VEGF, FGF2, and BMP4 regulate transitions of mesoderm to endothelium and blood cells in a human model of yolk sac hematopoiesis

Abstract

•Methylcellulose blast colony assay mimics yolk sac hematopoiesis.•FGF2 and VEGF are critical signals for the progression of mesoderm into endothelium.•BMP4 is required for subsequent generation of blood from hemogenic endothelium.•Blocking BMP4 signaling phenocopies aspects of RUNX1 gene deletion. Exogenous growth factors play an important role in mediating hematopoietic differentiation of human pluripotent stem cells. We explored the role of different factors in early human blood cell production using blast colony formation in methylcellulose as a surrogate assay for yolk sac hematopoiesis. A reporter cell line that read out endothelial (SOX17+) and hematopoietic (RUNX1C+) progenitors facilitated the identification of basic fibroblast growth and vascular endothelial growth factor as critical signals for the progression of mesoderm into endothelium. Bone morphogenetic protein 4 was needed for the subsequent generation of blood from hemogenic endothelium, and this was antagonized by Activin A or high concentrations of the WNT agonist CHIR-99021. Manipulations of the Hedgehog pathway or inhibition of Notch signaling reduced blast colony frequency but did not perturb cell differentiation. These data help to define distinct roles for prerequisite growth factors that commit mesoderm to hemogenic endothelium and subsequently allocate cells to blood lineages. Exogenous growth factors play an important role in mediating hematopoietic differentiation of human pluripotent stem cells. We explored the role of different factors in early human blood cell production using blast colony formation in methylcellulose as a surrogate assay for yolk sac hematopoiesis. A reporter cell line that read out endothelial (SOX17+) and hematopoietic (RUNX1C+) progenitors facilitated the identification of basic fibroblast growth and vascular endothelial growth factor as critical signals for the progression of mesoderm into endothelium. Bone morphogenetic protein 4 was needed for the subsequent generation of blood from hemogenic endothelium, and this was antagonized by Activin A or high concentrations of the WNT agonist CHIR-99021. Manipulations of the Hedgehog pathway or inhibition of Notch signaling reduced blast colony frequency but did not perturb cell differentiation. These data help to define distinct roles for prerequisite growth factors that commit mesoderm to hemogenic endothelium and subsequently allocate cells to blood lineages. The production of blood cells is regulated by multiple signaling pathways. The differentiation of human pluripotent stem cells is an experimentally tractable model allowing analysis of factors influencing early human development in vitro. A number of laboratories have published protocols that direct human pluripotent stem cell differentiation to early hematopoietic cells using the blast colony formation as a surrogate assay for extra-embryonic hematopoiesis from the yolk sac [1Kennedy M D'Souza SL Lynch-Kattman M Schwantz S Keller G Development of the hemangioblast defines the onset of hematopoiesis in human ES cell differentiation cultures.Blood. 2007; 109: 2679-2687Crossref PubMed Scopus (353) Google Scholar, 2Davis RP Costa M Grandela C et al.A protocol for removal of antibiotic resistance cassettes from human embryonic stem cells genetically modified by homologous recombination or transgenesis.Nat Protoc. 2008; 3: 1550-1558Crossref PubMed Scopus (36) Google Scholar, 3Yu QC Hirst CE Costa M et al.APELIN promotes hematopoiesis from human embryonic stem cells.Blood. 2012; 119: 6243-6254Crossref PubMed Scopus (42) Google Scholar, 4Bruveris FF Ng ES Leitoguinho AR et al.Human yolk sac-like haematopoiesis generates RUNX1-, GFI1- and/or GFI 1B-dependent blood and SOX17-positive endothelium.Development. 