HSC Development Research Articles

The histone acetyltransferase KAT6B is required for hematopoietic stem cell development and function

The histone lysine acetyltransferase KAT6B (MYST4, MORF, QKF) is the target of recurrent chromosomal translocations causing hematological malignancies with poor prognosis. Using Kat6b germline deletion and overexpression in mice, we determined the role of KAT6B in the hematopoietic system. We found that KAT6B sustained the fetal hematopoietic stem cell pool but did not affect viability or differentiation. KAT6B was essential for normal levels of histone H3 lysine 9 (H3K9) acetylation but not for a previously proposed target, H3K23. Compound heterozygosity of Kat6b and the closely related gene, Kat6a, abolished hematopoietic reconstitution after transplantation. KAT6B and KAT6A cooperatively promoted transcription of genes regulating hematopoiesis, including the Hoxa cluster, Pbx1, Meis1, Gata family, Erg, and Flt3. In conclusion, we identified the hematopoietic processes requiring Kat6b and showed that KAT6B and KAT6A synergistically promoted HSC development, function, and transcription. Our findings are pertinent to current clinical trials testing KAT6A/B inhibitors as cancer therapeutics.

Characterization of Niche Cells and Signals Regulating Stepwise Embryonic Hematopoiesis Using Slide-Seq and Merfish Spatial Transcriptomics

Stagewise HSC development initiates in the aorta-gonad-mesonephros (AGM) region during mouse embryonic day (E)9.5-E12.5 and progresses through stages including hemogenic endothelial cell (HEC) specification, the endothelial to hematopoietic transition (EHT), and maturation. The nascent HSCs (pre-HSCs) form in the AGM region and subsequently migrate to the fetal liver where they undergo further maturation and expansion. Recent advances in single cell technologies have significantly enhanced our understanding of intrinsic cellular programs involved in HSC development. However, our knowledge about niche cells and the signals they emanate in the AGM region, as well as their contribution to HSC development, remain limited. Here we've conducted spatial transcriptomics analysis using Slide-seq on mouse trunk tissues, including both the AGM region and the fetal liver, at E10.5, E11.5 and E12.5. In addition, we performed scRNA-seq on the cells collected from the AGM and trunk at E10.5, E11.5 and E12.5, as well as the fetal liver at E12.5. To integrate the Slide-seq and scRNA-seq data, we developed an algorithm that leverages capabilities from both spatial and in-depth transcriptional sequencing. As a result, we were able to successfully reconstitute the structures of the trunk tissues and obtain gene expression profiles of individual cells at 10 µm resolution. From the analysis, we identified 7 cell clusters surrounding the aorta, with one cluster comprised of Nfgr + cells predominantly occupying the dorsal aorta. The ventral part of aorta displayed a complex and multilayered cellular architecture, featuring pre-HSCs, endothelial cells, mesonephric cells, and macrophages. Notably, we identified a unique mesenchymal stromal cell (MSC) population that co-express Cdh2 (encodes N-cadherin or N-cad) and Pdgfra; thus, we call these cells N-cad + MSCs. These N-cad + MSCs were particularly enriched at the ventral part of the aorta, forming a single cell layer positioned between an inner layer of aortic endothelial cells and an outer layer of mesonephric cells. Next, we utilized several bioinformatics tools (CellphoneDB, CellChat, NicheChat, and StringDB) in effort to identify potential signals that could impact embryonic hematopoiesis. The results revealed intricate interactions between N-cad + MSCs and pre-HSCs, for example, involving JAG1-NOTCH1 and SDF-1 (encoded by Cxcl12)-CXCR4 ligand-receptor pairs. To validate the findings obtained by the Slide-seq data, we selected and designed customized RNA probes for 140 genes of interest, including cell markers and potential signals. We then conducted MERFISH (Multiplexed Error-Robust Fluorescence in situ Hybridization) on 18 E11.5 trunk sections. The MERFISH data enabled us to characterize the spatially resolved niche cells and their derived signals in the AGM region at the single-cell level, which largely corroborated the results obtained from the Slide-seq data. Specifically, the MERFISH data indicates that N-cad + MSCs are in direct contact with aortic endothelial cells and are the primary cellular source of Jag1 and Cxcl12 around the aorta. Given the unique position of N-cad + MSCs and their intricate interactions with pre-HSCs, we formulated the hypothesis that N-cad + MSCs play a crucial role in embryonic hematopoiesis. To functionally validate our hypothesis, we are conducting a series of experiments using Ncad CreER; Cxcl12 flox/floxand Ncad CreER; Jag1 flox/flox mouse models. We anticipate that embryos lacking N-cad + MSC derived Jag1 or Cxcl12 will exhibit a substantial decline in intra-aortic hematopoietic clusters, phenotypic pre-HSCs, and functional HSCs. In summary, our study employed sequencing-based (Slide-seq) and imaging-based (MERFISH) spatial transcriptomics technologies to successfully characterize dynamic, spatially resolved profiles of niche components and associated signaling in the AGM region at the single cell level. This work significantly enhances our understanding of niche signaling and its role in regulating embryonic hematopoiesis and may aid the development of novel methods for inducing the transition from pluripotent stem cells to HSCs.

