Methods and compositions for generating hematopoietic cells

#Any and all applications for which a foreign or domestic priority claim is identified in the Application Data Sheet as filed with the present application are hereby incorporated by reference under 37 CFR 1.57.

#The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a 7 kb file entitled EVHO001.004WO.TXT, created on Oct. 19, 2016, providing in electronic format subject matter which was present in the disclosure as originally filed. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

#Part of the present invention was disclosed by the inventors in Saxena et al., “Cyclic AMP Signaling through Epac Axis Modulated Human Hemogenic Endothelium and Enhances Hematopoietic Cell Generation,” Stem Cell Reports 6(5):692-703 (10 May 2016, online date 21 Apr. 2016).

#FIG. 1. A) Scheme of a transversal section of the AGM region of a human fetus. AGM—aorta-gonad-mesonephros; DA—dorsal aorta; UG—Urogenital Ridges; B) Representative micrographs of cellular growth following culture of AGM explants dissected from human fetuses: i: outgrowth with endothelial morphology; ii: outgrowth of stromal morphology; iii: outgrowth with neurosphere morphology from the DA region of the 42 days-old fetus; iv: outgrowth with neurosphere morphology from the DA region of the 58 days-old fetus. i and ii were taken from explants of the UG region of the 58 days-old fetus; C) Fluorescence microscopy pictures of the neurosphere-like structures stained for MAP2 and GFAP; D) Time-line of hPS-to-blood induction experiment, with neural-crest-conditioned media added for the last 4 days of the protocol (from time of emergence to endpoint) E) Gating strategy for the FACS analysis at the endpoint of the hES-to-blood differentiation protocol; F) Fold increase in percentage of the different hematopoietic subsets outlined in E, conditioned media versus control settings (n=1).

#FIG. 2. Norepinephrine added to hPS-to-blood at the time of emergence increases frequencies of HSC phenotype, CFU potential, and endothelial percursors. A) Time-line of the hPS-to-blood differentiation experiment, with Norepinephrine added at two different concentrations for the last 4 days of the protocol (from time of emergence to endpoint); B) Box and whisker plot of the percentage of cells, in the total viable population, with an HSC-like immunophenotype (n=7 to 12); C) Box and whisker plot of the fold difference (NE300 vs controls) of the HSC-like immunophenotype in the total viable population showing separately the different human ES (Hues2, Hues3, H1) and iPS (RB9-CB1) cell lines used in the experiments; D) Box and whisker plot of the percentage of CD43/45+ hematopoietic cells in the total viable population (n=7 to 12); E) Box and whisker plot of the percentage of CD34+ cells in the total CD43/45+ blood population (n=7 to 12) F) Box and whisker plot of the percentage of CD34+CD38+CD90+CD45RA− cells in the total CD43/45+ blood population (n=7 to 12); G) Box and whisker plot of the CFU counts per 20 000 plated cells in methylcellulose. (n=8); H) Bar graph of the average colony type distributions registered in the experiments shown in G. Error bars correspond to standard deviation (n=8); I) Gating strategy for assessing the frequency of CD34high CD90high endothelial precursor of definitive hematopoiesis in the total viable population; J) Box and whisker plot of the percentage of CD34high CD90high endothelial precursor in the total viable population (n=7 to 12); K) Box and whisker plot of the fold difference (NE300 vs controls) of the CD34-high CD90-high endothelial precursors in the total viable population showing separately the different human ES and iPS cell lines used in the experiments. Each data-point represents an independent experiment. * p<0.05 ** p<0.01; *** p<0.001; p values calculated with two-tailed paired Student t-tests

