Disruption of placental ACKR3 impairs growth and hematopoietic development of offspring.

ACKR3 scavenges and degrades the stem cell recruiting chemokine CXCL12, which is essential for proper embryonic and, in particular, haematopoietic development. Here, we demonstrate strong expression of ACKR3 on trophoblasts. Using a maternally administered pharmacological blocker and Cre-mediated genetic approaches, we demonstrate that trophoblast ACKR3 is essential for preventing movement of CXCL12 from the mother to the embryo, with elevated plasma CXCL12 levels being detected in embryos from ACKR3-blocker-treated mothers. Mice born to mothers treated with the blocker are lighter and shorter than those born to vehicle-treated mothers and, in addition, display profound anaemia associated with a markedly reduced bone marrow haematopoietic stem cell population. Importantly, although the haematopoietic abnormalities are corrected as mice age, our studies reveal a postnatal window during which offspring of ACKR3-blocker-treated mice are unable to mount effective inflammatory responses to inflammatory/infectious stimuli. Overall, these data demonstrate that ACKR3 is essential for preventing CXCL12 transfer from mother to embryo and for ensuring properly regulated CXCL12 control over the development of the haematopoietic system.

The impact of matched and mismatched donor-recipient genotypes for MDR1 polymorphisms (G2677TA, C1236T and C3435T) on the outcomes of patients after allogeneic haematopoietic stem cell transplantation.

In this study, we investigated whether matched and mismatched multidrug resistance gene (MDR1) genotypes (G2677TA, C1236T and C3435T) were associated with prognosis in patients after allogeneic haematopoietic stem cell transplantation (allo-HSCT). One hundred patients after transplantation and their donors were enrolled. Matched MDR1 G2677TA donor-recipient was associated with an increased risk of non-relapse mortality (NRM) (29.5% vs. 6.2%, p = 0.002), poor overall survival (OS) (51.7% vs. 63.8%, p = 0.024) and disease-free survival (DFS) (38.6% vs. 67%, p = 0.005). There were no differences in OS, DFS or NRM between MDR1 C1236T- and C3435T-matched and -mismatched groups. Subgroup analysis suggested that within the matched MDR1 G2677TA group, male gender, haematopoietic cell transplantation-specific comorbidity index ≥1, serum creatinine >137.2 µmol/L and post-transplantation thrombocytopenia were associated with poor survival. Our results demonstrated that patients receiving matched MDR1 G2677TA allo-HSCT experienced a poorer prognosis compared with the mismatched group. The potential mechanism may involve increased expression of P-glycoprotein, leading to decreased accumulation of antimicrobial agents and ultimately contributing to the progression of inflammation. This identification of MDR1 G2677TA genotype compatibility holds promise as a valuable molecular tool for selecting donors for allo-HSCT.

Current state of gene therapy in sickle cell disease.

Sickle cell disease (SCD) is a type of hemoglobinopathy due to an autosomal recessive genetic defect, causing significant red cell sickling, multi-organ damage and long-term severe morbidities. Due to its complicated care and the impact on quality of life, a curative treatment for SCD is highly desirable. In recent years, gene therapy is emerging as a curative option for SCD, where autologous haematopoietic stem cells are collected from SCD patients and genetically modified ex vivo to reduce its sickling tendency before reinfusion. Although still largely investigational, a limited number of gene therapy options have been recently granted approval for SCD patients. Published data are still currently limited, but early studies have so far demonstrated the intended outcomes of less vaso-occlusive crisis and haemolysis. Nonetheless, despite its curative potential, larger clinical trials and longer follow-up period are still necessary to evaluate the safety of this treatment option, especially the risk of unintended genetic modifications. Furthermore, SCD patients frequently have limited access to specialty care; hence, the issues of affordability and accessibility to SCD gene therapy must also be addressed for it to benefit the appropriate patient population.

CD34+ stromal cells/telocytes and their role in mouse lung development: Light microscopy, immunofluorescence, ultrastructural and scanning electron microscopy evidence.

