Perivascular niche cells sense thrombocytopenia and activate hematopoietic stem cells in an IL-1 dependent manner.

Hematopoietic stem cells (HSCs) residing in specialized niches in the bone marrow are responsible for the balanced output of multiple short-lived blood cell lineages in steady-state and in response to different challenges. However, feedback mechanisms by which HSCs, through their niches, sense acute losses of specific blood cell lineages remain to be established. While all HSCs replenish platelets, previous studies have shown that a large fraction of HSCs are molecularly primed for the megakaryocyte-platelet lineage and are rapidly recruited into proliferation upon platelet depletion. Platelets normally turnover in an activation-dependent manner, herein mimicked by antibodies inducing platelet activation and depletion. Antibody-mediated platelet activation upregulates expression of Interleukin-1 (IL-1) in platelets, and in bone marrow extracellular fluid in vivo. Genetic experiments demonstrate that rather than IL-1 directly activating HSCs, activation of bone marrow Lepr+ perivascular niche cells expressing IL-1 receptor is critical for the optimal activation of quiescent HSCs upon platelet activation and depletion. These findings identify a feedback mechanism by which activation-induced depletion of a mature blood cell lineage leads to a niche-dependent activation of HSCs to reinstate its homeostasis.

Highly efficient platelet generation in lung vasculature reproduced by microfluidics.

Platelets, small hemostatic blood cells, are derived from megakaryocytes. Both bone marrow and lung are principal sites of thrombopoiesis although underlying mechanisms remain unclear. Outside the body, however, our ability to generate large number of functional platelets is poor. Here we show that perfusion of megakaryocytes ex vivo through the mouse lung vasculature generates substantial platelet numbers, up to 3000 per megakaryocyte. Despite their large size, megakaryocytes are able repeatedly to passage through the lung vasculature, leading to enucleation and subsequent platelet generation intravascularly. Using ex vivo lung and an in vitro microfluidic chamber we determine how oxygenation, ventilation, healthy pulmonary endothelium and the microvascular structure support thrombopoiesis. We also show a critical role for the actin regulator Tropomyosin 4 in the final steps of platelet formation in lung vasculature. This work reveals the mechanisms of thrombopoiesis in lung vasculature and informs approaches to large-scale generation of platelets.

Low input capture Hi-C (liCHi-C) identifies promoter-enhancer interactions at high-resolution.

Long-range interactions between regulatory elements and promoters are key in gene transcriptional control; however, their study requires large amounts of starting material, which is not compatible with clinical scenarios nor the study of rare cell populations. Here we introduce low input capture Hi-C (liCHi-C) as a cost-effective, flexible method to map and robustly compare promoter interactomes at high resolution. As proof of its broad applicability, we implement liCHi-C to study normal and malignant human hematopoietic hierarchy in clinical samples. We demonstrate that the dynamic promoter architecture identifies developmental trajectories and orchestrates transcriptional transitions during cell-state commitment. Moreover, liCHi-C enables the identification of disease-relevant cell types, genes and pathways potentially deregulated by non-coding alterations at distal regulatory elements. Finally, we show that liCHi-C can be harnessed to uncover genome-wide structural variants, resolve their breakpoints and infer their pathogenic effects. Collectively, our optimized liCHi-C method expands the study of 3D chromatin organization to unique, low-abundance cell populations, and offers an opportunity to uncover factors and regulatory networks involved in disease pathogenesis.

STAT1 is essential for HSC function and maintains MHCIIhi stem cells that resist myeloablation and neoplastic expansion.

Adult hematopoietic stem cells (HSCs) are predominantly quiescent and can be activated in response to acute stress such as infection or cytotoxic insults. STAT1 is a pivotal downstream mediator of interferon (IFN) signaling and is required for IFN-induced HSC proliferation, but little is known about the role of STAT1 in regulating homeostatic hematopoietic stem/progenitor cells (HSPCs). Here, we show that loss of STAT1 altered the steady state HSPC landscape, impaired HSC function in transplantation assays, delayed blood cell regeneration following myeloablation, and disrupted molecular programs that protect HSCs, including control of quiescence. Our results also reveal STAT1-dependent functional HSC heterogeneity. A previously unrecognized subset of homeostatic HSCs with elevated major histocompatibility complex class II (MHCII) expression (MHCIIhi) displayed molecular features of reduced cycling and apoptosis and was refractory to 5-fluorouracil-induced myeloablation. Conversely, MHCIIlo HSCs displayed increased megakaryocytic potential and were preferentially expanded in CALR mutant mice with thrombocytosis. Similar to mice, high MHCII expression is a feature of human HSCs residing in a deeper quiescent state. Our results therefore position STAT1 at the interface of stem cell heterogeneity and the interplay between stem cells and the adaptive immune system, areas of broad interest in the wider stem cell field.

