Haematopoietic microenvironmental niches have been described as the 'gatekeepers' for the blood and immune systems. These niches change during ontogeny, with the bone marrow becoming the predominant site of haematopoiesis in post-natal life under steady state conditions. To determine the structure and function of different haematopoietic microenvironmental niches, it is essential to clearly define specific haematopoietic stem and progenitor cell subsets during ontogeny and to understand their temporal appearance and anatomical positioning. A variety of haematopoietic and non-haematopoietic cells contribute to haematopoietic stem and progenitor cell niches. The latter is reported to include endothelial cells and mesenchymal stromal cells (MSCs), skeletal stem cells and/or C-X-C motif chemokine ligand 12-abundant-reticular cell populations, which form crucial components of these microenvironments under homeostatic conditions. Dysregulation or deterioration of such cells contributes to significant clinical disorders and diseases worldwide and is associated with the ageing process. A critical appraisal of these issues and of the roles of MSC/C-X-C motif chemokine ligand 12-abundant-reticular cells and the more recently identified skeletal stem cell subsets in bone marrow haematopoietic niche function under homeostatic conditions and during ageing will form the basis of this research review. In the context of haematopoiesis, clinical translation will deal with lessons learned from the vast experience garnered from the development and use of MSC therapies to treat graft versus host disease in the context of allogeneic haematopoietic transplants, the recent application of these MSC therapies to treating emerging and severe coronavirus disease 2019 (COVID-19) infections, and, given that skeletal stem cell ageing is one proposed driver for haematopoietic ageing, the potential contributions of these stem cells to haematopoiesis in healthy bone marrow and the benefits and challenges of using this knowledge for rejuvenating the age-compromised bone marrow haematopoietic niches and restoring haematopoiesis.

Background: With haematology diagnostics being centralised over the past ten years in the UK and increasingly complex diagnostic pathways, it is important that the treating clinicians and reporters are speaking the “same language” in order that optimal treatment for patient can be achieved. We have reviewed the literature and there are studies looking at language use in general pathology, but to our knowledge nobody has looked at the language used in haemato-pathology reporting, in particular the descriptive terms around diagnoses. Sometimes the language used by reporters reflects the uncertainty in the diagnosis due to atypical appearances or other tests not being consistent, the fact that tests are incomplete or clinical information is missing from the referral. Service users may prefer more unequivocal language to definitively guide management. Supplementary communication in addition reports was often utilised to contextualise findings. Aims: We decided to review the language used in our regional haemato-oncology service Methods: We collected the most commonly used terms from the combined marrow smear and trephine morphology, flow cytometry, molecular and conventional genetics reports at our regional service. We then designed a survey to determine a percentage score for how certain a diagnosis was if it was preceded by the 8 commonly used descriptive terms: ‘suspicious of’, ‘compatible with’, ‘diagnostic of’, ‘suggestive of’, ‘in keeping with’, ‘consistent with’, ‘features are those of’ and ‘cannot be excluded’. Participants were also asked to rank the terms according to their certainty and to express if the term had more weighting if it was for a positive diagnosis or that of a remission. The was also a free text section for participants to express any terms they particularly liked or disliked. We asked both reporters and service users to complete the survey. Results: A total of 47 surveys were returned including 33 who described themselves as reporters and 14 who described themselves as service users. The mean percentage certainty was calculated for all responders as well as for the service user and reporter groups. The range was also calculated for these respective samples. We found that “diagnostic of” scored overall highest for degree of certainty at 97.8% and “cannot be excluded” lowest at 29.9%. The most interesting aspect was the large range of results in “suspicious of” and “cannot be excluded” where there was 70 percentage points between the highest and lowest answers. An unpaired t-test showed that for “suspicious of” and “compatible with” there was a statistically significant difference in mean certainty between reporters and service users. The largest difference between reporters and users was “suspicious of” with reporters rating 14% higher, 59% v 45%. Image:Summary/Conclusion: The survey shows how perceptions of reports can differ markedly between individual reporters and service users. This demonstrates the importance of the MDT in ensuring pathologists play an active role in the diagnosis and that their reports are not misinterpreted. Pathology services should try to standardise their language to reduce the amount of ambiguity in their reports. We would be very interested to see if these differences are replicated in other diagnostic services both within the UK and also in other languages. The next step in this project is to gather a focus group and gain more in-depth qualitative data. This would be to discuss not only the terms used in the questionnaire, but the wider themes of communication previously described.