Telocytes (TCs), a novel type of mesenchymal or interstitial cell with specific, very long and thin cellular prolongations, have been found in various mammalian organs and have potential biological functions. However, their existence during lung development is poorly understood. This study aimed to investigate the existence, morphological features, and role of CD34+ SCs/TCs in mouse lungs from foetal to postnatal life using primary cell culture, double immunofluorescence, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The immunofluorescence double staining profiles revealed positive expression of CD34 and PDGFR-α, Sca-1 or VEGFR-3, and the expression of these markers differed among the age groups during lung development. Intriguingly, in the E18.5 stage of development, along with the CD34+ SCs/TCs, haematopoietic stem cells and angiogenic factors were also significantly increased in number compared with those in the E14.5, E16.5, P0 and P7. Subsequently, TEM confirmed that CD34+ SCs/TCs consisted of a small cell body with long telopodes (Tps) that projected from the cytoplasm. Tps consisted of alternating thin and thick segments known as podomers and podoms. TCs contain abundant endoplasmic reticulum, mitochondria and secretory vesicles and establish close connections with neighbouring cells. Furthermore, SEM revealed characteristic features, including triangular, oval, spherical, or fusiform cell bodies with extensive cellular prolongations, depending on the number of Tps. Our findings provide evidence for the existence of CD34+ SCs/TCs, which contribute to vasculogenesis, the formation of the air‒blood barrier, tissue organization during lung development and homoeostasis.

Expression, Function, and Significance of Non B Cell-Derived Immunoglobulin in Haematological System.

Acute myeloid leukaemia (AML) is a collection of genetically diverse diseases characterised by abnormal proliferation of immature haematopoietic cells and disruption of normal haematopoiesis. Myeloid cells and lymphocytes originate from different haematopoietic precursors within the bone marrow. It has been traditionally assumed that myeloid cells cannot produce immunoglobulin (Ig), a marker of B cells and plasma cells. However, in recent years, all five Ig classes have been detected in CD34+ haematopoietic stem cells, mature monocytes and neutrophils, differentiated macrophages and tumour-associated macrophages, acute myeloid leukaemia cell lines, as well as myeloblasts of AML. The rearranged V(D)J sequences exhibit unique restricted or biased V gene usage and evidence of somatic mutation. Furthermore, AML-derived Igs could promote cell proliferation, induce apoptosis, and enhance migration. Elevated levels of Ig expression predict inferior clinical outcomes. These findings indicate that AML-derived Ig plays a role in AML pathogenesis and progression, and could serve as a novel biomarker for risk stratification, disease monitoring, and targeted therapy. In this chapter, we provide a comprehensive review of recent literature on the expression, function, and significance of non B cell-derived Ig in the haematological system, with a focus on AML.

Nestin expression in osteocytes following myeloablation and during bone marrow metastasis.

Nestin is an intermediate filament protein, which was originally detected in neuroepithelial stem cells. Besides its use as a phenotypic marker of mesenchymal stem cells in the hematopoeitic stem cell niche, the functional interpretation of nestin+ cells remains elusive. We investigated the cellular expression of nestin in bone marrow trephine biopsies of MPN patients, following myeloablation at a stage of hypocellularity during early regeneration. Here, nestin is highly expressed in mature osteocytes, arteriolar endothelial and perivascular cells and small capillaries within the bone marrow space, but not in sinusoid lining cells. This is in stark contrast to nestin expression pattern in myeloproliferative neoplasms that show hypercellularity due to oncogenic driver mutations. Here, nestin is expressed exclusively in endothelial cells of arterioles, but not in osteocytes or small capillaries. Thus, the pattern of nestin expression following myeloablation inversely correlates with cellularity in the bone marrow. This nestin expression pattern is mimicking early postnatal transcriptional programming during bone marrow development. We show that nestin expression in osteocytes occurs across different species following transplant and also in bone marrow metastasis.