Mapping the biogenesis of forward programmed megakaryocytes from induced pluripotent stem cells.

Platelet deficiency, known as thrombocytopenia, can cause hemorrhage and is treated with platelet transfusions. We developed a system for the production of platelet precursor cells, megakaryocytes, from pluripotent stem cells. These cultures can be maintained for >100 days, implying culture renewal by megakaryocyte progenitors (MKPs). However, it is unclear whether the MKP state in vitro mirrors the state in vivo, and MKPs cannot be purified using conventional surface markers. We performed single-cell RNA sequencing throughout in vitro differentiation and mapped each state to its equivalent in vivo. This enabled the identification of five surface markers that reproducibly purify MKPs, allowing us insight into their transcriptional and epigenetic profiles. Last, we performed culture optimization, increasing MKP production. Together, this study has mapped parallels between the MKP states in vivo and in vitro and allowed the purification of MKPs, accelerating the progress of in vitro-derived transfusion products toward the clinic.

CRLF3 plays a key role in the final stage of platelet genesis and is a potential therapeutic target for thrombocythemia.

The process of platelet production has so far been understood to be a 2-stage process: megakaryocyte maturation from hematopoietic stem cells followed by proplatelet formation, with each phase regulating the peripheral blood platelet count. Proplatelet formation releases into the bloodstream beads-on-a-string preplatelets, which undergo fission into mature platelets. For the first time, we show that preplatelet maturation is a third, tightly regulated, critical process akin to cytokinesis that regulates platelet count. We show that deficiency in cytokine receptor-like factor 3 (CRLF3) in mice leads to an isolated and sustained 25% to 48% reduction in the platelet count without any effect on other blood cell lineages. We show that Crlf3-/- preplatelets have increased microtubule stability, possibly because of increased microtubule glutamylation via the interaction of CRLF3 with key members of the Hippo pathway. Using a mouse model of JAK2 V617F essential thrombocythemia, we show that a lack of CRLF3 leads to long-term lineage-specific normalization of the platelet count. We thereby postulate that targeting CRLF3 has therapeutic potential for treatment of thrombocythemia.

Process analysis of pluripotent stem cell differentiation to megakaryocytes to make platelets applying European GMP.

Quality, traceability and reproducibility are crucial factors in the reliable manufacture of cellular therapeutics, as part of the overall framework of Good Manufacturing Practice (GMP). As more and more cellular therapeutics progress towards the clinic and research protocols are adapted to comply with GMP standards, guidelines for safe and efficient adaptation have become increasingly relevant. In this paper, we describe the process analysis of megakaryocyte manufacture from induced pluripotent stem cells with a view to manufacturing in vitro platelets to European GMP for transfusion. This process analysis has allowed us an overview of the entire manufacturing process, enabling us to pinpoint the cause and severity of critical risks. Risk mitigations were then proposed for each risk, designed to be GMP compliant. These mitigations will be key in advancing this iPS-derived therapy towards the clinic and have broad applicability to other iPS-derived cellular therapeutics, many of which are currently advancing towards GMP-compliance. Taking these factors into account during protocol design could potentially save time and money, expediting the advent of safe, novel therapeutics from stem cells.

Transfer to the clinic: refining forward programming of hPSCs to megakaryocytes for platelet production in bioreactors.

