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.

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.

Leukotriene E4 is a full functional agonist for human cysteinyl leukotriene type 1 receptor-dependent gene expression.

Leukotriene E4 (LTE4) the most stable of the cysteinyl leukotrienes (cysLTs) binds poorly to classical type 1 (CysLT1) and 2 (CysLT2) receptors although it induces potent responses in human airways in vivo, such as bronchoconstriction, airway hyperresponsiveness and inflammatory cell influx suggesting the presence of a novel receptor that preferentially responds to LTE4. To identify such a receptor two human mast cell lines, LAD2 and LUVA, were selected that differentially responded to LTE4 when analysed by intracellular signalling and gene expression. Comparative transcriptome analysis and recombinant gene overexpression experiments revealed CysLT1 as a receptor responsible for potent LTE4-induced response in LAD2 but not in LUVA cells, an observation confirmed further by gene knockdown and selective inhibitors. Lentiviral overexpression of CysLT1 in LUVA cells augmented intracellular calcium signalling induced by LTE4 but did not restore full agonist responses at the gene expression level. Our data support a model where both an increased expression of Gαq-coupled CysLT1, and sustained intracellular calcium mobilisation and extracellular signal-regulated kinase (Erk) activation, are required for LTE4-mediated regulation of gene expression in human cells. Our study shows for the first time that CysLT1 expression is critically important for responsiveness to LTE4 within a human cell system.

Human-specific epigenetic variation in the immunological Leukotriene B4 Receptor (LTB4R/BLT1) implicated in common inflammatory diseases.

BACKGROUND: Common human diseases are caused by the complex interplay of genetic susceptibility as well as environmental factors. Due to the environment's influence on the epigenome, and therefore genome function, as well as conversely the genome's facilitative effect on the epigenome, analysis of this level of regulation may increase our knowledge of disease pathogenesis. METHODS: In order to identify human-specific epigenetic influences, we have performed a novel genome-wide DNA methylation analysis comparing human, chimpanzee and rhesus macaque. RESULTS: We have identified that the immunological Leukotriene B4 receptor (LTB4R, BLT1 receptor) is the most epigenetically divergent human gene in peripheral blood in comparison with other primates. This difference is due to the co-ordinated active state of human-specific hypomethylation in the promoter and human-specific increased gene body methylation. This gene is significant in innate immunity and the LTB4/LTB4R pathway is involved in the pathogenesis of the spectrum of human inflammatory diseases. This finding was confirmed by additional neutrophil-only DNA methylome and lymphoblastoid H3K4me3 chromatin comparative data. Additionally we show through functional analysis that this receptor has increased expression and a higher response to the LTB4 ligand in human versus rhesus macaque peripheral blood mononuclear cells. Genome-wide we also find human species-specific differentially methylated regions (human s-DMRs) are more prevalent in CpG island shores than within the islands themselves, and within the latter are associated with the CTCF motif. CONCLUSIONS: This result further emphasises the exclusive nature of the human immunological system, its divergent adaptation even from very closely related primates, and the power of comparative epigenomics to identify and understand human uniqueness.

Characterisation of P2Y(12) receptor responsiveness to cysteinyl leukotrienes.

Leukotriene E4 (LTE4), the most stable of the cysteinyl leukotrienes (cysLTs), binds poorly to classical type 1 and 2 cysLT receptors although in asthmatic individuals it may potently induce bronchial constriction, airway hyperresponsiveness and inflammatory cell influx to the lung. A recent study has suggested that the purinergic receptor P2Y12 is required for LTE4 mediated pulmonary inflammation in a mouse model of asthma and signals in response to cysLTs. The aim of the study was to characterise the responsiveness of human P2Y12 to cysteinyl leukotrienes. Models of human CysLT1, CysLT2 and P2Y12 overexpressed in HEK293, CHO cells and human platelets were used and responsiveness to different agonists was measured using intracellular calcium, cAMP and ß-arrestin recruitment assays. CysLTs induced concentration dependent calcium mobilisation in cells overexpressing CysLT1 and CysLT2 but failed to induce any calcium response in cells expressing P2Y12 or P2Y12+ Gα16. In contrast, selective P2Y12 agonists ADP and 2-MeS-ADP induced specific calcium flux in cells expressing P2Y12+ Gα16. Similarly, specific response to 2-MeS-ADP, but not to cysLTs was also observed in cells expressing P2Y12 when intracellular cAMP and ß-arrestin signalling were analysed. Platelets were used as a model of human primary cells expressing P2Y12 to analyse potential signalling and cell activation through P2Y12 receptor or receptor heterodimers but no specific LTE4 responses were observed. These results show that LTE4 as well as other cysLTs do not activate intracellular signalling acting through P2Y12 and suggest that another LTE4 specific receptor has yet to be identified.