2020; 147dev193037Crossref PubMed Scopus (8) Google Scholar]. Mesodermal precursors of mammalian hematopoietic cells first arise from the posterior primitive streak [5Tam PP Behringer RR Mouse gastrulation: the formation of a mammalian body plan.Mech Dev. 1997; 68: 3-25Crossref PubMed Scopus (409) Google Scholar, 6Huber TL Kouskoff V Fehling HJ Palis J Keller G Haemangioblast commitment is initiated in the primitive streak of the mouse embryo.Nature. 2004; 432: 625-630Crossref PubMed Scopus (528) Google Scholar, 7Martyn I Siggia ED Brivanlou AH Mapping cell migrations and fates in a gastruloid model to the human primitive streak.Development. 2019; 146dev179564Crossref PubMed Scopus (29) Google Scholar]. This hematopoietically patterned mesoderm then develops into endothelium, from which a hemogenic subset undergoes an endothelial-to-hematopoietic transition to form blood lineages [8Lancrin C Sroczynska P Stephenson C Allen T Kouskoff V Lacaud G The haemangioblast generates haematopoietic cells through a haemogenic endothelium stage.Nature. 2009; 457: 892-895Crossref PubMed Scopus (477) Google Scholar, 9Eilken HM Nishikawa S Schroeder T Continuous single-cell imaging of blood generation from haemogenic endothelium.Nature. 2009; 457: 896-900Crossref PubMed Scopus (454) Google Scholar, 10Bertrand JY Chi NC Santoso B Teng S Stainier DY Traver D Haematopoietic stem cells derive directly from aortic endothelium during development.Nature. 2010; 464: 108-111Crossref PubMed Scopus (699) Google Scholar, 11Kissa K Herbomel P Blood stem cells emerge from aortic endothelium by a novel type of cell transition.Nature. 2010; 464: 112-115Crossref PubMed Scopus (656) Google Scholar]. We recently defined the requirements for RUNX1, GFI1, and/or GFI1B in this human pluripotent stem cell (hPSC) model of yolk sac-like human hematopoiesis [4Bruveris FF Ng ES Leitoguinho AR et al.Human yolk sac-like haematopoiesis generates RUNX1-, GFI1- and/or GFI 1B-dependent blood and SOX17-positive endothelium.Development. 2020; 147dev193037Crossref PubMed Scopus (8) Google Scholar]. A combination of gene deletion studies and small molecule inhibitors revealed that RUNX1 was required for all blood formation except an initial wave of GFI1/1B-dependent erythropoiesis. To complement this genetic analysis, here we examine the capacity of exogenous growth factors to regulate human blood and endothelial lineage specification in the blast colony model. Studies using hPSCs were approved by The Royal Children's Hospital (Reference No. 33001A) and Monash University (Reference No. 2002/225MC) Human Research Ethics Committees. The dual reporter SOX17mCHERRY/w RUNX1CGFP/w (SOX-RUNX) H9 and the RUNX1–/– (RUNX1-KO) PSC lines used in these studies have been previously described [4Bruveris FF Ng ES Leitoguinho AR et al.Human yolk sac-like haematopoiesis generates RUNX1-, GFI1- and/or GFI 1B-dependent blood and SOX17-positive endothelium.Development. 2020; 147dev193037Crossref PubMed Scopus (8) Google Scholar,12Ng ES Azzola L Bruveris FF et al.Differentiation of human embryonic stem cells to HOXA(+) hemogenic vasculature that resembles the aorta–gonad–mesonephros.Nat Biotechnol. 2016; 34: 1168-1179Crossref PubMed Scopus (94) Google Scholar]. Human PSCs were co-cultured with mouse embryonic fibroblasts [13Costa M Sourris K Hatzistavrou T Elefanty AG Stanley EG Expansion of human embryonic stem cells in vitro.Curr Protoc Stem Cell Biol. 2008; 5 (Chapter 1, Unit 1C.1.–1C.1.7)Crossref Scopus (39) Google Scholar], isolated from murine embryos according to Mouse Animal Ethics Approval A774 (Murdoch Children's Research Institute). The H9 hPSCs used in these studies were provided by ES Cell International and WiCell. Cell lines were regularly tested to exclude mycoplasma contamination and confirm genomic integrity. Human PSC culture and enzymatic passaging of lines were performed as previously described [14Ng ES Davis R Stanley EG Elefanty AG A protocol describing the use of a recombinant protein-based, animal product-free medium (APEL) for human embryonic stem cell differentiation as spin embryoid bodies.Nat Protoc. 