Deletion of the Artery Specific Enhancer Promotes B Cell and Impairs T Cell Development from the Hemogenic Endothelium

HSCs and HSC-independent lymphoid progenitors in the embryo arise from hemogenic endothelium (HE) at multiple anatomical locations. Although activation of arterial program in HE has been shown to play an important role in HSC development, the impact of the arterial program on specification of HSC-independent lymphoid cell remains unclear. During vascular development, arterial fate is specified following induction of DLL4 expression by VEGF by signaling through a highly conserved VEGF-responsive artery-specific enhancer located within the third intron of DLL4 ( DLL4-i3) which is controlled by ETS, SOXF and RBPJ factors. To investigate how the arterial program affects T and B cell lymphoid development, we generated DLL4-i3:Venus reporter and knockout ( DLL4 Di3/Di3) human pluripotent stem cells (hPSCs). Using a 3-dimensional, serum- and feeder-free spheroid differentiation system, we demonstrated that DLL4: i3 enhancer activity recapitulates endogenous DLL4 expression and arterial endothelial cell specification in human hPSC cultures. Moreover, DLL4-i3: Venus + HE and hematopoietic progenitors produced more myeloid CFCs and T cells as compared DLL4-i3: Venus - hemogenic cells. Deletion of DLL4i3 enhancer impaired arterial specification from hPSCs including formation of arterial DLL4 +CXCR4 +/- HE. In contrast to wild type (WT), hematopoietic progenitors (HPs) generated from DLL4 Di3/Di3hPSCs showed impaired CFC potential with significantly reduced G-CFCs and markedly diminished T cell potential. However, DLL4 Di3/Di3hPSCs showed efficient and highly reproducible B cell potential as compared to WT. To define the impact of artery-specific enhancer deletion on transcriptional program during endothelial-to-hematopoietic transition (EHT) and HP formation, we performed scRNAseq of all endothelial and hematopoietic cells from D5 and D8 differentiation cultures of DLL4-i3:Venus and DLL4 Di3/Di3 hPSCs. Through unsupervised clustering, 10 cell clusters (C0-C9) were distinguished and visualized using UMAP. Based on the expression panel of known pan-, arterial and hemogenic endothelial and hematopoietic markers we defined three clusters of HE with arterial features (AHE; high EFNB2, CDH5 and RUNX1 positive and SPN negative), two non-arterial HE clusters (low EFNB2, CDH5 and RUNX1 positive and SPN negative), two EHT clusters (CDH5 and SPN positive, high RUNX1, and GFI1b), and three HP clusters (SPN positive, high RUNX1 and CDH5 negative). GSEA of DLL4i3: Venus+ cells with active enhancer vs DLL4 Di3/Di3 cells revealed enrichment in ribosomes and oxidative phosphorylation pathways across all clusters and focal adhesion and tight junction pathways in HE and EHT clusters. DEG analysis demonstrated upregulation of EFNB2, MECOM HES4 in DLL4-i3: Venus+ cells within HE and EHT clusters, while DLL4 Di3/Di3 cells demonstrated upregulation of NRP2 and CDH13 genes. Interestingly, in EHT and HP clusters, we noticed that DLL4-i3 knockout significantly upregulates the expression of GAS5 lncRNAs known to be involved in growth arrest of human peripheral blood T cells. Using siRNA, we demonstrated that knockdown of GAS5 in D5 cultures of DLL4 Di3/Di3 cells rescued the T cell potential of the hematopoietic progenitors suggesting that NOTCH-mediated GAS5 suppression is essential for the T cell specification and expansion following EHT. In contrast to T cells, GAS5 downregulation had no effect on B cell specification of DLL4 Di3/Di3 HE. Overall, we established that DLL4-i3 enhancer is required for arterial programming of HE with T cell potential while deletion of this enhancers promotes B cell development. In addition, we demonstrated an important role of GAS5 lncRNA in the regulation of T cell development from HE.