Identification and protective role of CD34+ stromal cells/telocytes in experimental autoimmune encephalomyelitis (EAE) mouse spleen.

Experimental autoimmune encephalomyelitis (EAE) is a classical animal model of human multiple sclerosis (MS) that is most commonly used to study the neuropathology and therapeutic effects of the disease. Telocytes (TCs) are a specialized type of interstitial or mesenchymal cell first identified by Popescu in various tissues and organs. However, the existence, distribution and role of CD34+ stromal cells (SCs)/TCs in the EAE-induced mouse spleen remain to be elucidated. We conducted immunohistochemistry, immunofluorescence (double staining for CD34 and c-kit, vimentin, F4/80, CD163, Nanog, Sca-1, CD31 or tryptase) and transmission electron microscopy experiments to investigate the existence, distribution and role of CD34+ SCs/TCs in the EAE-induced mouse spleen. Interestingly, immunohistochemistry, double-immunofluorescence, and transmission electron microscopy results revealed that CD34+ SCs/TCs were significantly upregulated in the EAE mouse spleen. Immunohistochemical or double-immunofluorescence staining of CD34+ SCs/TCs showed positive expression for CD34, c-kit, vimentin, CD34/vimentin, c-kit/vimentin and CD34/c-kit, and negative expression for CD31 and tryptase. Transmission electron microscopy (TEM) results demonstrated that CD34+ SCs/TCs established close connections with lymphocytes, reticular cells, macrophages, endothelial cells and erythrocytes. Furthermore, we also found that M1 (F4/80) or M2 (CD163) macrophages, and haematopoietic, pluripotent stem cells were markedly increased in EAE mice. Our results suggest that CD34+ SCs/TCs are abundant and may play a contributing role in modulating the immune response, recruiting macrophages and proliferation of haematopoietic and pluripotent stem cells following injury to promote tissue repair and regeneration in EAE mouse spleens. This suggests that their transplantation combined with stem cells might represent a promising therapeutic target for the treatment and prevention of multiple autoimmune and chronic inflammatory disorders.

Adverse events caused by cord blood infusion in Japan during a 5-year period.

BACKGROUND AND OBJECTIVES: In Japan, cord blood is used for more than half of all unrelated stem cell transplantations. The public cord blood banks (CBBs) have been collecting information on cord blood transplantation-related adverse events from physicians on a voluntary basis, without common definitions of the adverse reactions. The aims of this study were to compare two classification systems to improve the reporting system and to clarify the actual risk from cord blood infusion, which can then provide the impetus to take appropriate measures to reduce adverse events. MATERIALS AND METHODS: We classified the reports according to existing criteria; one is the Proposed Standard Definitions for Surveillance of Non-Infectious Adverse Transfusion Reactions by the International Society of Blood Transfusion (ISBT) Working Party on Haemovigilance, and the other is the Common Terminology Criteria for Adverse Events (CTCAE). There were 140 cases with adverse events reported from April 2014 through March 2019. RESULTS: Twelve cases, such as donor-derived leukaemia/myelodysplastic syndromes (MDS) and chromosomal aberrations reported after engraftment, were excluded from this analysis. Of the 128 cases with adverse events at cord blood infusion, the CTCAE and ISBT criteria could not classify 6 cases and 68 cases, respectively. Classifying by the CTCAE, the most common side effect was hypertension in 35 cases, followed by anaphylaxis, allergic reactions, nausea, urticaria, etc. Serious adverse events (grades 4 and 5) were mainly anaphylaxis, with a frequency of 0.23%. CONCLUSION: It is necessary not only to provide information on adverse events but also to standardize the reporting of adverse events to support measures to reduce them.

Diversity in the bone marrow niche: Classic and novel strategies to uncover niche composition.

