Myeloid Potential Research Articles

A Dual-Site Inhibitor of CBP/p300 KIX is a Selective and Effective Modulator of Myb.

The protein-protein interaction between the KIX motif of the transcriptional coactivator CBP/p300 and the transcriptional activator Myb is a high-value target due to its established role in certain acute myeloid leukemias (AML) and potential contributions to other cancers. However, the CBP/p300 KIX domain has multiple binding sites, several structural homologues, many binding partners, and substantial conformational plasticity, making it challenging to specifically target using small-molecule inhibitors. Here, we report a picomolar dual-site inhibitor (MybLL-tide) of the Myb-CBP/p300 KIX interaction. MybLL-tide has higher affinity for CBP/p300 KIX than any previously reported compounds while also possessing 5600-fold selectivity for the CBP/p300 KIX domain over other coactivator domains. MybLL-tide blocks the association of CBP and p300 with Myb in the context of the proteome, leading to inhibition of key Myb·KIX-dependent genes in AML cells. These results show that MybLL-tide is an effective, modifiable tool to selectively target the KIX domain and assess transcriptional effects in AML cells and potentially other cancers featuring aberrant Myb behavior. Additionally, the dual-site design has applicability to the other challenging coactivators that bear multiple binding surfaces.

Delineating spatiotemporal and hierarchical development of human fetal innate lymphoid cells.

Whereas the critical roles of innate lymphoid cells (ILCs) in adult are increasingly appreciated, their developmental hierarchy in early human fetus remains largely elusive. In this study, we sorted human hematopoietic stem/progenitor cells, lymphoid progenitors, putative ILC progenitor/precursors and mature ILCs in the fetal hematopoietic, lymphoid and non-lymphoid tissues, from 8 to 12 post-conception weeks, for single-cell RNA-sequencing, followed by computational analysis and functional validation at bulk and single-cell levels. We delineated the early phase of ILC lineage commitment from hematopoietic stem/progenitor cells, which mainly occurred in fetal liver and intestine. We further unveiled interleukin-3 receptor as a surface marker for the lymphoid progenitors in fetal liver with T, B, ILC and myeloid potentials, while IL-3RA– lymphoid progenitors were predominantly B-lineage committed. Notably, we determined the heterogeneity and tissue distribution of each ILC subpopulation, revealing the proliferating characteristics shared by the precursors of each ILC subtype. Additionally, a novel unconventional ILC2 subpopulation (CRTH2– CCR9+ ILC2) was identified in fetal thymus. Taken together, our study illuminates the precise cellular and molecular features underlying the stepwise formation of human fetal ILC hierarchy with remarkable spatiotemporal heterogeneity.

A Human IPS Model Implicates Embryonic B-Myeloid Fate Restriction As a Developmental Susceptibility to ETV6-RUNX1

Childhood acute lymphoblastic leukemia (cALL) is clinically distinct from that in adults with higher incidence, better prognosis, a distinct mutational spectrum and that the pre-leukemic initiating events frequently occur in utero . One hypothesis for this difference is that childhood cancers arise in transient cell compartments unique to early human development. We have explored this in ETV6-RUNX1-associated cALL, where evidence from twin studies and neonatal heel prick testing has shown that this translocation occurs in utero and is an initiating event . Consistent with this, epidemiological evidence and changes in placental structure suggest that leukemic initiation occurs in the first trimester when both extra-embryonically and intra-embryonically-derived waves of hematopoiesis coexist. We therefore characterized B-lymphoid development in first trimester human embryos aiming to identify a candidate compartment vulnerable to the pre-leukemic initiating effect of ETV6-RUNX1. Using CD19 as a marker associated with definitive B lineage restriction in adult hematopoiesis, we established that the first CD19+ B cell progenitors emerged in the human fetal liver (FL) at Carnegie Stage (CS) 17 and were distinct from their cord blood or adult bone marrow counterparts in that the majority expressed surface IL7 receptor. Analysis of IL7R expression also uncovered a CD19-IL7R+ candidate B progenitor compartment. CD19-IL7R+ cells produced CD19+ B cells in vitro, possessed IGH DJ recombination, but also exhibited myeloid (but not megakaryocyte-erythroid) potential. Single cell analysis revealed co-expression of lymphoid and myeloid lineage programs in CD19-IL7R+ progenitors at CS20, whereas at CS17 their transcriptional programs appeared markedly more myeloid, indicating that lymphoid potential is first acquired in CD19-IL7R+ progenitors in this developmental window. Myeloid cells derived from CS20 IL7R progenitors harbored IGH DJ rearrangements, confirming their origins from a RAG-expressing lymphoid progenitor. We tested whether these distinct aspects of embryonic lymphoid development could be modeled using human pluripotent stem cells (hPSCs). In vitro B cell differentiation of hPSCs produced IL7R expressing CD19+ B cells as well as a CD19-IL7R+ progenitor that also “switched” from myeloid to B-myeloid programming during culture. At the global transcriptional level the hPSC-derived lymphoid hierarchy maps closely with that from the embryo, with both separating from the adult, suggesting that hPSCs provide a developmentally relevant model of early fetal B lymphopoiesis. We next used CRIPSR-directed homologous recombination to knock in the cDNA of RUNX1 into intron V of ETV6 resulting in expression of ETV6-RUNX1 under the endogenous ETV6 promoter. ETV6-RUNX1 hPSCs displayed a partial block in B lymphoid differentiation at the CD19-IL7R+ progenitor to proB cell transition and competitive co-culture demonstrated a selective advantage to the ETV6-RUNX1+ CD19-IL7R+ progenitors over their wild type counterparts. Single cell transcriptional analysis showed that the ETV6-RUNX1+ CD19+ B cells that do emerge express an abnormal B-myeloid gene expression signature akin to that seen in the CD19-IL7R+ progenitor. Consistent with this programming, when grown in myeloid cytokines, ETV6-RUNX1+ CD19+ B cells exhibited both a survival advantage and an aberrant ability to form IGH DJ rearranged macrophage colonies. Both the transcriptional and functional phenotypes were dependent on ETV6-RUNX1, as demonstrated by their reversion upon cre-mediated excision of the knock-in cassette. Our data support a model whereby ETV6-RUNX1 inhibits effective resolution of a lympho-myeloid program present in early fetal CD19-IL7R+ progenitors, impeding B lineage differentiation and resulting in the production of B cells with aberrant myeloid gene expression and potential. Such programming may explain the relatively high levels of myeloid antigen expression and lineage promiscuity seen in ETV6-RUNX1 and other cALLs. This hPSC model of ETV6-RUNX1 pre-leukemia will afford systematic evaluation of the contribution of second hit mutations seen in cALL and offers a tractable platform for drug screening. In conclusion we propose that a novel IL7R+ lympho-myeloid progenitor in the human embryonic fetal liver is a candidate target cell for in utero pre-leukemic initiation in childhood B-ALL. DisclosuresNo relevant conflicts of interest to declare.