A model for making investment decisions contributing to the heating supply companies’ development, taking into account the restrictions arising in the applying tariffs conditions with the “alternative boiler house” method

The study presents a model for making investment decisions that contribute to the development of heating supply companies (hereinafter referred to as HSC), taking into account the restrictions that arise in the conditions of applying tariffs using the “alternative boiler house” method. The study presents a generalized procedure for determining investment projects for the development of heating supply systems, considering the interests of participants in the heat supply of the territory, a curiosity assessment of the main participants in the heat supply of the territory. The weight of the criteria obtained as a result of the analysis is further used in the decision-making model. The simplex method was used as the basis for the implementation of the author’s model of investment decision-making. The simplex method is one of the methods for solving linear programming problems. An algorithm for optimizing the development function of the heating supply system by the simplex method and the index of the development of HSC in the conditions of applying tariffs using the “alternative boiler hous” method is proposed. The study presents a financing plan for a real project for the construction of a new boiler house.

Abstract 5467: Single-cell analysis of CD34-enriched blood cells reveals early prognostic markers of myelodysplastic syndromes

Abstract Myelodysplastic Syndromes (MDS) are a group of blood malignancies characterized by aberrant differentiation of hematopoietic stem and progenitor cells (HSPC) in the bone marrow that results in inefficient hematopoiesis and high risk of transformation to acute myeloid leukemia. In order to detect aberrant HSC differentiation in blood samples sensitively and accurately, we explored the use of scRNA-seq of CD34-enriched cells from peripheral blood in identifying differentiation trajectories that are abnormal in patient samples versus expected normal development. A total of 685,000 CD34-enriched PBMCs from 93 samples were analyzed using 10X 3’ v3 scRNA-seq library preparation. For increased efficiency, the samples were multiplexed in groups of 5 individuals and were later identified based on individual natural genetic variation using a custom genotyping assay or low-pass WGS. We then sequenced these 24 libraries on a UG100 sequencer to yield an average of 20,000 reads per cell. Six of these libraries were also sequenced on a NovaSeq. Comparison between scRNA profiles generated on the two different sequencers yielded highly similar results, including a similar ability to demultiplex the patient samples using genetic markers, accurately quantify gene modules and determine cell populations.To analyze the scRNA data, we used a novel computational framework, Metacell, to reconstruct metacell modules for both healthy and disease samples allowing us to find variability in the differentiation process between the disease samples and the normal baseline state. Two patients with MDS which had transformed to acute myelogenous leukemia (AML) demonstrated unique and distinct metacell clusters that were significantly separate from normal HSPC differentiation patterns, demonstrating distinct clusters with elevated levels of BCL2 which is an existing therapy target in secondary AML. Our results demonstrate that scRNA-seq analysis of peripheral blood HSPCs samples can be used to detect aberrations in HSC development in MDS patients and serve as a prognostic tool for stratification of patients with aggressive disease and drug response. Given the cost-efficiency of the entire process, we believe this is one of the first examples of the potential practical utility of single-cell analysis in a clinical setting. Citation Format: Eti Meiri, Nili Saar-Furer, Nimrod Rappoport, Sarah Pollock, Gila Lithwick-Yanai, Zohar Shipony, Nika Iremadze, Doron Lipson, Amos Tanay, Liran Shlush. Single-cell analysis of CD34-enriched blood cells reveals early prognostic markers of myelodysplastic syndromes. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5467.

Study within a review (SWAR).

Study within a review (SWAR).

Myh9 Plays an Essential Role in the Survival and Maintenance of Hematopoietic Stem/Progenitor Cells

Myosin heavy chain 9 (MYH9) gene encodes a protein named non-muscle heavy chain IIA (NMHC IIA), interacting with actin and participating in various biological processes. Mutations in MYH9 cause an array of autosomal dominant disorders, known as MYH9-related diseases (MYH9-RD). However, the role of MYH9 in normal hematopoiesis remains largely unexplored. By using Mx1-cre Myh9 conditional knockout mice, we established an inducible system to precisely inactivate Myh9 function in hematopoietic cells in vivo. The results showed that deletion of Myh9 led to severe defects in hematopoiesis, characterized by pancytopenia, drastic decreases of hematopoietic stem/progenitor cells (HSPC), and bone marrow failure, causing early lethality in mice. The defect in hematopoiesis caused by Myh9 ablation is cell autonomous. In addition, Myh9 deletion impairs HSPC repopulation capacity and increases apoptosis. RNA sequencing results revealed significant alterations in the expression of genes related to HSC self-renewal and maintenance, while multiple signal pathways were also involved, including genes for HSC and myeloid cell development, intrinsic apoptosis, targets of mTOR signaling, and maturity of hematopoietic cells. Our present study suggests an essential role for Myh9 in the survival and maintenance of HSPC in normal hematopoiesis.