Our view on the role and composition of the bone marrow (BM) has dramatically changed over time from a simple nutrient for the bone to a highly complex multicellular tissue that sustains haematopoiesis. Among these cells, multipotent haematopoietic stem cells (HSCs), which are predominantly quiescent, possess unique self-renewal capacity and multilineage differentiation potential and replenish all blood lineages to maintain lifelong haematopoiesis. Adult HSCs reside in specialised BM niches, which support their functions. Much effort has been put into deciphering HSC niches due to their potential clinical relevance. Multiple cell types have been implicated as HSC-niche components including sinusoidal endothelium, perivascular stromal cells, macrophages, megakaryocytes, osteoblasts and sympathetic nerves. In this review we provide a historical perspective on how technical advances, from genetic mouse models to imaging and high-throughput sequencing techniques, are unveiling the plethora of molecular cues and cellular components that shape the niche and regulate HSC functions.

Effect of expansion of human umbilical cord blood CD34 + cells on neurotrophic and angiogenic factor expression and function.

The use of CD34 + cell-based therapies has largely been focused on haematological conditions. However, there is increasing evidence that umbilical cord blood (UCB) CD34 + -derived cells have neuroregenerative properties. Due to low cell numbers of CD34 + cells present in UCB, expansion is required to produce sufficient cells for therapeutic purposes, especially in adults or when frequent applications are required. However, it is not known whether expansion of CD34 + cells has an impact on their function and neuroregenerative capacity. We addressed this knowledge gap in this study, via expansion of UCB-derived CD34 + cells using combinations of LDL, UM171 and SR-1 to yield large numbers of cells and then tested their functionality. CD34 + cells expanded for 14 days in media containing UM171 and SR-1 resulted in over 1000-fold expansion. The expanded cells showed an up-regulation of the neurotrophic factor genes BDNF, GDNF, NTF-3 and NTF-4, as well as the angiogenic factors VEGF and ANG. In vitro functionality testing showed that these expanded cells promoted angiogenesis and, in brain glial cells, promoted cell proliferation and reduced production of reactive oxygen species (ROS) during oxidative stress. Collectively, this study showed that our 14-day expansion protocol provided a robust expansion that could produce enough cells for therapeutic purposes. These expanded cells, when tested in in vitro, maintained functionality as demonstrated through promotion of cell proliferation, attenuation of ROS production caused by oxidative stress and promotion of angiogenesis.

Inflammation Regulates Haematopoietic Stem Cells and Their Niche.

Haematopoietic stem cells (HSCs) reside in the bone marrow and are supported by the specialised microenvironment, a niche to maintain HSC quiescence. To deal with haematopoietic equilibrium disrupted during inflammation, HSCs are activated from quiescence directly and indirectly to generate more mature immune cells, especially the myeloid lineage cells. In the process of proliferation and differentiation, HSCs gradually lose their self-renewal potential. The extensive inflammation might cause HSC exhaustion/senescence and malignant transformation. Here, we summarise the current understanding of how HSC functions are maintained, damaged, or exhausted during acute, prolonged, and pathological inflammatory conditions. We also highlight the inflammation-altered HSC niche and its impact on escalating the insults on HSCs.

Recapitulative haematopoietic development of human pluripotent stem cells in the absence of exogenous haematopoietic cytokines.

To improve the recapitulative quality of human pluripotent stem cell (hPSC) differentiation, we removed exogenous haematopoietic cytokines from the defined differentiation system. Here, we show that endogenous stimuli and VEGF are sufficient to induce robust hPSC-derived haematopoiesis, intensive generation of haematopoietic progenitors, maturation of blood cells and the emergence of definitive precursor cells including those that phenotypically identical to early human embryonic haematopoietic stem cells (HSCs). Moreover, the cytokine-free system produces significantly higher numbers of haematopoietic progenitors compared to the published protocols. The removal of cytokines revealed a broad developmental potential of the early blood cells, stabilized the hPSC-derived definitive precursors and led to spontaneous activation of inflammatory signalling. Our cytokine-free protocol is simple, efficient, reproducible and applicable for embryonic stem cells (ESCs) and induced PSCs. The spectrum of recapitulative features of the novel protocol makes the cytokine-free differentiation a preferred model for studying the early human haematopoietic development.