Single-Cell Transcriptome Analysis of Embryonic Erythro-Myeloid Progenitor Cells Reveals Lineage Heterogeneity

The paradigm that multipotential hematopoietic stem cells (HSC) differentiate through a hierarchical series of increasingly specified progenitors has recently been challenged by single cell transcriptome analyses, which suggest greater heterogeneity in progenitor populations and hierarchy than previously thought. These studies are complicated by the wide variety of hematopoietic progenitors at different stages of maturation concurrently present in the adult bone marrow. In the early mammalian embryo, unique hematopoietic progenitors arise prior to, and independently of, HSC. These include erythro-myeloid progenitors (EMP) that arise from hemogenic endothelium in the yolk sac and migrate to the fetal liver to jump-start definitive hematopoiesis before HSC emerge. In vitro, EMP can generate definitive erythroid, megakaryocyte and myeloid cells, although not B-cells, and include at least some single cells with both erythroid and myeloid potential (McGrath et al. 2015 Cell Rep. 11:1892). We have previously shown that day 9.5 (E9.5) murine EMP have a unique and uniform (kit+CD41+CD16/32+) immunophenotype in the yolk sac and circulation, which is maintained as they colonize the E10.5 liver. We performed single-cell transcriptome analysis of this apparently poised, embryonic-specific multi-potential hematopoietic progenitor population, asking whether it contains an underlying heterogeneity or intrinsic lineage hierarchy. High quality data, as assessed by transcriptomic complexity, read depth, and alignment distributions, were generated for 133 single-cell EMP (scEMP) derived from E9.5 mouse embryos. Use both of supervised and of semi-supervised analytical approaches uncovered transcriptome heterogeneity that allowed clustering of scEMP by expression patterns. Based on expression of known lineage-directing transcription factors and cell type enrichment analysis, clusters of cells were identified that could be annotated as multipotential hematopoietic, megakaryocyte/erythroid, myeloid, and a more specific macrophage lineage potential. A novel binary reduction data integration and transformation methods was developed and utilized to further refine and characterize single-cell classifications by facilitating cross platform comparisons with single cell RNA-seq data obtained from EMP progeny in E11.5 liver and with published gene expression datasets derived from adult hematopoietic progenitors and fetal macrophage populations. Predictions of lineage potential based on our analytical approaches were tested by sorting cells based on immunophenotypic markers differentially expressed in specific clusters and analyzing their potential using single cell colony-forming assays. These functional studies support the concept that immunophenotypically homogeneous EMP contain subpopulations of multipotential progenitors differentiating into erythro-megakaryocyte, myeloid, and macrophage-specific lineage fates. DisclosuresNo relevant conflicts of interest to declare.

Identification and Molecular Characterization of Endothelial Cells Associated with Hematopoietic Niche Formation in Humans Identifies IL-33 As an Anabolic Niche Factor

Mammalian bone marrow (BM) regeneration after myeloablation requires orchestrated interplay between osteogenesis, angiogenesis and hematopoiesis. Specific subsets of endothelial cells (ECs) have been identified in mice, including sinusoidal ECs (Hooper et al., 2009) and H-endothelium (Kusumbe et al., 2014, 2016; Ramasamy et al., 2014), that drive and coordinate these processes. Their relevance to human biology, however, remains largely elusive.Here, using a combinatory approach of cell purification and transcriptomics, we identify and characterize EC subsets associated with BM niche formation during ontogeny and regeneration in humans.Massive parallel RNA sequencing of FACS-purified BM ECs (CD45-CD235-7AAD-CD31+CD9+) from AML patients in the regenerating phase after chemotherapy (n=3) revealed upregulated endoglin (CD105) expression, in comparison to ECs at diagnosis or from normal BM (n=7) (FPKM fold-change increase (FCI): 10.1 ± 4.9; GLM LRT Edge R; p=0.001). FACS analysis confirmed a strong enrichment of endoglin-expressing ECs in regenerative marrow (n=48) compared to normal BM (n=9) (10.81% ± 2.14 vs 0.48% ± 0.24 of CD31+CD9+ cells; p=0.043).Similarly, in human fetal BM at 15-20 weeks after gestation (n=15), when angiogenesis, osteogenesis and hematopoiesis are tightly coupled, a striking predominance of endoglin-expressing ECs (62.8% ± 5.9 of CD31+CD9+ cells) was found, confirmed by in situ immunohistochemistry in comparison to adult core hip bone biopsies. Comprehensive transcriptional characterization by RNAseq of these endoglin-expressing ‘human regeneration-associated EC’ (hREC) from fetal bone (n=7) in comparison to endoglin- ECs revealed a remarkable phenotypic and molecular similarity to murine H-endothelium as well as activation of pathways and factors implicated in hematopoiesis, osteogenesis and angiogenesis. These included upregulated expression of CD31 (PECAM1) (FPKM FCI: 4.7 ± 1.0; p=0.04), mucin-like sialoglycoprotein endomucin (EMCN) (FPKM FCI: 435 ± 112; p <0.001), stromal cell-derived factor 1 (CXCL12) (FPKM FCI: 108 ± 34; p <0.001) and transcriptional activation of NOTCH (Normalized Enrichment score (NES): 1.51, False Discovery Rate-adjusted q-value (FDR): <0.10) and HIF1a (NES: 1.60, FDR <0.10) signaling pathways.IL-33 was found to be highly and significantly overexpressed in both fetal and regenerative marrow ECs in comparison to steady-state normal BM ECs (FPKM FCI: 587 ± 128 and 174 ± 86, respectively, both p <0.001). Expression of IL-33 in endoglin-expressing ECs was confirmed at the protein level in both humans and mice. Recombinant human IL-33 (1) promoted the expansion of distinct subsets of hematopoietic precursor cells, specifically immunophenotypic multipotent progenitors (2.5-FCI ± 0.43; p<0.05) and multilymphoid progenitors (2.7-FCI ± 0.2; p<0.01), (2) increased metabolic activity in HUVECs (PrestoBlue Cell Viability assay; mean fluorescence intensity 1.40-FCI ± 0.02; p=0.03), and (3) promoted osteogenic differentiation of BM-derived mesenchymal cells (Alizarin red-staining and colorimetric assessment of calcium deposition (3.61-FCI ± 0.63; p<0.02)). Repressing IL-33 in HUVEC by shRNAs impaired their expansion in culture dramatically (FC decrease 6-7 fold; p=0.01). In vivo, enrichment of an endoglin-expressing subset of BM ECs was observed in mice exposed to a myeloablative dose of 5-fluorouracil (n=5) compared to steady-state mice (n=3) (FCI: 9.0 ± 1.7; p=0.01). Administration of recombinant murine IL-33 to C57BL/6 wild-type mice recapitulated the expansion of hematopoietic progenitors with myeloid and lymphoid potential (HPC1 subset) (FCI 2.6 ± 0.5; p=0.003), and resulted in a relative increase in CD31+CD105+ BM ECs (FCI 3.5 ± 0.3; p=0.002) as well as bone progenitor (Lin-Ter119-CD51+Sca-) cells (FCI 2.9 ± 0.7; p=0.06) within the niche.Collectively, the data reveal the existence of an endothelial subset associated with BM regeneration and ontogeny in humans and identify IL-33 as a potential driver of marrow recovery. The molecular characterization of hREC is, thus, anticipated to instruct the discovery of novel determinants of EC-driven regeneration of the hematopoietic system after injury. DisclosuresRaaijmakers:Novartis: Consultancy.