Bclaf1 promotes hematopoietic stem cell repopulating capacity and self-renewal

Abstract Hematopoietic stem cells (HSC) balance differentiation and self-renewal to promote maintenance of mature blood cell lineages. The mechanisms that regulate HSC expansion during fetal hematopoietic development and adult HSC maintenance remain significant knowledge gaps. BCLAF1 (Bcl2-associated factor 1), a transcriptional regulator, is highly expressed in hematopoietic cells. We recently defined its function as a modulator of ETS-family transcription factor activity in developing B cells. Our objective in this study is to determine the molecular mechanism of BCLAF1 in regulation of fetal and adult HSCs. We used mouse models with selective deletion of Bclaf1 in hematopoietic cells to test whether BCLAF1 regulates HSC development and function. We find that loss of Bclaf1 results in significantly reduced numbers of fetal HSCs. The reduced HSC numbers persist in adult mice; but there is no additional decline or development of bone marrow failure with aging. In competitive transplant experiments, Bclaf1-deficient fetal and adult HSCs have defective repopulation and self-renewal capacity resulting in reduced reconstitution of all hematopoietic populations. Bclaf1-deficient HSCs do not have abnormalities in emergence, proliferation, cell death, or homing activity. Using single cell RNA-sequencing of fetal HSCs, we find that loss of BCLAF1 results in reduction of a subpopulation of HSCs and upregulation of the early response genes Jun, Fos, and Egr1. Collectively, these findings suggest BCLAF1 restrains activity of immediate early genes to preserve HSC self-renewal capacity and promote HSC expansion during both fetal development and repopulation of hematopoiesis after HSC transplant. Supported by grants from Children's Discovery Institute, St. Louis Children's Hospital Foundation, Siteman Cancer Center, and NIH (T32 AI007163).

Hematopoietic Stem Cell-Independent Differentiation of Mast Cells From Mouse Intraembryonic VE-Cadherin+ Cells.

Hematopoietic stem cell (HSC)-independent hematopoiesis from hemogenic endothelial cells (HECs) in the mouse embryo has been recognized as a source of tissue-resident hematopoietic cells in adult mice. Connective tissue mast cells (MCs) have been reported to originate from VE-cadherin (VE-cad)-expressing HECs in the yolk sac and embryo proper (EP) by a VE-cad-Cre-mediated lineage-tracing analysis. However, it remains unclear whether MCs are generated via a conventional HSC-dependent hematopoietic differentiation pathway, or whether through a fast-track pathway bypassing the emergence of HSCs. Here, we investigated whether EP-derived VE-cad+ cells differentiate into MCs independently of HSCs. VE-cad+ cells isolated from the embryonic day (E) 9.5-10.5 EP robustly formed connective tissue-type MCs in a newly established co-culture system using PA6 stromal cells. In contrast, bone marrow (BM) reconstitution assays of cultured cells indicated that E9.5 VE-cad+ cells did not differentiate into transplantable HSCs in this culture condition. Lymphoid-biased HSCs with a limited self-renewal capacity were occasionally detected in some cultures of E10.5 VE-cad+ cells, while MC growth was constantly observed in all cultures examined. HSCs purified from adult BM required a more extended culture period to form MCs in the PA6 co-culture than the embryonic VE-cad+ cells. Furthermore, E9.5-E10.5 VE-cad+ cells contributed to tissue-resident MCs in postnatal mice when transplanted into the peritoneal cavity of newborn mice. These results suggest that EP-derived VE-cad+ cells generate MCs independently of HSC development in vitro and possess the potential of generating connective tissue MCs in vivo, although the exact differentiation program remains unsolved.

ETV6/TEL1 Is Required for HSC Emergence during Embryonic Development.