Identification of CXCL12-abundant reticular cells in human adult bone marrow.

A population of mesenchymal stem cells, termed CXC chemokine ligand (CXCL)12-abundant reticular (CAR) cells or leptin receptor-expressing cells, are the major cellular component of niches for haematopoietic stem cells (HSCs) in murine bone marrow. CAR cells are characterized by several salient features, including much higher expression of CXCL12, stem cell factor (SCF), forkhead box C1 (FOXC1) and early B-cell factor 3 (EBF3), which are essential for HSC maintenance, than other cells. However, the human counterpart of CAR cells has not been fully described. Here, we show the presence of cells expressing much higher CXCL12 than other cells in human adult bone marrow using a flow cytometry-based in situ technique that enables high-throughput detection of mRNA at single-cell resolution. Most CXCL12hi cells expressed high levels of SCF, FOXC1 and EBF3 and had the potential to differentiate into adipocytes and osteoblasts. Histologically, the nuclei of CXCL12hi cells were identified and quantified by EBF3 expression in fixed marrow sections. CXCL12hi cells sorted from residual bone marrow aspirates of chronic myeloid leukaemia patients expressed reduced levels of CXCL12, SCF, FOXC1 and EBF3 in correlation with increased leukaemic burden. Together, we identified the human counterpart of CAR cells, enabling the evaluation of their alterations in various haematological disorders by flow cytometric and histological analyses.

Poor mobilization of autologous CD34+ peripheral blood stem cells in haematology patients undergoing autologous stem cell transplantation is associated with the presence of variants in genes implicated in clonal haematopoiesis of indeterminant potential.

Clonal haematopoiesis of indeterminant potential (CHIP) increases in frequency with age. The effect of CHIP on the mobilization of autologous CD34+ peripheral blood stem cells (PBSC) has not been reported. This study uses a DNA-based targeted candidate gene approach to identify the presence of somatic mutations in ASXL1, DNMT3A, JAK2, SF3B1, TET2 and TP53 in CD34+ haematopoietic progenitor cell-apheresis products of 96 patients who undergo PBSC mobilization for autologous stem cell transplantation (ASCT). Variants were identified in a significantly greater proportion of patients who experience poor CD34+ PBSC mobilization. A DNA-based targeted candidate gene array is able to predict poor CD34+ PBSC mobilization and may be deployed pre-emptively to minimize mobilization and graft failures.

Eltrombopag preferentially expands haematopoietic multipotent progenitors in human aplastic anaemia.

Eltrombopag has been added to first-line treatment of immune aplastic anaemia (AA), resulting in higher responses. We analysed marrow samples of AA patients who responded to immunosuppressive therapy (IST) alone or in combination with eltrombopag for the composition of the haematopoietic stem and progenitor cell (HSPC) compartment. The number of CD34+ cells and multipotent progenitors was higher in patients treated with eltrombopag (P < 0·005; P < 0·05; respectively), but not the number of stem cells. No aberrant phenotype was observed. These results indicate that eltrombopag augments CD34+ cells in vivo and preferentially expands multipotent progenitors, but not stem cells.

Clonal tracking of haematopoietic cells: insights and clinical implications.

Recent advances in high-throughput genomics have enabled the direct tracking of outputs from many cell types, greatly accelerating the study of developmental processes and tissue regeneration. The capacity for long-term self-renewal with multilineage differentiation potential characterises the cellular dynamics of a special set of developmental states that are critical for maintaining homeostasis. In haematopoiesis, the archetypal model for development, lineage-tracing experiments have elucidated the roles of haematopoietic stem cells to ongoing blood production and the importance of long-lived immune cells to immunological memory. An understanding of the biology and clonal dynamics of these cellular fates and states can provide clues to the response of haematopoiesis to ageing, the process of malignant transformation, and are key to designing more efficacious and durable clinical gene and cellular therapies.

Protein phosphatase, Mg2+/Mn2+-dependent 1D (PPM1D) mutations in haematological cancer.