The production of in vitro-derived platelets has great potential for transfusion medicine. Here, we build on our experience in the forward programming (FoP) of human pluripotent stem cells (hPSCs) to megakaryocytes (MKs) and address several aspects of the complex challenges to bring this technology to the bedside. We first identify clinical-grade hPSC lines that generate MKs efficiently. We design a bespoke media to maximize both production and maturity of MKs and improve platelet output. Crucially, we transition the lentiviral-based FoP of hPSCs to a nonviral inducible system. We also show how small molecules promote a definitive hematopoiesis phenotype during the differentiation process, thereby increasing the quality of the final product. Finally, we generate platelets using a bioreactor designed to reproduce the physical cues that promote platelet production in the bone marrow. We show that these platelets are able to contribute to both thrombus formation in vitro and have a hemostatic effect in thrombocytopenic mice in vivo.

The placenta protects the fetal circulation from anxiety-driven elevations in maternal serum levels of brain-derived neurotrophic factor.

Brain-derived neurotrophic factor (BDNF) plays crucial roles in brain function. Numerous studies report alterations in BDNF levels in human serum in various neurological conditions, including mood disorders such as depression. However, little is known about BDNF levels in the blood during pregnancy. We asked whether maternal depression and/or anxiety during pregnancy were associated with altered serum BDNF levels in mothers (n = 251) and their new-born infants (n = 212). As prenatal exposure to maternal mood disorders significantly increases the risk of neurological conditions in later life, we also examined the possibility of placental BDNF transfer by developing a new mouse model. We found no association between maternal symptoms of depression and either maternal or infant cord blood serum BDNF. However, maternal symptoms of anxiety correlated with significantly raised maternal serum BDNF exclusively in mothers of boys (r = 0.281; P = 0.005; n = 99). Serum BDNF was significantly lower in male infants than female infants but neither correlated with maternal anxiety symptoms. Consistent with this observation, we found no evidence for BDNF transfer across the placenta. We conclude that the placenta protects the developing fetus from maternal changes in serum BDNF that could otherwise have adverse consequences for fetal development.

Interferon-γ signaling in human iPSC-derived neurons recapitulates neurodevelopmental disorder phenotypes.

Maternal immune activation increases the risk of neurodevelopmental disorders. Elevated cytokines, such as interferon-γ (IFN-γ), in offspring's brains play a central role. IFN-γ activates an antiviral cellular state, limiting viral entry and replication. Moreover, IFN-γ is implicated in brain development. We tested the hypothesis that IFN-γ signaling contributes to molecular and cellular phenotypes associated with neurodevelopmental disorders. Transient IFN-γ treatment of neural progenitors derived from human induced pluripotent stem cells increased neurite outgrowth. RNA sequencing analysis revealed that major histocompatibility complex class I (MHCI) genes were persistently up-regulated through neuronal differentiation-an effect that was mediated by IFN-γ-induced promyelocytic leukemia protein (PML) nuclear bodies. Critically, IFN-γ-induced neurite outgrowth required both PML and MHCI. We also found evidence that IFN-γ disproportionately altered the expression of genes associated with schizophrenia and autism, suggesting convergence between genetic and environmental risk factors. Together, these data implicate IFN-γ signaling in neurodevelopmental disorder etiology.

Remodeling of Bone Marrow Hematopoietic Stem Cell Niches Promotes Myeloid Cell Expansion during Premature or Physiological Aging.

Hematopoietic stem cells (HSCs) residing in the bone marrow (BM) accumulate during aging but are functionally impaired. However, the role of HSC-intrinsic and -extrinsic aging mechanisms remains debated. Megakaryocytes promote quiescence of neighboring HSCs. Nonetheless, whether megakaryocyte-HSC interactions change during pathological/natural aging is unclear. Premature aging in Hutchinson-Gilford progeria syndrome recapitulates physiological aging features, but whether these arise from altered stem or niche cells is unknown. Here, we show that the BM microenvironment promotes myelopoiesis in premature/physiological aging. During physiological aging, HSC-supporting niches decrease near bone but expand further from bone. Increased BM noradrenergic innervation promotes ß2-adrenergic-receptor(AR)-interleukin-6-dependent megakaryopoiesis. Reduced ß3-AR-Nos1 activity correlates with decreased endosteal niches and megakaryocyte apposition to sinusoids. However, chronic treatment of progeroid mice with ß3-AR agonist decreases premature myeloid and HSC expansion and restores the proximal association of HSCs to megakaryocytes. Therefore, normal/premature aging of BM niches promotes myeloid expansion and can be improved by targeting the microenvironment.

Flow cytometry for near-patient testing in premature neonates reveals variation in platelet function: a novel approach to guide platelet transfusion.