Diverse and Distinct Human Hematopoietic Differentiation Programs Generate By Bone Marrow and G-CSF Mobilized Hematopoietic Stem and Progenitors Cells

Several studies have highlighted murine hematopoietic stem cell (HSC) heterogeneity using single cell transplantation, clonal tracking barcoding analysis as well as RNAseq single cell analysis. Here we have used data from a gene therapy trial to treat Wiskott-Aldrich syndrome(WAS) to explore hematopoiesis in humans. In the trial, the therapeutic vector (lentivirus) integrates into the genome at unique positions in each hematopoietic stem and progenitor cell (HSPCs) and is consequently transmitted to all its progeny. Each integration sites (IS) corresponds to a particular stem/progenitor cell clone, for which we can quantify its contribution in each cell lineage. Thus hematopoietic ontogeny in humans can be inferred by tracking the appearance of unique IS in fractionated blood cell populations.We concentrated on four WAS patients treated by gene therapy with two distinct sources of autologous HSPC : bone marrow (BM) or mobilized peripheral blood (MPB) (collected by apheresis following administration of granulocyte colony-stimulating factor (G-CSF)). In these patients, we have sorted 5 cell types from peripheral blood at two timepoints of follow up (1 year and 3 years): myeloid (granulocytes and monocytes) and lymphoid subpopulations (T, B and NK cells), and analyzed their IS profile (using our new optimized pipeline, INSPIIRED, Berry et al Mol. Ther. Methods Clin. Dev. 2017 and Sherman et al Mol. Ther. Methods Clin. Dev. 2017). Using this approach, we have characterized up to tens of thousands IS per patients. We used statistical methods to account for sparse sampling and imperfect cell purifications. Using clustering algorithms, we identified different groups of IS clones corresponding to different human hematopoietic differentiation programs. In patients grafted with BM, we showed that at least a third of IS clones correspond to multipotent HSPCs, contributing to all 5 lineages, while other IS clones are mostly oligopotent or unipotent HSPCs with either myeloid or lymphoid potential. In patients treated with MPB, we also observed a diversity of IS clones but corresponding mainly to unipotent or oligopotent HSPCs and very rare multipotent clones. Longitudinal analysis of clonal dynamics, still ongoing, suggests the maintenance of this heterogeneity of HSPC over time.This study demonstrates the heterogeneity of human HSPC, with a diversity of inferred hematopoietic programs contributing to human blood homeostasis. Importantly these data also highlight distinct hematopoietic differentiation programs depending on the source of HSPC , G-CSF mobilized HSPC or BM resident HSPC. These new findings and approaches provide unique data on human hematopoiesis and should help defining the best source of HSPC and mobilizing agent for future cell and gene therapy clinical trials. DisclosuresThrasher:Orchard Therapeutics: Consultancy; Rocket Pharmaceuticals Ltd: Consultancy; 4bio Ventures Management Ltd: Other: Advisory Board; Torus Therapeutics, Inc: Other: Advisory Board.

The origin of blood cells and myeloid cells

In human hematopoiesis, cells of various lineages exist, such as neutrophils, lymphocytes, and erythrocytes. Unveiling the pathway from stem cells to the various lineages helps us understand the blood disorders and develop therapies for them. We have studied the developmental pathway of hematopoiesis for decades and found that myeloid potential is retained just before the differentiation into each lineage of the various lineage progenitors. This uniqueness of myeloid cells might reflect the character of mixed-phenotype leukemia and provide a very important clue in determining the evolutional history of blood cells. Recent studies concerning the differentiation pathways of megakaryocytes and granulocytes as well as the findings on the hemocytes of invertebrates have strongly supported the concept of the uniqueness of myeloid cells and enabled us to propose insights into the evolutional history of blood. In this paper, we discuss the origin of blood cells in the context of developmental pathways during ontogeny and phylogeny.

Decitabine shows anti-acute myeloid leukemia potential via regulating the miR-212-5p/CCNT2 axis.