2008; 3: 768-776Crossref PubMed Scopus (213) Google Scholar]. To identify blast colonies, cells were differentiated toward extra-embryonic yolk sac hematopoietic lineages using the spin embryoid body (EB) method in STEMdiff APEL medium (StemCell Technologies, Vancouver, BC, Canada) [14]. Cultures were supplemented for 2.5–3 days with 10–20 ng/mL recombinant human (rh) Activin A (R&D Systems, Minneapolis, MN), 20 ng/mL rh bone morphogenetic protein 4 (BMP4, R&D Systems), 30 ng/mL rh vascular endothelial growth factor (VEGF, PeproTech, Rocky Hill, NJ), and 40 ng/mL stem cell factor (SCF, PeproTech) before dissociation and input into methylcellulose assays. For analysis, EBs and methylcellulose cultures were harvested and dissociated into a single-cell suspension using TrypLE Select (Invitrogen, Waltham, MA) and passed through a 40-µm cell strainer. Blast colony-forming cells were detected by culturing 3 × 103 dissociated cells from day 2.5–3 EBs in a formulation designated MC-APEL (1% methylcellulose in APEL medium) supplemented with 100 ng/mL rh SCF, 2 U/mL rh erythropoietin (EPO, PeproTech), 50 ng/mL rh VEGF, 50 ng/mL rh interleukin (IL)-3 (PeproTech), 50 ng/mL rh IL-6, 50 ng/mL rh thrombopoietin (TPO, PeproTech), 20 ng/mL rh BMP4, and 10 ng/mL rh fibroblast growth factor 2 (FGF2, PeproTech). Colony formation was scored after 9–11 days of differentiation. Where indicated, methylcellulose cultures also contained 5–10 mmol/L SU5402 (Sigma-Aldrich, St. Louis, MO), 0.7 mmol/L DMH-1 (Sigma-Aldrich), 10 ng/mL Activin A, 4 mmol/L SB431542 (Cayman Chemical Co., Ann Arbor, MI), 3–6 mmol/L CHIR-99021 (Tocris Bioscience, Bristol, UK), 5–10 mmol/L IWR-1 (Sigma-Aldrich), 5–10 mmol/L XAV939 (Sigma-Aldrich), 0.25-1 mmol/L SANT-1 (Sigma-Aldrich), 1–3 mmol/L purmorphamine (Sigma-Aldrich), and 10–20 mmol/L tert-butyl (2S)-2-[[(2S)-2-[[2-(3,5-difluorophenyl)acetyl]-amino]propanoyl]amino]-2-phenylacetate (DAPT) (Sigma-Aldrich). Antibodies directed against the following cell surface antigens, listed in Table 1, were used to stain dissociated cells for flow cytometric analysis. Platelet-derived growth factor receptor α (PDGFRα) and TIE2/TEK were detected with secondary antibodies conjugated with allophycocyanin (APC) or phycoerythrin-cyanine 7 (PE-Cy7). Flow cytometric analysis was performed using a BD Biosciences (Franklin Lakes, NJ) LSR Fortessa analyzer. Flow sorting used a BD Biosciences Influx or BD Biosciences FACSAria Fusion cell sorter. Samples were gated using forward scatter area (FSC-A) and forward scatter height (FSC-H) to exclude doublets. Live cells were selected by FSC and propidium iodide exclusion. Positive gates were determined by comparing stained samples with unstained samples.Table 1Flow cytometry antibodiesAntigenProducerFluorochromeCatalogue no.Antibody cloneDilutionPlatelet-derived growth factor receptor α (PDGFRα)BD Pharmingen556001aR11:100TIE2/TEKBD Pharmingen557039c331:100CD31BioLegendAPC303115WM591:50BioLegendBV421303123WM591:30CD34BioLegendPE-Cy7435155811:100CD43BioLegendAPC34320610G71:50BD PharmingenBV4215629161G101:30CD45BioLegendBV421304032H1301:30C-X-C chemokine receptor 4 (CXCR4)BioLegendBV42130651812G51:30BioLegendPE-Cy730651312G51:100Glycophorin A (GYPA)BD PharmingenAPC551336GA-R2 (HIR2)1:2,500Vascular endothelial growth factor receptor 2 (VEGFR2/KDR)BioLegendAlexa Fluor-647338909HKDR-11:10APC goat anti-mouse IgGBD PharmingenAPC550826Poly 12701:100BioLegendAPC405308Poly 40531:100PE-Cy7 goat anti-mouse IgGBioLegendPE-Cy7405315Poly 40531:100 Open table in a new tab The RNA sequencing data presented in this article were described previously [4Bruveris FF Ng ES Leitoguinho AR et al.Human yolk sac-like haematopoiesis generates RUNX1-, GFI1- and/or GFI 1B-dependent blood and SOX17-positive endothelium.Development. 