ETV6/TEL1 (hereinafter referred to as TEL1) is an ets family transcription factor that is frequently involved in chromosomal translocations associated with leukaemia, including t(12:21) which generates the TEL-AML1 fusion gene, the most common genetic abnormality in childhood cancer. The role of TEL1 in normal haemopoiesis has been extensively studied in the mouse where knock-out studies have indicated that TEL1 is not required for development of primitive (ie yolk sac-derived) haemopoiesis. TEL1-knockout mice die at day E10.5 due to a defect in angiogenesis precluding direct analysis of the definitive (ie intraembryonically-derived) haematopoietic system. Analysis of chimaeric mice generated using TEL1−/− ES cells suggest that TEL1 is not required for generation of the definitive haemopoietic system during ontogeny but is required for HSC survival perinatally at bone marrow stages. Conditional knockout studies have subsequently identified a role for TEL1 in the survival of adult bone marrow-derived HSC. We now report the analysis of TEL1 function in amphibian (Xenopus) embryos where haemopoietic development can be studied even in embryos with defective angiogenesis. We characterised the TEL1 expression pattern and knocked down its function using antisense morpholino oligonucleotide injection. Consistent with the mouse knockout, specification of the primitive haemopoietic system was unaffected, however a defect in maturation of primitive erythroid cells, not previously reported in the mouse, was also apparent. We next analysed the development of the definitive haemopoietic system, thought to arise as clusters of HSCs in the floor of the dorsal aorta. But, no clusters of HSCs were found. Formation of the dorsal aorta however, proceeded normally, indicating that TEL1 controls the decision between the arterial and HSC programmes. Given, the critical role for TEL1 in the development of HSC indicated by these studies, a temporal analysis of emergence of intraembryonic HSC in TEL-deficient mice may be warranted.

A "Switch" in Time through Genes Aligned: Unraveling the Genomic Landscape of HSC Development.

Seeking to define the "switch" from fetal to adult hematopoiesis, Li etal. (2020) performed extensive genomic and epigenomic profiling of hematopoietic stem and progenitor cells across ontogeny (as explored in this issue of Cell Stem Cell). Gradual and stochastic changes in genomic and epigenomic regulation suggest the absence of any specific regulator.

Multi-layered Spatial Transcriptomics Identify Secretory Factors Promoting Human Hematopoietic Stem Cell Development.

SummaryHematopoietic stem cells (HSCs) first emerge in the embryonic aorta-gonad-mesonephros (AGM) region. Studies of model organisms defined intersecting signaling pathways that converge to promote HSC emergence predominantly in the ventral domain of the dorsal aorta. Much less is known about mechanisms driving HSC development in humans. Here, to identify secreted signals underlying human HSC development, we combined spatial transcriptomics analysis of dorsoventral polarized signaling in the aorta with gene expression profiling of sorted cell populations and single cells. Our analysis revealed a subset of aortic endothelial cells with a downregulated arterial signature and a predicted lineage relationship with the emerging HSC/progenitor population. Analysis of the ventrally polarized molecular landscape identified endothelin 1 as an important secreted regulator of human HSC development. The obtained gene expression datasets will inform future studies on mechanisms of HSC development in vivo and on generation of clinically relevant HSCs in vitro.

SOX17 Is Essential for Integration of Arterial and HOXA Programs in Hemogenic Endothelium

Recent advances in understanding the major bottlenecks in derivation of engraftable HSCs and lymphoid cells from pluripotent stem cells (PSC), have identified deficiencies in NOTCH and HOXA signaling as contributing factors to the observed functional deficits of PSC-derived hematopoietic progenitors. However, little is known about the mechanisms that are essential for establishing these pathways during PSC differentiation. Here, we revealed the critical role of SOX17 in linking HOXA and NOTCH-mediated arterial programs in hemogenic endothelium (HE) and specification of definitive lympho-myeloid hematopoiesis. Using SOX17-knockout (SOX17-/-) and SOX17 DOX-inducible (iSOX17) hESCs, we found that SOX17-deficiency substantially reduces formation of CD144+CD43-CD73-DLL4+CXCR4+/- arterial HE and definitive lympho-myeloid hematopoiesis, while SOX17 upregulation at mesodermal stage of development causes the opposite effect. Molecular profiling of HE generated from iSOX17 hESCs in DOX+ and DOX- conditions using RNAseq, SOX17 ChIPseq and ATACseq, revealed that SOX17 overexpression upregulates 522 genes enriched in NOTCH, TGFb, HEDGEHOG and WNT signaling, including DLL1, DLL4, NOTCH4, LFNG, WNT5a, WNT5b, GLI3, and genes associated with HSC development, CXCR4,KITLG and ALDH1A2. In addition, we noted significant upregulation of HOXA7,HOXA9, HOXA10, HOXB8, HOXC4 and CDX2 homeobox genes in SOX17-induced cultures, with no expression of HOXA genes observed in HE from SOX17-/- cells. ChIPSeq analysis revealed DOX+ specific SOX17 binding at transcriptional start sites (TSS) of 316 significantly upregulated genes, including ALDH1A2, CDX2, DLL1, DLL4, HEY1, HOXA7, HOXB8, HOXC4 and KITLG, suggesting that upregulation of these genes could be explained by their direct activation by SOX17. Since ALDH1A2 and CDX2 are known to play a role in the activation of HOXA genes, we investigated whether SOX17's effect on HOXA expression could also be mediated by ALDH1A2 and CDX2. We found that adding ALDH1 inhibitor to DOX+ cultures had no effect on arterial HE development and HOXA expression. In contrast, transfection of iSOX17 hPSCs cultures with CDX2 shRNA significantly decreased arterial HE formation and downregulated HOXA7, HOXA9, and HOXA10 expression. Overall, our studies indicate that SOX17 plays a critical role in the activation and integration of arterial and HOXA programs in HE, which is mediated by CDX2. These findings will be important for designing a strategy for direct HSC fate programming from hPSCs. Disclosures Uenishi: Casebia Therapeutics: Employment. Slukvin:Cynata Therapeutics: Consultancy, Other: Founder and Stockholder.