Previous research has revealed the involvement of microRNA-212-5p (miR-212-5p) and cyclin T2 (CCNT2) in acute myeloid leukemia (AML). However, whether the miR-212-5p/CCNT2 axis is required for the function of decitabine in AML has not been well elucidated. Quantitative reverse transcription-polymerase chain reaction was used to examine enrichment of miR-212-5p. The relationship between CCNT2 and miR-212-5p was verified by the luciferase reporter assay. Cell apoptosis was evaluated by flow cytometry and western blot. CCK-8 assay was performed to determine cell viability. Decitabine significantly repressed cell viability, while promoted cell apoptosis. Meanwhile, the expression levels of cyclinD1, CDK4, and Bcl-2 were suppressed in cells with decitabine exposure, but Bax and caspase-3 expression levels were upregulated. Besides, miR-212-5p upregulation had the similar function with decitabine in AML cell proliferation and apoptosis. Subsequently, restoration of CCNT2 attenuated miR-212-5p overexpression-induced effects in Kasumi-1 and SKNO-1 cells. In addition, miR-212-5p depletion reversed decitabine-induced CCNT2 downregulation. The miR-212-5p/CCNT2 axis had an implication in the anti-leukemic effect of decitabine in AML.

Generation of Retinoic Acid-Dependent Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

The generation of the hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) is a major goal for regenerative medicine. In the embryo, HSCs derive from a HOXA+ population known as hemogenic endothelium (HE) in a retinoic acid (RA)-dependent manner. Using hPSCs, we have previously identified a KDR+CD235a− mesodermal population that gives rise to a clonally multipotent HOXA+ definitive HE. However, this HE lacks HSC-like capacity in the absence of exogenous transgenes and is functionally unresponsive to RA treatment. Thus, the specification of an RA-dependent hematopoietic program from hPSCs has remained elusive. Through single cell RNA-seq (scRNA-seq) analyses, we identified that 2 distinct KDR+CD235a− populations exist prior to HE specification, distinguishable by CXCR4 expression. Interestingly, KDR+CD235a−CXCR4− mesoderm expressed CYP26A1, an RA degrading enzyme, and harbored definitive hematopoietic potential within hPSC differentiation cultures in the absence of RA signaling, indicating the HE specified from CXCR4− mesoderm as RA-independent (RAi). In sharp contrast, KDR+CD235a−CXCR4+ mesoderm exclusively expressed ALDH1A2, the key enzyme in the synthesis of RA, but lacked hematopoietic potential under the same culture conditions. However, the stage-specific application of RA signaling to CXCR4+ mesoderm resulted in the robust specification of CD34+HOXA+ HE with definitive erythroid, myeloid, and lymphoid hematopoietic potential, establishing this HE as RA-dependent (RAd). Furthermore, while RAi HE entirely failed to persist following murine hematopoietic xenografts, RAd HE transiently persisted within the peripheral blood and bone marrow of murine hosts. To assess whether these functionally distinct hPSC mesodermal progenitors are physiologically relevant to human embryonic development, we integrated scRNA-seq datasets from the hPSC mesodermal cultures and a gastrulating human embryo. These analyses revealed that in vivo, distinct KDR+CXCR4−CYP26A1+ and KDR+CXCR4+ALDH1A2+ populations can be found at the stage of emergent mesoderm, following patterning of nascent mesoderm. Additional comparison to later stage human embryos demonstrated that RAd HE has a more fetal-like HOXA expression pattern than RAi HE. Scoring of single fetal HE cells against hPSC-derived HE revealed that while some early fetal HE cells were similar to RAi HE, the late fetal HE cells, which are hypothesized to give rise to HSCs, were more similar to RAd HE. Lastly, as HSC-competent HE is expected to express arterial genes, we found a subset of late fetal HE with this phenotype that were exclusively similar to RAd HE. Collectively, these data represent the first ever characterization of RA-dependent hPSC-derived definitive hematopoiesis and its mesodermal progenitor. Additionally, we provide evidence for in vivo mesodermal and HE correlates for both RAi and RAd hematopoietic programs within human embryos. This novel insight into human hematopoietic development will serve as an important tool for modeling development and ultimately provide the basis for de novo specification of HSCs. Disclosures No relevant conflicts of interest to declare.

Hematopoietic Stem Cells Are Endowed with Erythroid Signature in Beta-Thalassemia