2020; 147dev193037Crossref PubMed Scopus (8) Google Scholar]. Briefly, differentiated SOX-RUNX cultures were harvested from spin EBs at day 2 and from methylcellulose blast colony assays at days 2+2 and 2+3, and fractioned by flow cytometry on expression of PDGFRα, CD34, CD43, and mCHERRY (SOX17) using a BD Biosciences Influx or BD Biosciences FASCAria Fusion cell sorter. Total RNA was isolated from the sorted fractions using the RNA Isolate II Mini or Micro Kits (Bioline, London, UK) as specified by the manufacturer. cDNA was reverse transcribed using random hexamer priming and the Tetro cDNA synthesis (Bioline) kit in accordance with the manufacturers’ instructions. Total RNA was sequenced and analyzed at the Murdoch Children's Research Institute as described [4Bruveris FF Ng ES Leitoguinho AR et al.Human yolk sac-like haematopoiesis generates RUNX1-, GFI1- and/or GFI 1B-dependent blood and SOX17-positive endothelium.Development. 2020; 147dev193037Crossref PubMed Scopus (8) Google Scholar]. These data have been deposited with Gene Expression Omnibus under Accession No. GSE124086 SuperSeries, GSE124085 SubSeries. Total RNA was isolated from differentiated hPSCs using the RNA Isolate II Mini or Micro Kits (Bioline) as specified by the manufacturer. cDNA was reverse transcribed using random hexamer priming and the Tetro cDNA synthesis (Bioline) kit in accordance with the manufacturer's instructions. TaqMan gene expression probes (Applied Biosystems, Waltham, MA) and Bioline reagents were used for quantitative real-time polymerase chain reaction using GAPDH as the reference gene to normalize the data. TaqMan assays directed toward the following target sequences were used to detect gene expression: RUNX1 (Hs00231079_m1). To dissect the early segregation of human blood and endothelial lineages, we used a SOX17mCHERRY/w RUNX1CGFP/w dual-reporter hPSC line (termed SOX-RUNX cells) [4Bruveris FF Ng ES Leitoguinho AR et al.Human yolk sac-like haematopoiesis generates RUNX1-, GFI1- and/or GFI 1B-dependent blood and SOX17-positive endothelium.Development. 2020; 147dev193037Crossref PubMed Scopus (8) Google Scholar,12Ng ES Azzola L Bruveris FF et al.Differentiation of human embryonic stem cells to HOXA(+) hemogenic vasculature that resembles the aorta–gonad–mesonephros.Nat Biotechnol. 2016; 34: 1168-1179Crossref PubMed Scopus (94) Google Scholar] to track the emergence of SOX17+ endothelial and RUNX1C+ blood progenitors in combination with cell surface antigens indicative of lineage assignment. We differentiated SOX-RUNX cells for 2.5–3 days as spin EBs [14Ng ES Davis R Stanley EG Elefanty AG A protocol describing the use of a recombinant protein-based, animal product-free medium (APEL) for human embryonic stem cell differentiation as spin embryoid bodies.Nat Protoc. 2008; 3: 768-776Crossref PubMed Scopus (213) Google Scholar] and subsequently transferred the dissociated cells to a methylcellulose-based blast colony-forming cell (BL-CFC) assay (Figure and day 3 differentiated cells the (Figure from which (Figure RP Costa M Grandela C et al.A protocol for removal of antibiotic resistance cassettes from human embryonic stem cells genetically modified by homologous recombination or transgenesis.Nat Protoc. 2008; 3: 1550-1558Crossref PubMed Scopus (36) Google Scholar]. blast and blood SOX17-positive and endothelium, and cells (Figure QC Hirst CE Costa M et al.APELIN promotes hematopoiesis from human embryonic stem cells.Blood. 2012; 119: 6243-6254Crossref PubMed Scopus (42) Google FF Ng ES Leitoguinho AR et al.Human yolk sac-like haematopoiesis generates RUNX1-, GFI1- and/or GFI 1B-dependent blood and SOX17-positive endothelium.Development. 