A Tailored Habits-based Dietary Intervention Coupled with Oral Rehabilitation on the Nutritional Status of Partially Dentate Older Adults (P04-018-19)

ObjectivesThis study developed and tested a habit-based tailored dietary intervention, in conjunction with oral rehabilitation amongst partially dentate older adults. MethodsA randomised controlled trial was conducted on 56 patients recruited from the Centre for Dentistry, Queens University Belfast (mean age = 71.8 years). Participants were randomised to an intervention group (habits-based dietary intervention) or a control group and followed up for 8 months. The intervention group attended four meetings with a trained researcher to discuss individual areas of their dietary intake (fruit/vegetables, wholegrains, healthy proteins). Habit formation was assessed using the Self-Report Behavioural Automaticity Index (SRBAI) and the primary outcome measure was self-reported automaticity for healthy food choices. ResultsPreliminary results showed that change in automaticity and habit adherence in the intervention group for all tailored dietary habits was significant between baseline and follow up visits (P < 0.001). There were also statistically significant and strong correlations between automaticity and habit adherence (Fruit/vegetables rho = 0.68: Wholegrains rho = 0.98: Healthy Proteins rho = 0.86) for the intervention group. There was an increase in automaticity in the intervention group of 4.1 (7.8 SD) SRBAI points (scale 0–7) for fruit/vegetable intake compared to 2.1 (5.1 SD) for the control group. Similar trends were detected for wholegrain and healthy protein automaticity but, other than wholegrain (P = 0.047), between group differences were non-significant (P > 0.05). BMI decreased in the intervention group (29.5 to 28.6 kg/m2) compared to a slight increase in the control group (27.9 to 28.0 kg/m2). There were improvements in Mini Nutritional Assessment (0.75 intervention: 0.53 control) mean change scores for both groups, however between-group differences were not statistically significant (P = 0.7). ConclusionsBased on preliminary results, this study demonstrated the success of a habit-based dietary intervention coupled with oral rehabilitation in positively influencing dietary behaviours and other nutritional outcomes in partially dentate older adults. Funding SourcesNorthern Ireland HSC Research and Development Office and the Health Service Executive Awards on Ageing.

Pro-inflammatory Aorta-Associated Macrophages Are Involved in Embryonic Development of Hematopoietic Stem Cells

SummaryHematopoietic stem cells (HSCs) are generated from specialized endothelial cells of the embryonic aorta. Inflammatory factors are implicated in regulating mouse HSC development, but which cells in the aorta-gonad-mesonephros (AGM) microenvironment produce these factors is unknown. In the adult, macrophages play both pro- and anti-inflammatory roles. We sought to examine whether macrophages or other hematopoietic cells found in the embryo prior to HSC generation were involved in the AGM HSC-generative microenvironment. CyTOF analysis of CD45+ AGM cells revealed predominance of two hematopoietic cell types, mannose-receptor positive macrophages and mannose-receptor negative myeloid cells. We show here that macrophage appearance in the AGM was dependent on the chemokine receptor Cx3cr1. These macrophages expressed a pro-inflammatory signature, localized to the aorta, and dynamically interacted with nascent and emerging intra-aortic hematopoietic cells (IAHCs). Importantly, upon macrophage depletion, no adult-repopulating HSCs were detected, thus implicating a role for pro-inflammatory AGM-associated macrophages in regulating the development of HSCs.

P3HT:spiro-OMeTAD blending system as a hole conductor for solid-state hybrid solar cells with a dendritic TiO2/Sb2S3 nanorod composite structure

A dendritic TiO2/Sb2S3 nanorod/blend P3HT:spiro-OMeTAD system with considerable absorption and fluorescence quenching may have potential for HSC development.