Background Beta-thalassemia (Bthal) is a genetic disorder due to mutations in the ß-globin gene, leading to a reduced or absent production of HbA, which interferes with erythroid cell maturation and limits normal red cell production. Patients are affected by severe anemia, hepatosplenomegaly, and skeletal abnormalities due to rapid expansion of the erythroid compartment in bone marrow (BM) caused by ineffective erythropoiesis. In a classical view of hematopoiesis, the blood cell lineages arise via a hierarchical scheme starting with multipotent stem cells that become increasingly restricted in their differentiation potential through oligopotent and then unipotent progenitors. In human, novel purification strategies based on differential expression of CD49f and CD90 enrich for long-term (49f+) and short-term (49f−) repopulating hematopoietic stem cells (HSCs), with distinct cell cycle properties, but similar myeloid (My) and lymphoid (Ly) potential. In this view, it has been proposed that erythroid (Ery) and megakaryocytic (Mk) fates branch off directly from CD90-/49f− multipotent progenitors (MPPs). Recently, a new study suggested that separation between multipotent (Ery/My/Ly) long-term repopulating cells (Subset1, defined as CLEC9AhighCD34low) and cells with only My/Ly and no Ery potential (Subset2, defined as CLEC9AlowCD34high)occurs within the phenotypic HSC/MPP and CD49f+ HSCs compartment. Aims A general perturbed and stress condition is present in the thalassemic BM microenvironment. Since its impact on the hematopoietic cell subpopulations is mostly unknown, we will investigate which model of hematopoiesis/erythropoiesis occurs in Bthal. Moreover, since Beta-Thalassemia is an erythropoietic disorder, it could be considered as a disease model to study the 'erythroid branching' in the hematopoietic hierarchy. Methods We defined by immunophenotype and functional analysis the lineage commitment of most primitive HSC/MPP cells in patients affected by this pathology compared to healthy donors (HDs). Furthermore, in order to delineate the transcriptional networks governing hematopoiesis in Beta-thalassemia, RNAseq analysis was performed on sorted hematopoietic subpopulations from BM of Bthal patients and HDs. By droplet digital PCR on RNA purified from mesenchymal stromal cells of Bthal patients, we evaluated the expression levels of some niche factors involved in the regulation of hematopoiesis and erythropoiesis. Moreover, the protein levels in the BM plasma were analyzed by performing ELISA. Results Differences in the primitive compartment were observed with an increased proportion of multipotent progenitors in Bthal patients compared to HDs. The Subset1 compartment is actually endowed with an enhanced Ery potential. Focusing on progenitors (CD34+ CD38+) and using a new sorting scheme that efficiently resolved My, Ery, and Mk lineage fates, we quantified the new My (CD71-BAH1-/+) and Ery (CD71+ BAH1-/+) subsets and found a reduction of Ery subset in Bthal samples. We can hypothesize that the erythroid-enriched subsets are more prone to differentiate quickly due to the higher sensitivity to Epo stimuli or other bone marrow niche signals. Gene set enrichment analysis, perfomed on RNAseq data, showed that Bthal HSC/MPP presented negative enrichment of several pathways related to stemness and quiescence. Cellular processes involved in erythropoiesis were found altered in Bthal HSC. Moreover, some master erythroid transcription factors involved were overrepresented in Bthal across the hematopoietic cascade. We identified the niche factors which affect molecular pathways and the lineage commitment of Bthal HSCs. Summary/Conclusions Overall, these data indicate that Bthal HSCs are more cycling cells which egress from the quiescent state probably towards an erythroid differentiation, probably in response to a chronic BM stimulation. On the other hand,some evidences support our hypothesis of an 'erythroid branching' already present in the HSC pool, exacerbated by the pathophysiology of the disease. Disclosures No relevant conflicts of interest to declare.

A new lymphoid-primed progenitor marked by Dach1 downregulation identified with single cell multi-omics.

A classical view of blood cell development is that multipotent hematopoietic stem and progenitor cells (HSPCs) become lineage-restricted at defined stages. Lin-c-Kit+Sca-1+Flt3+ cells, termed lymphoid-primed multipotent progenitors (LMPPs), have lost megakaryocyte and erythroid potential but are heterogeneous in their fate. Here, through single-cell RNA sequencing, we identify the expression of Dach1 and associated genes in this fraction as being coexpressed with myeloid/stem genes but inversely correlated with lymphoid genes. Through generation of Dach1-GFP reporter mice, we identify a transcriptionally and functionally unique Dach1-GFP- subpopulation within LMPPs with lymphoid potential with low to negligible classic myeloid potential. We term these 'lymphoid-primed progenitors' (LPPs). These findings define an early definitive branch point of lymphoid development in hematopoiesis and a means for prospective isolation of LPPs.

Molecular docking studies, anti-Alzheimer’s disease, antidiabetic, and anti-acute myeloid leukemia potentials of narcissoside

In this research, we explored their capacity for Narcissoside antioxidant and anticholinergic, antidiabetic, and anti-acute myeloid leukaemia. Narcissoside’s antioxidant activities were elucidated by the use of various bioanalytical assays. Narcissoside’s radical scavenging activities were evaluated by DPPH • and ABTS • + scavenging activities. On the other hand, IC50 values were calculated for DPPH • , and ABTS • + scavenging, acetylcholinesterase, and α-glucosidase inhibition effects of narcissoside. IC50 values narcissoside, as 11.54 nM for AChE and 65.58 nM for α-glucosidase were calculated with % Activity-[Inhibitory] graphs. Then, ADME/T analysis of narcissoside molecule was performed to calculate the drug becoming parameters.

Augmenting emergency granulopoiesis with CpG conditioned mesenchymal stromal cells in murine neutropenic sepsis

AbstractPatients with immune deficiencies from cancers and associated treatments represent a growing population within the intensive care unit with increased risk of morbidity and mortality from sepsis. Mesenchymal stromal cells (MSCs) are an integral part of the hematopoietic niche and express toll-like receptors, making them candidate cells to sense and translate pathogenic signals into an innate immune response. In this study, we demonstrate that MSCs administered therapeutically in a murine model of radiation-associated neutropenia have dual actions to confer a survival benefit in Pseudomonas aeruginosa pneumo-sepsis that is not from improved bacterial clearance. First, MSCs augment the neutrophil response to infection, an effect that is enhanced when MSCs are preconditioned with CpG oligodeoxynucleotide, a toll-like receptor 9 agonist. Using cytometry by time of flight, we identified proliferating neutrophils (Ly6GlowKi-67+) as the main expanded cell population within the bone marrow. Further analysis revealed that CpG-MSCs expand a lineage restricted progenitor population (Lin−Sca1+C-kit+CD150−CD48+) in the bone marrow, which corresponded to a doubling in the myeloid proliferation and differentiation potential in response to infection compared with control. Despite increased neutrophils, no reduction in organ bacterial count was observed between experimental groups. However, the second effect exerted by CpG-MSCs is to attenuate organ damage, particularly in the lungs. Neutrophils obtained from irradiated mice and cocultured with CpG-MSCs had decreased neutrophil extracellular trap formation, which was associated with decreased citrullinated H3 staining in the lungs of mice given CpG-MSCs in vivo. Thus, this preclinical study provides evidence for the therapeutic potential of MSCs in neutropenic sepsis.

