularly important when treating adults or when administering repeat doses of cells. To overcome this, UCB cell expansion is being explored to increase cell numbers. The current focus of UCB cell expansion is CD34+ haematopoietic stem cells (HSCs) for which the main application is treatment of haematological conditions. Currently there are 36 registered clinical trials that are examining the efficacy of expanded UCB cells with 31 of these being for haematological malignancies. Early data from these trials suggest that expanded UCB cells are a safe and feasible treatment option and show greater engraftment potential than unexpanded UCB. Outside of the haematology research space, expanded UCB has been trialled as a therapy in only two preclinical studies, one for spinal cord injury and one for hind limb ischemia. Proteomic analysis of expanded UCB cells in these studies showed that the cells were neuroprotective, anti-inflammatory and angiogenic. These findings are also supported by in vitro studies where expanded UCB CD34+ cells showed increased gene expression of neurotrophic and angiogenic factors compared to unexpanded CD34+ cells. Preclinical evidence demonstrates that unexpanded CD34+ cells are a promising therapy for neurological conditions where they have been shown to improve multiple indices of injury in rodent models of stroke, Parkinson's disease and neonatal hypoxic ischemic brain injury. This review will highlight the current application of expanded UCB derived HSCs in transplant medicine, and also explore the potential use of expanded HSCs as a therapy for neurological conditions. It is proposed that expanded UCB derived CD34+ cells are an appropriate cellular therapy for a range of neurological conditions in children and adults.