In reach into Nursing Homes Education, Training and Development Programme

When nursing home residents become acutely unwell they are often referred to hospital for sometimes aimless and sometimes distressing treatment. RCN (RCN 2015) conducted a NI regional survey across the independent sector; One key recommendation was that in partnership with HSC trusts, a learning and development pathway should be developed for independent sector registrants to promote their on-going development, and support maintenance of their clinical skills and competence. A greater cohesion between the statutory and independent sector was advocated providing then an in reach service of specialist support and a greater accessibility to shared learning and development opportunities. A statistical study of ED attendances to Antrim Area Hospital (AAH) from 20 nursing homes was undertaken over six months (2013/14) which identified that of the 497 attendances, approximately 30% patients were discharged not requiring admission and potentially with appropriate skills training could be nursed within their care home environment. The key aim of this initiative was to empower nursing home registrant staff through a coaching, education, training and development programme which would enhance and enable skills and knowledge using a facilitative anticipatory care model so to maintain residents safely in their care home for as long as possible. This small cycle of change (PDSA) quality improvement initiative reflected Northern Ireland’ s regional strategy ‘Transforming Your Care’ (DHSSPS, 2011) highlighting older people should be cared for in the right place at the right time with the best possible outcome. In February 2015, 20 nursing homes completed a patient profile to help inform the education programme content and identified champions to undertake the education programme which included; long term conditions management, dementia care, recognising /managing the deteriorating patient, medicines optimisation and end of life advance care planning. They also attended a clinical skills programme in catheter management, Peg Tube management, syringe driver management and venepuncture. Champions were equipped and empowered to cascade training to other staff in the home. The Trust practice development facilitator provided visible support to the home connecting them to other specialist services such as the dementia home support team, medication review pharmacy team and the voluntary sector (Alzheimer’s, Parkinson’s UK, Age NI. Expediting patient discharge from hospital to the care home became a feature when additional care home nurse training was required. The evaluative process included both qualitative and quantitative methodologies. Underpinned by the triple aim IHI improvement framework this initiative reduced avoidable ED attendances by 31%; reducing cost per capita (approx £319K), reduced reliance on community services, improved the care home population health, and improved the resident and registrant nurse experience. The success has resulted in extension to a further 20 homes in NHSCT and the model is sustained through the permanency of the integrated partnership between independent sector and primary /secondary care and is transferable to other trusts in NI/UK.

Developing HSCs become Notch independent by the end of maturation in the AGM region

AbstractThe first definitive hematopoietic stem cells (dHSCs) in the mouse emerge in the dorsal aorta of the embryonic day (E) 10.5 to 11 aorta-gonad-mesonephros (AGM) region. Notch signaling is essential for early HSC development but is dispensable for the maintenance of adult bone marrow HSCs. How Notch signaling regulates HSC formation in the embryo is poorly understood. We demonstrate here that Notch signaling is active in E10.5 HSC precursors and involves both Notch1 and Notch2 receptors, but is gradually downregulated while they progress toward dHSCs at E11.5. This downregulation is accompanied by gradual functional loss of Notch dependency. Thus, as early as at final steps in the AGM region, HSCs begin acquiring the Notch independency characteristic of adult bone marrow HSCs as part of the maturation program. Our data indicate that fine stage-dependent tuning of Notch signaling may be required for the generation of definitive HSCs from pluripotent cells.

Ubiquitylation of the Paf oncoprotein and interaction with PCNA is essential for hematopoietic stem cell function and development.

Abstract Hematopoietic stem cells (HSCs) are critical for the lifelong production and maintenance of all blood cell types. The molecular mechanisms that guide this process remain poorly understood. The 15kDa proliferating cell nuclear antigen (PCNA) associated factor (Paf) is a potent oncogene that is over-expressed in most cancers. We have previously shown that Paf is essential for HSC and progenitor function and development. Paf deficient mice (Paf−/−) are leukopenic due to reduced number of HSCs and committed progenitors. Paf−/− HSCs failed to maintain quiescence, to self-renew and to support long term hematopoietic reconstitution. To determine the in vivo molecular interactions and pathways by which Paf functions to mediate hematopoiesis, we introduced mutant versions of Paf into the Paf−/− mice. These unique mouse models allowed us to show that Paf-PCNA interactions and Paf ubiquitylation are both essential for hematopoiesis. Furthermore, biochemical analyses of cells from these mice showed that Paf interaction with PCNA was essential for nuclear localization and proper ubiquitylation of Paf. Collectively, therefore, our studies show that Paf function is dependent upon the ability to interact with PCNA and that the ubiquitylation of Paf regulates Paf’s function during hematopoiesis. Analyses are ongoing to further delineate the molecular mechanism by which Paf mediates HSC function and development.