3022 – GENERATION OF RETINOIC ACID-DEPENDENT HEMOGENIC ENDOTHELIAL PROGENITORS FROM HUMAN PLURIPOTENT STEM CELLS

The generation of the hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) is a major goal for regenerative medicine. In the embryo, HSCs derive from a HOXA+ population known as hemogenic endothelium (HE) in a retinoic acid (RA)-dependent manner. Using hPSCs, we have previously identified a KDR+CD235a- mesodermal population that gives rise to a clonally multipotent definitive HE. However, this HE lacks HSC-like capacity in the absence of exogenous transgenes, and is functionally unresponsive to RA treatment. Thus, the specification of a RA-dependent hematopoietic program from hPSCs has remained elusive. Through single cell RNAseq analyses, we identified that two distinct KDR+CD235a- populations exist prior to HE specification, distinguishable by CXCR4 expression. Interestingly, CYP26A1, a RA degrading enzyme, was expressed in KDR+CD235a-CXCR4- mesoderm, and this was the only mesodermal population harboring definitive hematopoietic potential. In sharp contrast, KDR+CD235a-CXCR4+ mesoderm expressed ALDH1A2, a key enzyme in the synthesis of RA and lacked any hematopoietic potential. However, the stage-specific application of retinoic acid signaling to this CXCR4+ mesodermal population resulted in the robust specification of CD34+ HE with multi-lineage definitive erythroid, myeloid and lymphoid hematopoietic potential. Further, in vivo correlates to these functionally-distinct mesodermal precursors could be found within a scRNA-seq dataset of gastrulating murine embryos. Additional comparison to human embryos demonstrated that CD34+ cells specified with retinol from CXCR4+ mesoderm have fetal-like HOXA expression. Collectively, this represents the first ever characterization of stage-specific RA-dependent hPSC-derived definitive hematopoiesis, and its mesodermal progenitor. This novel insight into human hematopoietic development will ultimately provide the basis for the de novo specification of HSCs.

1018 – KLF6 MEDIATES AGE-RELATED DYSFUNCTION IN HUMAN HEMATOPOIETIC STEM AND PROGENITOR CELLS.

With aging there is a gradual decline in normal HSC function, which is accompanied by an increased risk for the development of hematological malignancies. We recently reported that <i>KLF6</i>, a Krüpper-like transcription factor, is one of the top genes downregulated with aging in human Lin⁻CD34⁺CD38⁻ cells. In order to test whether loss of <i>KLF6</i> may be implicated in age-related HSC dysfunction, we performed CRISPR-Cas9-based genome editing and transcriptional activation of the <i>KLF6</i> locus in human CD34+ cells. KLF6-deficientcells displayed in vitro block in myeloid and erythroid differentiation as well as increased colony-forming capacity on methylcellulose assays. Importantly, CRISPR targeting of the nearest putative enhancer to the <i>KLF6</i> locus (-20kb), which resulted in >75% downregulation of the <i>KLF6</i> transcript, recapitulated the differentiation block and colony-forming phenotypes. RNA-seq analysis confirmed that in young CD34⁺ cells both targeting <i>KLF6</i> and its putative enhancer, results in gene expression signature enriched not only for our previously reported human aging HSC signature, but also for several leukemia-associated gene signatures. Next, we sought to determine if re-expression of KLF6 in aged CD34+ cells could reverse the aging phenotype. <i>KLF6</i> induction in these cells using a dCas9-VP64 fusion system led to a normalization of their myeloid differentiation potential in vitro, resulting in aged CD34+ cells behaving similar to their younger counterpart controls. Finally, both inactivation of the KLF6 or the -20kb enhancer resulted an aged-like in vivo hematopoiesis when engrafted into NSGS mice. This was evidenced by an increased myeloid output in peripheral blood compared to control (wks 8-14), which was accompanied by a decrease in lymphoid output. Moreover, analysis of the bone marrow composition at week 14 showed increased frequency of HSC and progenitor components. Our findings demonstrate that KLF6 is essential for normal hematopoietic function and that reactivation of KLF6 in aged HSPCs leads to an attenuation of the aging HSPC phenotype in vitro.

Single-Cell RNA-Seq Mapping of Human Thymopoiesis Reveals Lineage Specification Trajectories and a Commitment Spectrum in T Cell Development

The challenges in recapitulating invivo human Tcell development in laboratory models have posed a barrier to understanding human thymopoiesis. Here, we used single-cell RNA sequencing (sRNA-seq) to interrogate the rare CD34+ progenitor and the more differentiated CD34- fractions in the human postnatal thymus. CD34+ thymic progenitors were comprised of a spectrum of specification and commitment states characterized by multilineage priming followed by gradual Tcell commitment. The earliest progenitors in the differentiation trajectory were CD7- and expressed a stem-cell-like transcriptional profile, but had also initiated Tcell priming. Clustering analysis identified a CD34+ subpopulation primed for the plasmacytoid dendritic lineage, suggesting an intrathymic dendritic specification pathway. CD2 expression defined Tcell commitment stages where loss of B cell potential preceded that of myeloid potential. These datasets delineate gene expression profiles spanning key differentiation events in human thymopoiesis and provide a resource for the further study of human Tcell development.

Multipotent RAG1+ progenitors emerge directly from haemogenic endothelium in human pluripotent stem cell-derived haematopoietic organoids.

Defining the ontogeny of the human adaptive immune system during embryogenesis has implications for understanding childhood diseases including leukaemias and autoimmune conditions. Using RAG1:GFP human pluripotent stem cell reporter lines, we examined human T-cell genesis from pluripotent-stem-cell-derived haematopoietic organoids. Under conditions favouring T-cell development, RAG1+ cells progressively upregulated a cohort of recognized T-cell-associated genes, arresting development at the CD4+CD8+ stage. Sort and re-culture experiments showed that early RAG1+ cells also possessed B-cell, myeloid and erythroid potential. Flow cytometry and single-cell-RNA-sequencing data showed that early RAG1+ cells co-expressed the endothelial/haematopoietic progenitor markers CD34, VECAD and CD90, whereas imaging studies identified RAG1+ cells within CD31+ endothelial structures that co-expressed SOX17+ or the endothelial marker CAV1. Collectively, these observations provide evidence for a wave of human T-cell development that originates directly from haemogenic endothelium via a RAG1+ intermediate with multilineage potential.