The past five decades have seen significant progress in our understanding of human hematopoiesis. This has in part been due to the unprecedented development of advanced technologies, which have allowed the identification and characterization of rare subsets of human hematopoietic stem and progenitor cells and their lineage trajectories from embryonic through to adult life. Additionally, surrogate in vitro and in vivo models, although not fully recapitulating human hematopoiesis, have spurred on these scientific advances. These approaches have heightened our knowledge of hematological disorders and diseases and have led to their improved diagnosis and therapies. Here, we review human hematopoiesis at each end of the age spectrum, during embryonic and fetal development and on aging, providing exemplars of recent progress in deciphering the increasingly complex cellular and molecular hematopoietic landscapes in health and disease. This review concludes by highlighting links between chronic inflammation and metabolic and epigenetic changes associated with aging and in the development of clonal hematopoiesis.

© Annals of Blood. All rights reserved. Ann Blood 2020 | http://dx.doi.org/10.21037/aob-20-82 Platelet concentrates are transfused for life-saving prophylactic or therapeutic purposes. These include treatment for those thrombocytopenic patients with acute or chronic blood loss, or those undergoing hematopoietic stem cell (HSC) or organ transplantation (1). In 2017, European blood banks issued over 2.9 million platelet concentrates, of which just over 2.28 million were transfused into 317,898 recipients (2). More recent estimates [although these figures are affected by and evolving during the current 2020 SARSCoV2 pandemic (3)] indicate that over 2 million platelet concentrates (around 7,000 per day) are administered per annum to patients in the USA alone (4,5). Although guidelines have been developed for platelet transfusions (1,5,6), and revised recently in the light of a potential scarcity of platelets during the SARS-CoV2 pandemic (3), controversies still exist as to best practice. In less challenging times, one such controversy relates to defining or developing the best platelet product for transfusion. While such platelets are generally sourced from healthy blood donors by apheresis or after isolation and pooling from whole blood (1), they possess a short shelflife (5–7 days at room temperature), have restrictive storage conditions, rely on donor availability, are susceptible to pathogen contamination, and can lead to allo-immunisation to HLA Class I antigens (1,3,5). The prospect that platelet donations may not meet future demands has spurred on research to generate platelets from alternative sources. These include the production of megakaryocytes and platelets from a variety of stem and progenitor cells ex vivo and the development of technologies to manufacture artificial platelets (7-9). Needless to say, platelets once generated ex vivo from stem and progenitor cells would undoubtedly face similar, or possibly even further, limitations related to shelf-life and storage conditions as those sourced directly from donors. However, advantages cited include a lack of reliance on immediate blood donor availability, reduction in the potential risk of pathogen (e.g., viral or variant Creutzfeldt-Jakob disease) transmission, and the potential to reduce their immunogenicity through, for example, genetic modification of the originating stem and progenitor cells (7,8). A number of stem cell studies have concentrated on the use of ES (embryonic stem) or iPS (induced pluripotent stem) cells or immortalised cell lines with megakaryocytic potential for sustained platelet production, incorporating such techniques as forward programing to enhance megakaryocyte and platelet production (7,8). Safety concerns include the propensity for tumor formation through cross-contamination of the platelet products by the originating lines (7,8). An alternative approach is to manufacture and amplify megakaryocytes and their precursors ex vivo after sourcing hematopoietic stem and progenitor cells (HSPCs) from healthy human donors prior to infusion into patients, with platelet production then proceeding in vivo. Indeed, modelling studies in murine or non-human primate models indicate that megakaryocytes Editorial Commentary

