Identification of novel regulators of developmental hematopoiesis using Endoglin regulatory elements as molecular probes.

Enhancers are the primary determinants of cell identity, and specific promoter/enhancer combinations of Endoglin (ENG) have been shown to target blood and endothelium in the embryo. Here, we generated a series of embryonic stem cell lines, each targeted with reporter constructs driven by specific promoter/enhancer combinations of ENG, to evaluate their discriminative potential and value as molecular probes of the corresponding transcriptome. The Eng promoter (P) in combination with the -8/+7/+9-kb enhancers, targeted cells in FLK1 mesoderm that were enriched for blast colony forming potential, whereas the P/-8-kb enhancer targeted TIE2+/c-KIT+/CD41- endothelial cells that were enriched for hematopoietic potential. These fractions were isolated using reporter expression and their transcriptomes profiled by RNA-seq. There was high concordance between our signatures and those from embryos with defects at corresponding stages of hematopoiesis. Of the 6 genes that were upregulated in both hemogenic mesoderm and hemogenic endothelial fractions targeted by the reporters, LRP2, a multiligand receptor, was the only gene that had not previously been associated with hematopoiesis. We show that LRP2 is indeed involved in definitive hematopoiesis and by doing so validate the use of reporter gene-coupled enhancers as probes to gain insights into transcriptional changes that facilitate cell fate transitions.National Health and Medical Research Council of Australia, Australian Research Council, Dr Tom Bee Stem Cell Research Fund, Cancer Research UK, Biotechnology and Biological Sciences Research Council, Leukaemia and Lymphoma Research, The Leukaemia and Lymphoma Society, core support grants by the Wellcome Trust to the Cambridge Institute for Medical Research and Wellcome Trust - MRC Cambridge Stem Cell Institute (Grant IDs: R01 HL04880, P015PO1HL32262-32, 5P30 DK49216, 5R01 DK53298, 5U01 HL10001-05, R24 DK092760)This is the author accepted manuscript. The final version is available from the American Society of Hematology via http://dx.doi.org/10.1182/blood-2016-02-69787

The JAK-STAT signaling pathway is differentially activated in CALR-positive compared with JAK2V617F-positive ET patients.

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Combining single-cell tracking and omics improves blood stem cell fate regulator identification

Molecular programs initiating cell fate divergence (CFD) are difficult to identify. Current approaches usually compare cells long after CFD initiation, therefore missing molecular changes at its start. Ideally, single cells that differ in their CFD molecular program but are otherwise identical are compared early in CFD. This is possible in diverging sister cells, which were identical until their mother's division and thus differ mainly in CFD properties. In asymmetrically dividing cells, divergent daughter fates are prospectively committed during division, and diverging sisters can thus be identified at the start of CFD. Using asymmetrically dividing blood stem cells, we developed a pipeline (ie, trackSeq) for imaging, tracking, isolating, and transcriptome sequencing of single cells. Their identities, kinship, and histories are maintained throughout, massively improving molecular noise filtering and candidate identification. In addition to many identified blood stem CFD regulators, we offer here this pipeline for use in CFDs other than asymmetric division.ISSN:0006-4971ISSN:1528-002

Notch ligand Dll4 impairs cell recruitment to aortic clusters and limits blood stem cell generation

International audienceHematopoietic stem cells (HSCs) develop from the hemogenic endothelium in cluster structures that protrude into the embryonic aortic lumen. Although much is known about the molecular characteristics of the developing hematopoietic cells, we lack a complete understanding of their origin and the three-dimensional organization of the niche. Here, we use advanced live imaging techniques of organotypic slice cultures, clonal analysis, and mathematical modeling to show the two-step process of intra-aortic hematopoietic cluster (IACH) formation. First, a hemogenic progenitor buds up from the endothelium and undergoes division forming the monoclonal core of the IAHC. Next, surrounding hemogenic cells are recruited into the IAHC, increasing their size and heterogeneity. We identified the Notch ligand Dll4 as a negative regulator of the recruitment phase of IAHC. Blocking of Dll4 promotes the entrance of new hemogenic Gfi1+ cells into the IAHC and increases the number of cells that acquire HSC activity. Mathematical modeling based on our data provides estimation of the cluster lifetime and the average recruitment time of hemogenic cells to the cluster under physiologic and Dll4-inhibited conditions

Notch ligand Dll4 impairs cell recruitment to aortic clusters and limits blood stem cell generation.

Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium in cluster structures that protrude into the embryonic aortic lumen. Although much is known about the molecular characteristics of the developing hematopoietic cells, we lack a complete understanding of their origin and the three-dimensional organization of the niche. Here, we use advanced live imaging techniques of organotypic slice cultures, clonal analysis, and mathematical modeling to show the two-step process of intra-aortic hematopoietic cluster (IACH) formation. First, a hemogenic progenitor buds up from the endothelium and undergoes division forming the monoclonal core of the IAHC. Next, surrounding hemogenic cells are recruited into the IAHC, increasing their size and heterogeneity. We identified the Notch ligand Dll4 as a negative regulator of the recruitment phase of IAHC. Blocking of Dll4 promotes the entrance of new hemogenic Gfi1+ cells into the IAHC and increases the number of cells that acquire HSC activity. Mathematical modeling based on our data provides estimation of the cluster lifetime and the average recruitment time of hemogenic cells to the cluster under physiologic and Dll4-inhibited conditions.Wellcome Trust, MR

Adaptation to<i>ex vivo</i>culture drives human haematopoietic stem cell loss of repopulation capacity in a cell cycle independent manner

Loss of long-term haematopoietic stem cell function (LT-HSC) hampers the success of ex vivo HSC gene therapy and expansion procedures, but the kinetics and the mechanisms by which this occurs remain incompletely characterized. Here through time-resolved scRNA-Seq, matched in vivo functional analysis and the use of a reversible in vitro system of early G 1 arrest, we define the sequence of transcriptional and functional events occurring during the first ex vivo division of human LT-HSCs. We demonstrate that contrary to current assumptions, loss of long-term repopulation capacity during culture is independent of cell cycle progression. Instead it is a rapid event that follows an early period of adaptation to culture, characterised by transient gene expression dynamics and constrained global variability in gene expression. Cell cycle progression however contributes to the establishment of differentiation programmes in culture. Our data have important implications for improving HSC gene therapy and expansion protocols

The human GFI136N variant induces epigenetic changes at the Hoxa9 locus and accelerates K-RAS driven myeloproliferative disorder in mice

Item does not contain fulltextThe coding single nucleotide polymorphism GFI136N in the human gene growth factor independence 1 (GFI1) is present in 3%-7% of whites and increases the risk for acute myeloid leukemia (AML) by 60%. We show here that GFI136N, in contrast to GFI136S, lacks the ability to bind to the Gfi1 target gene that encodes the leukemia-associated transcription factor Hoxa9 and fails to initiate histone modifications that regulate HoxA9 expression. Consistent with this, AML patients heterozygous for the GFI136N variant show increased HOXA9 expression compared with normal controls. Using ChipSeq, we demonstrate that GFI136N specific epigenetic changes are also present in other genes involved in the development of AML. Moreover, granulomonocytic progenitors, a bone marrow subset from which AML can arise in humans and mice, show a proliferative expansion in the presence of the GFI136N variant. In addition, granulomonocytic progenitors carrying the GFI136N variant allele have altered gene expression patterns and differ in their ability to grow after transplantation. Finally, GFI136N can accelerate a K-RAS driven fatal myeloproliferative disease in mice. Our data suggest that the presence of a GFI136N variant allele induces a preleukemic state in myeloid precursors by deregulating the expression of Hoxa9 and other AML-related genes