Loss of KLF6 Recapitulates Molecular and Functional Changes Associated with Aging in Human Hematopoietic Stem and Progenitor Cells

With aging there is a gradual decline in normal HSC function, which is accompanied by an increased risk for the development of hematological malignancies. While a lot of work has been done in mice to understand this functional decline, less is known about human HSC biology with aging. We recently reported that KLF6, a Krüpper-like transcription factor, is one of the top genes downregulated with aging in human Lin-CD34+CD38- cells, and that this downregulation correlates with loss of H3K27ac at several KLF6 upstream putative enhancer regions. Therefore, we hypothesized that age-acquired epigenetic deregulation at the KLF6 locus resulting in loss of expression may be implicated in age-related HSC dysfunction and increased risk of malignant transformation. In order to test this, we isolated CD34+ hematopoietic stem and progenitor cells (HSPCs) from healthy individuals and performed CRISPR-Cas9-based genome editing and transcriptional activation of the KLF6 locus. KLF6-deficient cells were evaluated in terms of their function by colony-forming potential, in vitro differentiation, and hematopoietic reconstitution in immunocompromised mice. Myeloid and erythroid in vitro differentiation assays in liquid culture revealed that KLF6 knock-out (KO) in healthy, young HSPC results in persistent CD34+ expression (n=5, p&amp;lt;0.01) and strong reduction of the CD11b, CD15 and CD33 myeloid markers (n=5, p&amp;lt;0.05 for all markers), and the CD71 and CD235a erythroid markers (n=5, p&amp;lt;0.05 for both markers), indicating that loss of KLF6 leads to a block in the differentiation programs of HSPCs. Moreover, KLF6 KO cells plated on methylcellulose exhibited an increase in the total number of colonies (n=5, p=0.02) with a strong increase in the formation of granulocyte-monocyte colonies (n=5, p=0.014) as well as an increase in erythroid burst-forming units (n=5, p=0.034), indicating increased progenitor potential in these cells. Importantly, CRISPR targeting of the nearest putative enhancer to the KLF6 locus (-25kb), which resulted in &amp;gt;75% downregulation of the KLF6 transcript, recapitulated the differentiation block and colony-forming phenotypes. Next, in order to define if KLF6 genomic inactivation results in an expression profile similar to that observed in healthy aged donors, we performed RNA-seq analysis. This confirmed that in young CD34+ cells both targeting KLF6 and its putative enhancer, results in gene expression signature enriched not only for our previously reported human aging HSC signature (GSEA NES=1.25 &amp; FDR&amp;lt;0.01 for genes up with aging and NES=-1.17 and FDR&amp;lt;0.1 for genes down with aging), but also for several leukemia-associated gene signatures. Next, we sought to determine if re-expression of KLF6 in aged CD34+ cells could reverse the aging phenotype. KLF6 induction in these cells using a dCas9-VP64 fusion system led to a decrease in their myeloid differentiation potential, compared to unmanipulated and non-targeting control (NTC). This decrease in the in vitro myeloid output brought aged CD34+ cells to a behavior closer to their younger counterpart controls. Finally, to determine the impact that KLF6 inactivation may have in the hematopoietic system in vivo, we engrafted KLF6 knock-out (KO) (n=7) and NTC (n=7) cells into immunodeficient NSGS recipients. Analysis of KLF6 KO recipients revealed an increased myeloid output in peripheral blood compared to NTC (weeks 8 to 14), which was accompanied by a decrease in lymphoid output. Moreover, analysis of the bone marrow composition at week 14 showed increased frequency of CD34+CD38-CD45RA-CD90+CD49f+ HSC and CD34+CD38+ progenitor components (p=0.02, and p=0.04, respectively). In summary, our findings demonstrate that KLF6 is essential for normal in vitro and in vivo hematopoietic function, and that loss of this transcription factor recapitulates both the expression profile of aged HSC as well as several of the functional characteristics of aged hematopoiesis. These observations were further validated by the reactivation of KLF6 in aged HSPCs, which resulted in an attenuation of the aging HSPC phenotype in vitro. Finally, changes in gene expression in KLF6 KO cells indicate that it may be essential for regulation of gene expression programs involved in malignant transformation, such that age-related loss of this transcription factor may contribute to predisposition to myeloid malignancies. Disclosures No relevant conflicts of interest to declare.

Adenylate Kinase 2 Links Energy Metabolism and Cell Fate in Hematopoietic Stem and Progenitor Cells

While hematopoietic stem and progenitor cells (HSPCs) were thought to rely mainly on glycolysis for energy supply, emerging evidence suggests that defects in mitochondrial functions can impact HSPC development with respect to self-renewal, differentiation and aging. The exact mechanisms underlying metabolic reprogramming and cell fate decisions during human hematopoiesis, however, remain elusive. Biallelic mutations in the mitochondrial enzyme adenylate kinase 2 (AK2), cause reticular dysgenesis (RD), one of the most profound forms of severe combined immunodeficiency (SCID). AK2 catalyzes the interconversion between adenine nucleotides and thereby controls the availability of ADP for oxidative phosphorylation. Clinically, RD patients not only present with profound lymphopenia, typical for classic SCID, but also suffer from severe congenital neutropenia. The developmental arrest across the T, NK and granulocytic lineages suggests that AK2 deficiency causes a metabolic defect with global impact on hematopoiesis. Our prior work in induced pluripotent stem cells (iPSCs) from RD patients has shown that maturation-arrested iPSC-derived HSPCs exhibit increased oxidative stress and an energy-depleted adenine nucleotide profile, suggesting that AK2-regulated mitochondrial bioenergetics play an integral role in HSPC differentiation. Therefore, RD serves as an excellent model to study the impact of mitochondrial metabolism during human HSPC development. Methods: Since iPSCs do not recapitulate definitive hematopoiesis, we developed an AK2 biallelic knock-out model in primary human HSPCs using CRISPR/Cas9 gene editing. Employing a homologous recombination-mediated dual color reporter strategy, we were able to select for HSPCs with biallelic AK2 knock-out. HSPCs edited at the safe harbor AAVS1 site were used as a control. FACS purified AK2-/- and AAVS1-/- HSPCs were in vitro differentiated along the granulocytic lineage, and cells at various differentiation stages were sorted for RNA-seq and metabolomics analysis. Results: We analyzed the myeloid differentiation potential of AK2-/- HSPCs in vitro. Compared to AAVS1-/- controls, AK2-/- HSPCs displayed a severely decreased colony forming potential of both myeloid and erythroid lineages. In addition, AK2-/- HSPCs showed a granulocytic maturation arrest at the HLA-DR-, CD117+ promyelocyte stage, consistent with the characteristic phenotype observed in RD patients. We then performed RNA-seq studies on in vitro differentiated promyelocyte and neutrophil subpopulations derived from AK2-/- and control HSPCs. The RNA-seq analysis showed differential gene expression in glutathione metabolism and IL-10 signaling pathways, suggesting an increase in oxidative stress and inflammation, respectively, caused by AK2 deficiency. In addition, genes implicated in antimicrobial function and granule synthesis were downregulated in AK2-/- neutrophils, suggesting a functional defect. Liquid chromatography-mass spectrometry (LC-MS/MS) studies to delineate differences in metabolite profile conferred by AK2 deficiency at different stages of HSPC development are currently in progress. Conclusions: We have established the first cell-traceable biallelic AK2 CRISPR knock-out model in primary human HSPCs that recapitulates the myeloid phenotype of RD patients. This model allows us to profile AK2 knock-out cells at different developmental stages. AK2-/- granulocyte precursors showed a transcriptional signature suggestive of worsening oxidative stress, inflammation and defective effector cell functions during maturation. To understand the mechanistic underpinnings for these observations we are now using a global metabolomics approach to profile the changes in energy metabolites that occur during development in AK2-deficient and control HSPC subpopulations. Understanding how metabolism governs differentiation and self-renewal of human HSPCs has important translational implications to improve hematopoietic stem cell products and transplantation outcomes. Disclosures Morrison: Frequency Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; OncoMed Pharmaceuticals: Equity Ownership; GI Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Kolon Gene Therapeutics: Consultancy; Protein Fluidics: Other: Stock Options.