Accurately defining hierarchical relationships between human stem cells and their progeny, and using this knowledge for new cellular therapies, will undoubtedly lead to further successful treatments for life threatening and chronic diseases, which represent substantial burdens on patient quality of life and to healthcare systems globally. Clinical translation relies in part on appropriate biomarker, in vitro manipulation and transplantation strategies. CD164 has recently been cited as an important biomarker for enriching both human haematopoietic and skeletal stem cells, yet a thorough description of extant human CD164 monoclonal antibody (Mab) characteristics, which are critical for identifying and purifying these stem cells, was not discussed in these articles. Here, we highlight earlier but crucial research describing these relevant characteristics, including the differing human CD164 Mab avidities and their binding sites on the human CD164 sialomucin, which importantly may affect subsequent stem cell function and fate.

Humanized immune system (HIS) mouse models are useful tools for the in vivo investigation of human hematopoiesis. However, the majority of HIS models currently in use are biased towards lymphocyte development and fail to support long-term multilineage leucocytes and erythrocytes. Those that achieve successful multilineage reconstitution often require preconditioning steps which are expensive, cause animal morbidity, are technically demanding, and poorly reproducible. In this study, we address this challenge by using HSPC-NBSGW mice, in which NOD,B6.SCID IL-2rγ-/-KitW41/W41 (NBSGW) mice are engrafted with human CD133+ hematopoietic stem and progenitor cells (HSPCs) without the need for preconditioning by sublethal irradiation. These HSPCs are enriched in long-term hematopoietic stem cells (LT-HSCs), while NBSGW mice are permissive to human hematopoietic stem cell (HSC) engraftment, thus reducing the cell number required for successful HIS development. B cells reconstitute with the greatest efficiency, including mature B cells capable of class-switching following allogeneic stimulation and, within lymphoid organs and peripheral blood, T cells at a spectrum of stages of maturation. In the thymus, human thymocytes are identified at all major stages of development. Phenotypically distinct subsets of myeloid cells, including dendritic cells and mature monocytes, engraft to a variable degree in the bone marrow and spleen, and circulate in peripheral blood. Finally, we observe human erythrocytes which persist in the periphery at high levels following macrophage clearance. The HSPC-NBSGW model therefore provides a useful platform for the study of human hematological and immunological processes and pathologies.

The ontogeny of the human haematopoietic system during fetal development has previously been characterized mainly through careful microscopic observations1. Here we reconstruct a phylogenetic tree of blood development using whole-genome sequencing of 511 single-cell-derived haematopoietic colonies from healthy human fetuses at 8 and 18 weeks after conception, coupled with deep targeted sequencing of tissues of known embryonic origin. We found that, in healthy fetuses, individual haematopoietic progenitors acquire tens of somatic mutations by 18 weeks after conception. We used these mutations as barcodes and timed the divergence of embryonic and extra-embryonic tissues during development, and estimated the number of blood antecedents at different stages of embryonic development. Our data support a hypoblast origin of the extra-embryonic mesoderm and primitive blood in humans. PMID: 33981037 Funding information This work was supported by: Wellcome Trust, United Kingdom Grant ID: 203151/Z/16/Z European Research Council, International

The myelodysplastic syndromes (MDS) are common myeloid malignancies characterized by ineffective hematopoiesis and blood cytopenias, with patients showing increasing bone marrow blasts with disease progression [1].Mutations in genes involved in pre-mRNA splicing (SF3B1, SRSF2, U2AF1, and ZRSR2) are the most common mutations found in MDS, occurring in over 50% of all cases [2-4].There is evidence that some spliceosome components play a role in the maintenance of genomic stability [5].Splicing is a transcription coupled process; splicing factor mutations affect transcription and may lead to the accumulation of Rloops (RNA-DNA hybrids with a displaced single stranded DNA) [6].Mutations in the splicing factors SRSF2 and U2AF1 have been recently shown to increase R-loop formation in leukemia cell lines, resulting in increased DNA damage, replication stress, and activation of the ATR-Chk1 pathway [7,8].SF3B1 is the most frequently mutated