Cdx2 Cooperates with Flt3-ITD to Induce Acute Myeloid Leukaemia in Mice

The caudal-related homeobox gene CDX2 is ectopically expressed in 90% of human acute myeloid leukaemia (AML), but not normal haematopoietic stem and progenitor cells (HSPC). Retroviral expression of Cdx2 causes a highly penetrant, lethal AML in mouse models, while short hairpin RNA-mediated targeted knockdown of CDX2 in vitro impairs growth and clonogenic potential of AML cell lines (Scholl et al. 2007). These findings implicate Cdx2 overexpression as a clinically relevant model of myeloid leukaemogenesis, however existing studies have been limited by the requirement for ex vivo manipulation, retroviral overexpression and transplantation.To understand the role of Cdx2 expression in de novo leukaemic transformation of HSC, we generated an inducible transgenic mouse model of Cdx2 expression linked to the mCherry fluorescent reporter. Cdx2 was specifically activated in adult HSC using the tamoxifen-inducible SclCreERT:Cdx2 model and compared with LysMCre:Cdx2 mice that selectively activated Cdx2 in the myeloid lineage, commencing at committed myeloid progenitors. SclCreERT:Cdx2 mice developed a lethal myelodysplastic phenotype with a long latency (median survival, 184 days) characterised by leukopenia, anaemia, thrombocytopenia and reduced marrow cellularity. Bone marrow histology revealed prominent megakaryocytic dysplasia. Competitive transplantation assays showed dramatic loss of long-term HSC self-renewal after tamoxifen induction of Cdx2 in the secondary recipients. Conversely, LysMCre:Cdx2 mice developed a myeloproliferative phenotype with leukocytosis, splenomegaly and extramedullary haematopoiesis. Interestingly, both cohorts exhibited marked neutrophil hypersegmentation compared to non-Cdx2 controls, a characteristic that is compatible with myelodysplasia, and suggests that Cdx2 not only controls HSC development and differentiation, but is required for granulocyte maturation. In contrast to retroviral models, AML was never observed with Cdx2 expression alone.FLT3-internal tandem duplication (ITD) represents a tyrosine kinase mutation that is present in 35% of AML patients, making it one of the most common genetic alterations found in AML. Transgenic Flt3ITD mice develop myelodysplastic syndrome (MDS)/myeloproliferative neoplasm (MPN) with long latency (approx. 10-15 months) but do not develop AML (Lee et al. 2007), suggesting that additional mutations are required for complete leukaemogenesis. Preliminary data has shown a correlation between CDX2 and FLT3 expression in AML. We therefore crossed SclCreERT:Cdx2 with Flt3ITD/+ and Flt3ITD/ITD mice. SclCreERT:Cdx2xFlt3ITD/+ mice developed a rapidly progressive lethal MDS with marked anaemia and thrombocytopenia, splenomegaly, morphological dysplasia and a shorter latency (median survival, 45 days) with a low incidence of transformation to AML. Strikingly, SclCreERT:Cdx2xFlt3ITD/ITD mice developed a fully penetrant AML characterised by marked leukocytosis, splenomegaly and hepatomegaly, and >20% circulating blast cells, demonstrating that Cdx2 cooperates with Flt3ITD to induce AML in a Flt3ITD gene dosage-dependent manner (Fig 1). Transplantation experiments of these samples into lethally irradiated, syngeneic wildtype (WT) recipients revealed that cells from SclCreERT:Cdx2xFlt3ITD/+ or SclCreERT:Cdx2xFlt3ITD/ITD but not WT, Flt3ITD/+ or Flt3ITD/ITD resulted in direct phenocopy of the primary disease with a short latency lethality, demonstrating the disease is intrinsic to transformed HSPC populations.Mechanistically, SclCreERT:Cdx2xFlt3ITD/+ and SclCreERT:Cdx2xFlt3ITD/ITD samples showed marked depletion of long-term HSC, abnormal cell cycle regulation and widespread gene expression changes leading to the activation of a leukaemia stem cell (LSC) program, including the aberrant expression of several Hox genes.Altogether, this work demonstrates that conditional Cdx2 expression has a critical role in the transformation of HSPC populations to AML LSC. This is a novel, inducible model of de novo leukaemic transformation and reflects common genetic aberrations seen in human AML. Thus, this model may help to identify tractable susceptibilities to target LSC populations and improve clinical outcomes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.