2020; 147dev193037Crossref PubMed Scopus (8) Google Scholar]. reporter small of SOX17+ cells were at day 3 the cells were into methylcellulose but RUNX1C+ cells were not 2 days of methylcellulose culture (Figure previously described [4Bruveris FF Ng ES Leitoguinho AR et al.Human yolk sac-like haematopoiesis generates RUNX1-, GFI1- and/or GFI 1B-dependent blood and SOX17-positive endothelium.Development. 2020; 147dev193037Crossref PubMed Scopus (8) Google Scholar], blood progenitors and a small of hematopoietic cells were after 3 days of methylcellulose culture (Figure of the cells in with hemogenic [4Bruveris FF Ng ES Leitoguinho AR et al.Human yolk sac-like haematopoiesis generates RUNX1-, GFI1- and/or GFI 1B-dependent blood and SOX17-positive endothelium.Development. 2020; 147dev193037Crossref PubMed Scopus (8) Google Scholar] (Figure the emergence and of cells revealed that blast of all a SOX17+ after days in methylcellulose (Figure 5 days of methylcellulose of contained SOX17+ cells (Figure analysis of surface expression revealed the initial endothelial of blast with high and cells at by expression of and in at (Figure expression was from (Figure This progression the of hematopoietic from endothelial after the first day of methylcellulose culture (Figure The from this of are with the progression of mesoderm to endothelium and blood cells in the blast colony formation assay [4Bruveris FF Ng ES Leitoguinho AR et al.Human yolk sac-like haematopoiesis generates RUNX1-, GFI1- and/or GFI 1B-dependent blood and SOX17-positive endothelium.Development. 2020; 147dev193037Crossref PubMed Scopus (8) Google Scholar]. SOX-RUNX cells were differentiated to hematopoietic mesoderm for 2.5–3 days in medium supplemented with BMP4, Activin A, VEGF, and were and cells were in methylcellulose supplemented with hematopoietic differentiation (Figure We or growth factors and inhibitors VEGF, and Notch to on blast colony formation and differentiation. Blast were and differentiation was analyzed by flow cytometry (Figure and of SOX-RUNX cultures revealed blast colony generation blast with SOX17+ endothelium and RUNX1C+ blood cells. The blast a of blood cells (Figure at are required for murine mesoderm with mouse embryonic stem cells hematopoietic colony formation P A et fibroblast growth factor hematopoietic PubMed Google K M J is required for embryonic growth and during mouse Dev. PubMed Scopus Google Scholar]. VEGF signaling is for and with embryonic in from in vascular and hematopoietic development P V G et blood development and in embryos a VEGF PubMed Scopus Google K et embryonic by of the VEGF PubMed Scopus Google Scholar]. removal of exogenous and VEGF from methylcellulose blast colony frequency was reduced for and VEGF (Figure with the of an [1Kennedy M D'Souza SL Lynch-Kattman M Schwantz S Keller G Development of the hemangioblast defines the onset of hematopoiesis in human ES cell differentiation cultures.Blood. 2007; 109: 2679-2687Crossref PubMed Scopus (353) Google Scholar]. the of blood and endothelial were not in the of at that is required for commitment to colony formation for differentiation. studies of blast colony sorted fractions [4Bruveris FF Ng ES Leitoguinho AR et al.Human yolk sac-like haematopoiesis generates RUNX1-, GFI1- and/or GFI 1B-dependent blood and SOX17-positive endothelium.Development. 2020; 147dev193037Crossref PubMed Scopus (8) Google Scholar] the that the of are by or production of by blast colony precursors and at the of VEGF (Figure at or with dual inhibition of and VEGF signaling by the small molecule colony formation was and hematopoietic and endothelial expression was (Figure at with cells in the cultures at and the VEGF are during blast colony the of VEGF is for blast colony the for exogenous VEGF at the for endothelium and blood development at we the Activin A and BMP4, of early mesoderm differentiation. BMP4 is for human blast colony formation [1Kennedy M D'Souza SL Lynch-Kattman M Schwantz S Keller G Development of the hemangioblast defines the onset of hematopoiesis in human ES cell differentiation cultures.Blood. 