BACKGROUND: Neonatal haemorrhaging is often co-observed with thrombocytopenia; however, no evidence of a causal relationship with low platelet count has been reported. Regardless, the administration of a platelet transfusion is often based upon this parameter. Accurate measurement of platelet function in small volumes of adult blood samples by flow cytometry is well established and we propose that the use of the same technology could provide complementary information to guide the administration of platelet transfusions in premature neonates. METHODS: In 28 neonates born at 27-41 weeks gestation, platelet function after stimulation agonists was measured using fibrinogen binding and P-selectin expression (a marker of degranulation). RESULTS: Platelets of neonates with gestation of ≤36 weeks (n = 20) showed reduced fibrinogen binding and degranulation with ADP, and reduced degranulation with CRP-XL. Degranulation Scores of 7837 ± 5548, 22,408 ± 5301 and 53,131 ± 12,102 (mean ± SEM) identified significant differences between three groups: <29, 29-36 and >36 weeks gestation). Fibrinogen binding and degranulation responses to ADP were significantly reduced in suspected septic neonates (n = 6) and the Fibrinogen Binding scores clearly separated the septic and healthy group (88.2 ± 10.3 vs 38.6 ± 12.2, P = 0.03). CONCLUSIONS: Flow cytometric measurement of platelet function identified clinically different neonatal groups and may eventually contribute to assessment of neonates requiring platelet transfusion.

Using genome editing to engineer universal platelets.

Genome editing technologies such as zinc finger nucleases, TALENs and CRISPR/Cas9 have recently emerged as tools with the potential to revolutionise cellular therapy. This is particularly exciting for the field of regenerative medicine, where the large-scale, quality-controlled editing of large numbers of cells could generate essential cellular products ready to move towards the clinic. This review details recent progress towards generating HLA Class I null platelets using genome editing technologies for ß2-microglobulin deletion, generating a universally transfusable cellular product. In addition, we discuss various methods for megakaryocyte (MK) production from human pluripotent stem cells and subsequent platelet production from the MKs. As well as simply producing platelets, differentiating MK cultures can enable us to understand megakaryopoiesis in vivo and take steps towards ameliorating bleeding disorders or deficiencies in MK maturation in patients. Thus by intersecting both these areas of research, we can produce optimised differentiation systems for the production of universal platelets, thus offering a stable supply of platelets for difficult-to-match patients and providing areas with transmissible disease concerns or an unpredictable supply of platelets with a steady supply of quality-controlled platelet units.

Platelet α-granules modulate the inflammatory response under systemic lipopolysaccharide injection in mice.

BACKGROUND: Beyond their role in hemostasis and thrombosis, platelets are also important mediators of inflammation by the release of hundreds of factors stored in their α-granules. Mutations in Nbeal2 cause gray platelet syndrome (GPS), characterized by the lack of platelet α-granules. This study aims to evaluate the immunological (proinflammatory) effects of platelet α-granules. STUDY DESIGN AND METHODS: We performed an experiment using Nbeal2-/- mice, the mouse model of GPS. Systemic inflammation was induced by intravenous injection of lipopolysaccharide (LPS). Inflammatory response was assessed by quantification of inflammatory soluble factors and platelet biological response modifiers. RESULTS: The lack of Nbeal2 (in Nbeal2 -/- mice, compared with controls) significantly reduced the recruitment of circulating neutrophils and monocytes. Moreover, after LPS injection, there was a significant increase in neutrophil and monocyte counts in control animals, compared with Nbeal2 -/- mice. The control of inflammation, evaluated by the production of anti-inflammatory cytokines, appeared to be greater in Nbeal2-/- mice compared with controls. Conversely, the production of certain inflammatory-soluble mediators known to characterize normal platelet secretion, such as soluble CD40 ligand (sCD40L), was decreased under experimental inflammation in Nbeal2 -/- mice. CONCLUSIONS: These results show that α-granules play a direct role in platelet-mediated inflammation balance, confirming the need to further investigate platelet-associated inflammatory pathophysiology and inflammatory adverse events related to blood transfusion.

Sphingolipid dysregulation due to lack of functional KDSR impairs proplatelet formation causing thrombocytopenia.