#FIG. 3: Cyclic AMP Induction Increases HSC-like Frequency During hPSC-to-Hematopoietic Differentiation. A) Conditions and timeline applied to differentiate hPSCs towards mesoderm commitment and hematopoietic differentiation; B) Flow cytometric analysis of hematopoietic cells at day 14 of differentiation. Representative flow cytometry plots (biexponential axis) of cells cultured in control medium (Mesototal), and cells treated with Forskolin, IBMX, or Forskolin+IBMX are shown; C) Percent of the hematopoietic surface phenotypes indicated in (B). Data represents mean±S.E.M. of three independent experiments. Statistical analysis was performed using the t-test. Significance is shown compared to the control setting. *, p<0.05, ***, p<0.001, n.s., not significant; D) Frequency of the putative HSC-like cells (in viable fraction) is shown. Data represents mean±S.E.M. of three independent experiments. Statistical analysis was performed using the t-test. Significance is shown compared to the control setting. *, p<0.05, n.s., not significant; E) Total CFU numbers after 12-day colony-forming unit (CFU) assay of differentiated hematopoietic cells treated with Forskolin, IBMX and Forskolin+IBMX. The CFU distribution of 3 independent experiments is shown as total number of colonies. CFU-G (granulocyte), CFU-M (macrophage), CFU-E (erythroid), CFU-GM (granulocyte/macrophage). Statistical analysis was performed using the t-test. Significance is shown compared to the control setting. *, p<0.05, **, p<0.01, ***, p<0.001; F) Cell numbers obtained per well (per 1×104 seeded cells) after CFU assay are shown. Data represents mean±S.E.M. of three independent experiments. Statistical analysis was performed using the t-test. Significance is shown compared to the control setting. **, p<0.01, ***, p<0.001, n.s., not significant. See also FIGS. 5 and 6.

#Activation of specific G-protein coupled receptors by catecholamines, as well as neurotransmitters, growth factors and hormones activates the cyclic AMP (cAMP) signaling pathway, and is followed by cell-type dependent responses mediated by cAMP effectors Protein Kinase A (PKA) and Exchange proteins activated by cAMP (Epac). Epac have been shown to modulate endothelial cell remodeling, enhances endothelial cell adhesion, and regulates the integrity of endothelial cell junctions, however, the role of Epac in the endothelial-to-hematopoietic transition (EHT) was previously unknown. While norepinephrine signaling is clearly involved in hematopoietic stem cell function, the mechanism of catecholamine signaling, through its second messenger, cyclic AMP (3′-5′-cyclic adenosine mono phosphate), and its downstream signaling pathways had not previously been evaluated in the context of hematopoietic development. Moreover, the importance of cAMP in in vitro systems for the generation of de novo blood e.g., pluripotent stem cell differentiation, and the direct preprogramming of somatic cells to hematopoietic cells or hematopoietic precursors had previously not been demonstrated.

#Cyclic AMP-mediated regulation of adult hematopoiesis has been suggested in studies showing that: cAMP increases C—X—C chemokine receptor type 4 (CXCR4) expression and motility of hematopoietic progenitors, HSCs from Gsα-deficient mice do not engraft, and Gsα-deficient osteocytes alter the BM niche, leading to defective hematopoiesis. In human hematopoietic cells, Prostaglandin E2 (PGE2)-mediated cAMP activation enhances human cord blood engraftment. Recently, cAMP has been shown to regulate hematopoietic emergence and homing in studies where cAMP was upregulated by adenosine in zebrafish and mouse, by PGE2 in zebrafish and mouse, and by shear stress in murine AGM. However, the role and mechanism of cAMP signaling, as mediated through PKA and Epac, in regulating human developmental hematopoiesis had not been adequately studied, and no study had previously been performed on the role of cAMP in the human hematopoietic developmental context.

#Human pluripotent stem cells (hPSC), including human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC), can be used in the in vitro generation of de novo blood for therapeutic purposes. We have shown that hPSC-derived HSC-like cells possess lymphoid and myeloid differentiation ability, a key feature of HSCs. Recent studies have functionally demonstrated an endothelial precursor to blood (hemogenic endothelium) from hPSC differentiation cultures, further establishing hPSCs as a suitable model to study human hematopoietic cell development. However, the signals regulating EHT in human hematopoiesis remain undefined. Additionally, for functional transplantable HSCs, it is vital to reduce reactive oxygen species (ROS) and oxidative stress, as reduced ROS is crucial for HSC functionality.