Potently Cytotoxic Natural Killer Cell Potential Initially Emerges from Erythro-Myeloid Progenitors during Mammalian Development

Natural killer (NK) cells are innate immune cells that target and kill virally infected and malignant cells, making them an attractive target for adoptive immunotherapies. An alternative to donor-derived NK cells is the use of human pluripotent stem cell (hPSC)-derived NK cells, as a renewable "off the shelf" product. Previous studies have identified hPSC-derived NK cells as potently cytotoxic, compared to donor-derived NK cells. As the differentiation of hPSCs mimics early embryonic development, this raises the possibility that hPSC-derived NK cells are ontogenically distinct from adult NK cells. NK cells are present during embryonic hematopoiesis, but their ontogenic origins are poorly understood. NK cells are thought to arise from a common lymphoid progenitor (CLP), lying downstream of hematopoietic stem cells (HSCs), but evidence exists that NK cells may arise from HSC-independent progenitors as NK cells are found in the early murine fetal liver, and NK cell progenitors are found in the early human yolk sac (YS). In this study, we investigated the emergence of NK cells during murine and human embryonic hematopoietic development. During murine embryogenesis, overlapping HSC-independent waves of hematopoietic progenitors occur in the YS that give rise to hematopoietic cells prior to HSC emergence at E10.5. The "primitive" wave occurs at E7.5, followed by an "erythro-myeloid progenitor" (EMP) wave at E8.5. To study NK cell potential during murine YS hematopoiesis, we cultured total YS and sorted hematopoietic progenitors under NK cell promoting conditions. Strikingly, we found that the YS contains NK cell potential. Further, sorted E8.5 kit+CD41+CD16/32+ EMP progenitors, but not primitive hematopoietic progenitors, contain robust NK cell potential. EMP-derived NK (EMP-NK) cells were larger and more granular than adult CLP-derived NK cells. Additionally, NK cells from the E15.5 fetal liver were larger and more granular than NK cells from the adult spleen. Both EMP-NK cells and E15.5 fetal liver NK cells had a more robust degranulation response than their HSC-derived counterparts. Together, these data support the concept that EMP in the YS serve as an initial source of physiologically relevant, functional embryonic NK cells that are phenotypically and functionally distinct from adult NK cells. As hPSC-derived NK cells were described as potently cytotoxic, and we observed that murine HSC-independent NK cells robustly degranulate, we next asked whether NK cell development from hPSCs recapitulates that found in the murine embryo. We have demonstrated previously, using a stage-specific WNT signal manipulation approach that specifies ontogenically distinct hematopoietic progenitors, that hPSC-derived NK cell progenitors can be obtained from two distinct progenitors in vitro. In this study, we sought to better understand the development and function of these two NK cell populations. Stage-specific WNT inhibition (WNTi) during hPSC mesodermal patterning yielded extra-embryonic-like HOXA-/low CD34+ populations that possessed erythroid, myeloid and NK cell potential, but lacked T cell potential. The CD56+ NK cells in these cultures co-emerged with CD15+ granulocytes, indicating that these NK cells may arise from a committed myeloid progenitor. In contrast, HOXA+ CD34+ cells, obtained in a WNT-dependent (WNTd) manner, harbored erythro-myelo-lymphoid multi-lineage potential, including NK cell potential. Phenotypically, WNTi-NK cells were larger, more granular and more mature, compared to WNTd-NK and cord blood (CB)-derived NK cells, reminiscent of murine EMP-NK cells. Further, following multiple stimulation assays, WNTi-NK and WNTd-NK cells had different effector biases. WNTi-NK cells are biased for potent cytotoxic degranulation and exhibited superior cell killing in an ADCC assay. In contrast, WNTd-NK and CB-NK had an attenuated degranulation response, but robustly produced inflammatory cytokines. Finally, RNA-seq analysis demonstrated that WNTd-NK cells were most similar to CB-NK cells. Collectively, these studies identify for the first time that the murine EMP harbor NK cell potential, and these NK cells are functionally unique. These observations raise new questions regarding which ontogenic origin of NK cells should be used in future hPSC-derived adoptive immunotherapy strategies. Disclosures Fehniger: Cyto-Sen Therapeutics: Consultancy; Horizon Pharma PLC: Other: Consultancy (Spouse). Palis:Rubius Therapeutics: Consultancy.