Sphingolipids are fundamental to membrane trafficking, apoptosis, and cell differentiation and proliferation. KDSR or 3-keto-dihydrosphingosine reductase is an essential enzyme for de novo sphingolipid synthesis, and pathogenic mutations in KDSR result in the severe skin disorder erythrokeratodermia variabilis et progressiva-4 Four of the eight reported cases also had thrombocytopenia but the underlying mechanism has remained unexplored. Here we expand upon the phenotypic spectrum of KDSR deficiency with studies in two siblings with novel compound heterozygous variants associated with thrombocytopenia, anemia, and minimal skin involvement. We report a novel phenotype of progressive juvenile myelofibrosis in the propositus, with spontaneous recovery of anemia and thrombocytopenia in the first decade of life. Examination of bone marrow biopsies showed megakaryocyte hyperproliferation and dysplasia. Megakaryocytes obtained by culture of CD34+ stem cells confirmed hyperproliferation and showed reduced proplatelet formation. The effect of KDSR insufficiency on the sphingolipid profile was unknown, and was explored in vivo and in vitro by a broad metabolomics screen that indicated activation of an in vivo compensatory pathway that leads to normalization of downstream metabolites such as ceramide. Differentiation of propositus-derived induced pluripotent stem cells to megakaryocytes followed by expression of functional KDSR showed correction of the aberrant cellular and biochemical phenotypes, corroborating the critical role of KDSR in proplatelet formation. Finally, Kdsr depletion in zebrafish recapitulated the thrombocytopenia and showed biochemical changes similar to those observed in the affected siblings. These studies support an important role for sphingolipids as regulators of cytoskeletal organization during megakaryopoiesis and proplatelet formation.

Transcription Factor Levels after Forward Programming of Human Pluripotent Stem Cells with GATA1, FLI1, and TAL1 Determine Megakaryocyte versus Erythroid Cell Fate Decision.

The production of blood cells and their precursors from human pluripotent stem cells (hPSCs) in vitro has the potential to make a significant impact upon healthcare provision. We demonstrate that the forward programming of hPSCs through overexpression of GATA1, FLI1, and TAL1 leads to the production of a population of progenitors that can differentiate into megakaryocyte or erythroblasts. Using "rainbow" lentiviral vectors to quantify individual transgene expression in single cells, we demonstrate that the cell fate decision toward an erythroblast or megakaryocyte is dictated by the level of FLI1 expression and is independent of culture conditions. Early FLI1 expression is critical to confer proliferative potential to programmed cells while its subsequent silencing or maintenance dictates an erythroid or megakaryocytic fate, respectively. These committed progenitors subsequently expand and mature into megakaryocytes or erythroblasts in response to thrombopoietin or erythropoietin. Our results reveal molecular mechanisms underlying hPSC forward programming and novel opportunities for application to transfusion medicine.

Inhibition of profibrotic microRNA-21 affects platelets and their releasate.

Fibrosis is a major contributor to organ disease for which no specific therapy is available. MicroRNA-21 (miR-21) has been implicated in the fibrogenetic response, and inhibitors of miR-21 are currently undergoing clinical trials. Here, we explore how miR-21 inhibition may attenuate fibrosis using a proteomics approach. Transfection of miR-21 mimic or inhibitor in murine cardiac fibroblasts revealed limited effects on extracellular matrix (ECM) protein secretion. Similarly, miR-21-null mouse hearts showed an unaltered ECM composition. Thus, we searched for additional explanations as to how miR-21 might regulate fibrosis. In plasma samples from the community-based Bruneck Study, we found a marked correlation of miR-21 levels with several platelet-derived profibrotic factors, including TGF-ß1. Pharmacological miR-21 inhibition with an antagomiR reduced the platelet release of TGF-ß1 in mice. Mechanistically, Wiskott-Aldrich syndrome protein, a negative regulator of platelet TGF-ß1 secretion, was identified as a direct target of miR-21. miR-21-null mice had lower platelet and leukocyte counts compared with littermate controls but higher megakaryocyte numbers in the bone marrow. Thus, to our knowledge this study reports a previously unrecognized effect of miR-21 inhibition on platelets. The effect of antagomiR-21 treatment on platelet TGF-ß1 release, in particular, may contribute to the antifibrotic effects of miR-21 inhibitors.