Introduction: Humanised mouse models provide a platform to investigate human immune biology in vivo, reducing the need for large animal studies. Importantly they can overcome biological differences between humans and other species, reducing the uncertainty in progressing to clinical studies. In transplantation research, humanised immune system (HIS) models have provided pre-clinical evidence for human-specific therapies such as biologics and regulatory T cell therapy, which are now in clinical trials. Most HIS models lack multilineage leukocyte development, limiting accurate representation of the full alloresponse in transplantation. To address this, we assessed immune reconstitution and human skin transplant rejection in NOD,B6.SCID Il2rγ−/− KitW41/W41 (NBSGW) mice, which are capable of long-term multilineage haemopoiesis without irradiation due to the presence of an inactivating mutation in the stem cell growth factor receptor. Materials and Methods: Human cord blood CD133+ haematopoietic stem cells (HSCs) were injected intravenously into non-irradiated NBSGW mice (n=20). Engraftment in blood and lymphoid organs was assessed over 20 weeks. In a separate group, HSC-engrafted mice (HIS-NBSGW) underwent allogeneic human skin transplantation 10-12 weeks post-injection (Figure 1).At the point of skin transplant rejection (or after 100 days) blood, spleen, bone marrow (BM), thymus and skin were analysed by multiparameter flow cytometry and NanoString gene expression analysis. Results and Discussion: High levels of human CD45+ cell chimerism developed reliably in the blood, BM and spleen after HSC transplantation in a dose-dependent manner (Figure 2).Multilineage engraftment was achieved, consisting of human myeloid cells, B and T lymphocytes at multiple recognised stages of development. Expression of Human Leukocyte Antigen (HLA) Class I and II molecules was identified on CD3- thymocytes. Antigen-presenting cells (APCs), B cells and T cells were found to be functional in in vitro assays. In mice with multilineage human leukocyte reconstitution, allogeneic human skin grafts rejected. Rejection was associated with RNA markers of upregulated immune function and elevated production of IgM, IgG and Th1, Th2 and Th17-related cytokines (Figure 3).Conclusion: The HIS-NBSGW model provides a technically straightforward research system utilising adult mice, which does not require irradiation, co-transplantation of human haematopoietic stromal tissues, or provision of human cytokines. By producing functional multilineage immune cells, it is a useful method for assessing HSC function in vivo and a framework for assessing human transplant responses encompassing the breadth of innate, cellular and humoural immunity. Clarendon Fund, University of Oxford. MRC Discovery Award, Ref: MC_PC_15069.

Although cytokine-mediated expansion of human hematopoietic stem cells (HSCs) can result in high yields of hematopoietic progenitor cells, this generally occurs at the expense of reduced bone marrow HSC repopulating ability, thereby limiting potential therapeutic applications. Because bromodomain-containing proteins (BCPs) have been demonstrated to regulate mouse HSC self-renewal and stemness, we screened small molecules targeting various BCPs as potential agents for ex vivo expansion of human HSCs. Of 10 compounds tested, only the bromodomain and extra-terminal motif inhibitor CPI203 enhanced the expansion of human cord blood HSCs without losing cell viability in vitro. The expanded cells also demonstrated improved engraftment and repopulation in serial transplantation assays. Transcriptomic and functional studies showed that the expansion of long-term repopulating HSCs was ac-companied by synchronized expansion and maturation of megakaryocytes consistent with CPI203-mediated reprogramming of cord blood hematopoietic stem and progenitor cells. This approach may therefore prove beneficial for ex vivo gene editing, for enhanced platelet production, and for the improved usage of cord blood for transplantation research and therapy.