Until recently, the protein phosphatase, Mg2+/Mn2+-dependent 1D (PPM1D) gene had not been examined in haematological cancer, but several studies have now explored the functional role of this gene and its aberrations. It is often mutated in the context of clonal haemopoiesis (including in patients with lymphoma, myeloproliferative neoplasms and myelodysplastic syndrome) and mutations have been associated with exposure to cytotoxic and radiation therapy, development of therapy-related neoplasms and inferior survival. The vast majority of PPM1D mutations found in haematopoietic cells are of the nonsense or frameshift type and located within terminal exon 6. These genetic defects are rarely found in the blood of healthy individuals. PPM1D encodes the PPM1D phosphatase [also named wild-type p53-induced phosphatase 1 (WIP1)], which negatively regulates signalling molecules within the DNA damage response pathway, including tumour suppressor p53. Clonal expansion of PPM1D mutant haematopoietic cells can potentially be prevented with inhibitors; however, human trials are awaited. In the present review, we provide a review of the literature regarding PPM1D and its role in haematological cancer.

Stem cell concepts in myelodysplastic syndromes: lessons and challenges.

According to the cancer stem cell (CSC) hypothesis, CSCs are the only cancer cells that can give rise to and sustain all cells that constitute a cancer as they possess inherent or acquired self-renewal potential, and their elimination is required and potentially sufficient to achieve a cure. Whilst establishing CSC identity remains challenging in most cancers, studies of low-intermediate risk myelodysplastic syndromes (MDS), other chronic myeloid malignancies and clonal haematopoiesis of indeterminant potential (CHIP) strongly support that the primary target cell usually resides in the rare haematopoietic stem cell (HSC) compartment. This probably reflects the unique self-renewal potential of HSCs in normal human haematopoiesis, combined with the somatic initiating genomic driver lesion not conferring extensive self-renewal potential to downstream progenitor cells. Mutational 'fate mapping' further supports that HSCs are the only disease-propagating cells in low-intermediate risk MDS, but that MDS-propagating potential might be extended to progenitors upon disease progression. The clinical importance of MDS stem cells has been highlighted through the demonstration of selective persistence of MDS stem cells in patients at complete remission in response to therapy. This implies that MDS stem cells might possess unique resistance mechanisms responsible for relapses following otherwise efficient treatments. Specific surveillance of MDS stem cells should be considered to assess the efficiency of therapies and as an early indicator of emerging relapses in patients in clinical remission. Moreover, further molecular characterization of purified MDS stem cells should facilitate identification and validation of improved and more stem cell-specific therapies for MDS.

Different subsets of haematopoietic cells and immune cells in bone marrow between young and older donors.

Young donors are reported to be associated with better transplant outcomes than older donors in allogeneic hematopoietic stem cell transplantation (allo-HSCT), but the mechanism is still unclear. The current study compared the different subsets of haematopoietic stem cells (HSCs) and their progenitors as well as immune cells in bone marrow (BM) between young and older donors. The frequencies of HSCs, multipotent progenitors (MPPs) and myeloid progenitors, including common myeloid progenitors (CMPs) and megakaryocyte-erythroid progenitors (MEPs), were decreased, whereas those of lymphoid progenitors, including multi-potent lymphoid progenitors (MLPs) and common lymphoid progenitors (CLPs), were increased in the BM of young donors compared with in that of older donors. Lower reactive oxygen species (ROS) levels were observed in BM HSCs and six progenitor lines in young donors. Furthermore, young donors demonstrated higher frequencies of naive T cells and immune suppressor cells, such as alternative macrophages (M2) and lower frequencies of memory T cells and immune effectors, including T helper-1 and T cytotoxic-1 cells, in BM than older donors. Multivariate analysis demonstrated that donor age was independently correlated with BM HSC frequency. Although further validation is required, our results suggest that the differences in the frequency and immune differentiation potential of HSCs in BM between young donors and older donors may partly explain the different outcomes of allo-HSCT.

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.