Gene expression profile of DLL4+ and DLL4- Hemato-Endothelial Progenitors (HEPs) subpopulations
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ABSTRACT: In hESCs, expression of the Notch ligand DLL4 parallels the emergence of bipotent hematoendothelial progenitors (HEPs) and promotes their hematopoietic differentiation. During differentiation, DLL4 is only expressed in a subpopulation of HEPs. To study the developmental fate of the two subpopulations of HEPs identified by DLL4 expression, we FACS-isolated DLL4high and DLL4low/- HEPs at day 15 of differentiation and performed gene expression analysis using microarrays
Project description:In hESCs, expression of the Notch ligand DLL4 parallels the emergence of bipotent hematoendothelial progenitors (HEPs) and promotes their hematopoietic differentiation. During differentiation, DLL4 is only expressed in a subpopulation of HEPs. To study the developmental fate of the two subpopulations of HEPs identified by DLL4 expression, we FACS-isolated DLL4high and DLL4low/- HEPs at day 15 of differentiation and performed gene expression analysis using microarrays 10^5 DLL4high and DLL4low/- HEPs purified by FACS from H9 and AND1 hEBs at day 15 of hematopoietic differentiation were used for gene expression analysis using Whole Human Genome Oligo Microarray chips (Agilent Technologies).
Project description:During hematopoietic differentiation of hESCs, HOXA9 expression parallels hematopoietic development but is restricted to the hemogenic precursors (HEP, CD31+CD34+CD45-), and diminishes as HEPs differentiate into blood cells (CD45+). Enforced expression of Hoxa9 in hESCs robustly promoted differentiation into primitive (CD34+CD45+) and total (CD45+) blood cells with higher clonogenic (CFU) potential. To identify patterns of gene expression that could explain at the molecular level the developmental impact of HOXA9 in hematopoietic commitment of hESCs, we performed gene expression profile in FACS-purified EV- and HOXA9-HEPs. 10^5 HoxA9 over expressing and EV control HEPs purified by FACS from H9 and AND1 hEBs at day 15 of hematopoietic differentiation were used for gene expression analysis using Whole Human Genome Oligo Microarray chips (Agilent Technologies).
Project description:During hematopoietic differentiation of hESCs, HOXA9 expression parallels hematopoietic development but is restricted to the hemogenic precursors (HEP, CD31+CD34+CD45-), and diminishes as HEPs differentiate into blood cells (CD45+). Enforced expression of Hoxa9 in hESCs robustly promoted differentiation into primitive (CD34+CD45+) and total (CD45+) blood cells with higher clonogenic (CFU) potential. To identify patterns of gene expression that could explain at the molecular level the developmental impact of HOXA9 in hematopoietic commitment of hESCs, we performed gene expression profile in FACS-purified EV- and HOXA9-HEPs.
Project description:Coronaries are essential for myocardial growth and heart function. Notch is crucial for mouse embryonic angiogenesis, but its role in coronary development remains uncertain. We show Jag1, Dll4 and activated Notch1 receptor expression in sinus venosus (SV) endocardium. Endocardial Jag1 removal blocks SV capillary sprouting, while Dll4 inactivation stimulates excessive capillary growth, suggesting that ligand antagonism regulates coronary primary plexus formation. Later endothelial ligand removal, or forced expression of Dll4 or the glycosyltransferase MFng, blocks coronary plexus remodeling, arterial differentiation, and perivascular cell maturation. Endocardial deletion of Efnb2 phenocopies the coronary arterial defects of Notch mutants. Angiogenic rescue experiments in ventricular explants, or in primary human endothelial cells, indicate that EphrinB2 is a critical effector of antagonistic Dll4 and Jag1 functions in arterial morphogenesis. Thus, coronary arterial precursors are specified in the SV prior to primary coronary plexus formation and subsequent arterial differentiation depends on a Dll4-Jag1-EphrinB2 signaling cascade.
Project description:The purpose of this experiment was to compare the transcriptome of FoP-Heps, with undifferentiated hiPSCs, HLCs generated by direct differentiation, and primary samples (adult and fetal). FoP-Heps where generated in vitro from hESCs by forward programming (FoP) using a combination of 4 transcription factors (HNF1A, FOXA3, HNF6, RORc). Human fetal liver samples where obtained from first trimester embryos.
Project description:Molecular mechanism underline immune cell type population shift upon anti-DLL4 treatment C57BL/6 mice were injected with anti-DLL4, or an isotype control antibody as controls. Two weeks later mice were sacrificed, and thymi was harvested from 4 anti-DLL4 and 4 control animals. Total thymocytes, DN cells (CD4-CD8-) and DN1(CD4-CD8- CD44+CD25-) cells were isolated. Samples included in this data set are: 3 Thymocytes-anti-DLL4; 3 Thymocytes-isotype control; 3 DN1-anti-DLL4; 2 DN1-isotype controls; 3 DN-anti-DLL4; 3 DN-isotyoe control.
Project description:The t(4;11)(q21;q23) translocation is associated with high-risk infant pro-B-cell acute lymphoblastic leukemia (B-ALL) and arises prenatally during embryonic/fetal hematopoiesis. The developmental/pathogenic contribution of the t(4;11)-resulting MLL-AF4 (MA4) and AF4-MLL (A4M) fusions remains enigmatic; MA4 is always expressed in t(4;11)+B-ALL patients, but the reciprocal fusion A4M is expressed in only 50% of patients. Because prenatal leukemogenesis manifests as impaired early hematopoietic differentiation, we took advantage of well-established human embryonic stem cell-based hematopoietic differentiation models to study whether the A4M fusion co-operates with MA4 during early human hematopoietic development. Co-expression of A4M and MA4 strongly promoted the emergence of hemato-endothelial precursors (HEPs), both endothelial-primed and hemogenic-primed. Double fusion-expressing HEPs specified into significantly higher numbers of both hematopoietic and endothelial-committed cells, irrespective of the differentiation protocol used and without hijacking survival/proliferation. Functional analysis of differentially expressed genes and differentially enriched H3K79me3 genomic regions by RNA-seq and H3K79me3 ChIP-seq, respectively, confirmed a hematopoietic/endothelial cell differentiation signature in double fusion-expressing HEPs. Importantly, ChIP-seq analysis revealed a significant enrichment of H3K79 methylated regions specifically associated with HOX-A cluster genes in double fusion expressing differentiating hematopoietic cells. Overall these results establish a functional and molecular cooperation between MA4 and A4M fusions during human hematopoietic development.
Project description:Transcriptional profiling of retinas extracted from mouse pups 24 hours after IVT injection (at P8) of 1 microgram VEGFA, 4 micrograms Dll4-Fc, or 4 microgram hFc alone. Goal was to determine the mechanism by which inhibiting Dll4/Notch pathway is vasoprotective during retinal development. Three-condition experiment: VEGF-A treated vs hFc, Dll4-Fc vs hFc. Four replicates each for Dll4-Fc and hFc, five for VEGF-A.