P0-related protein (PZR), a Noonan and LEOPARD syndrome target, is a member of the transmembrane Immunoglobulin superfamily. Its cytoplasmic tail contains two immune-receptor tyrosine-based inhibitory motifs (ITIMs), implicated in adhesion-dependent signaling and regulating cell adhesion and motility. PZR promotes cell migration on the extracellular matrix (ECM) molecule, fibronectin, by interacting with SHP-2 (Src homology-2 domain-containing protein tyrosine phosphatase-2), a molecule essential for skeletal development and often mutated in Noonan and LEOPARD syndrome patients sharing overlapping musculoskeletal abnormalities and cardiac defects. To further explore the role of PZR, we assessed the expression of PZR and its ITIM-less isoform, PZRb, in human bone marrow mesenchymal stromal cells (hBM MSC), and its ability to facilitate adhesion to and spreading and migration on various ECM molecules. Furthermore, using siRNA knockdown, confocal microscopy, and immunoprecipitation assays, we assessed PZR and PZRb interactions with β1 integrins. PZR was the predominant isoform in hBM MSC. Migrating hBM MSCs interacted most effectively with fibronectin and required the association of PZR, but not PZRb, with the integrin, VLA-5(α5β1), leading to modulation of focal adhesion kinase phosphorylation and vinculin levels. This raises the possibility that dysregulation of PZR function may modify hBM MSC migratory behavior, potentially contributing to skeletal abnormalities.

might be perturbed in SCD and thalassemia. SCD is an inflammatory condition 9 , which results in chronic leukocytosis, particularly neutrophilia and monocytosis and this chronic inflammatory state may perturb HSPC function 10,11 . SCD is also associated with persistently elevated plasma erythropoietin (Epo) 12 and transferrin receptor 13 levels. Murine models show that high levels of Epo reprogram the transcriptome of HSPCs, supporting the development of committed erythroid progenitors 14 . This results in an erythroid lineage bias during differentiation and a decreased myeloid output. Interestingly, murine models also show that the increased reactive oxygen species in SCD perturb haemopoietic stem cell function 15 .

BACKGROUNDUmbilical cord blood (UCB) is a source of hematopoietic stem cells for transplantation, offering an alternative for patients unable to find a matched adult donor. UCB is also a versatile source of hematopoietic stem and progenitor cells (hCD34 + HSPCs) for research into hematologic diseases, in vitro expansion, ex vivo gene therapy, and adoptive immunotherapy. For these studies, there is a need to isolate hCD34 + HSPCs from cryopreserved units, and protocols developed for isolation from fresh cord blood are unsuitable.STUDY DESIGNThis study describes a modified method for isolating hCD34 + HSPCs from cryopreserved UCB. It uses the Plasmatherm system for thawing, followed by CD34 microbead magnetic‐activated cell sorting isolation with a cell separation kit (Whole Blood Columns, Miltenyi Biotec). hCD34 + HSPC phenotypes and functionality were assessed in vitro and hematologic reconstitution determined in vivo in immunodeficient mice.RESULTSTotal nucleated cell recovery after thawing and washing was 44.7 ± 11.7%. Recovery of hCD34 + HSPCs after application of thawed cells to Whole Blood Columns was 77.5 ± 22.6%. When assessed in two independent laboratories, the hCD34+ cell purities were 71.7 ± 10.7% and 87.8 ± 2.4%. Transplantation of the enriched hCD34 + HSPCs into NSG mice revealed the presence of repopulating hematopoietic stem cells (estimated frequency of 0.07%) and multilineage engraftment.CONCLUSIONThis provides a simplified protocol for isolating high‐purity human CD34 + HSPCs from banked UCB adaptable to current Good Manufacturing Practice. This protocol reduces the number of steps and associated risks and thus total production costs. Importantly, the isolated CD34 + HSPCs possess in vivo repopulating activity in immunodeficient mice, making them a suitable starting population for ex vivo culture and gene editing.