2007; 109: 2679-2687Crossref PubMed Scopus (353) Google Scholar], and provided by Activin A, is required for primitive hematopoietic development in mouse and human PSC cultures Keller P and signaling regulate distinct in the pathway from embryonic stem cells to Stem 2008; PubMed Scopus Google Scholar, S Sroczynska P Lacaud G Kouskoff V The specification of embryonic stem cells to hematopoietic is by to A, and 2008; PubMed Scopus Google Scholar, M G et the emergence of hematopoietic progenitors in human pluripotent stem cell differentiation 2012; PubMed Scopus Google Scholar, A G M Keller G signaling the specification of and primitive hematopoiesis from human pluripotent stem Biotechnol. PubMed Scopus Google Scholar]. To BMP4 we exogenous BMP4 from the methylcellulose and with a pathway This SOX17+ endothelium at the of hematopoietic cells and (Figure at To the we used Activin A as a surrogate for inhibition was the type I Activin 5 small molecule SB431542 cultures supplemented with Activin A a in hematopoietic differentiation and colony formation as with of BMP4 with did not the of endothelial and hematopoietic lineages, colony frequency (Figure at This with the for Activin A for the formation of precursors at day 2 of yolk differentiation from hPSCs A G M Keller G signaling the specification of and primitive hematopoiesis from human pluripotent stem Biotechnol. PubMed Scopus Google Scholar]. the differentiation to in cultures also Activin A (Figure revealed of on hematopoietic cell that or blood cell formation on the of C et inhibition the generation of hematopoietic progenitors from human ES hemogenic endothelial cells using a 2012; PubMed Scopus Google T of erythropoietin and inhibition on development in human pluripotent stem Cell PubMed Scopus Google Scholar]. These are with the that a for Activin A signaling in hematopoiesis J Stanley EG Elefanty AG embryonic stem cells through that to exogenous and 2010; PubMed Scopus Google Scholar]. to mesoderm formation days and the generation of primitive hematopoietic progenitors A G M Keller G signaling the specification of and primitive hematopoiesis from human pluripotent stem Biotechnol. PubMed Scopus Google Scholar], and to cultures 4 and the molecule or hematopoietic cell C et inhibition the generation of hematopoietic progenitors from human ES hemogenic endothelial cells using a 2012; PubMed Scopus Google T of erythropoietin and inhibition on development in human pluripotent stem Cell PubMed Scopus Google Scholar]. WNT have roles in hematopoiesis in mouse and human PSCs Keller P and signaling regulate distinct in the pathway from embryonic stem cells to Stem 2008; PubMed Scopus Google J Stanley EG Elefanty AG embryonic stem cells through that to exogenous and 2010; PubMed Scopus Google Scholar, S L in stem PubMed Scopus Google Scholar, T early lineage specification of human embryonic stem cells by the of and 2008; PubMed Scopus Google Scholar]. WNT pathway through methylcellulose with the CHIR-99021 reduced hematopoietic cell generation and blast colony in a (Figure at These were with previously from K Hirst CE QC et promotes hematopoietic or differentiation from on the of Cell PubMed Scopus Google Scholar] and A G M Keller G signaling the specification of and primitive hematopoiesis from human pluripotent stem Biotechnol. PubMed Scopus Google Scholar], WNT pathway reduced blast colony formation and primitive hematopoietic differentiation. WNT inhibition the IWR-1 and XAV939 also blast colony frequency and blood cell SOX17+ endothelial expression (Figure at the for but not of WNT The Hedgehog pathway is required for primitive and hematopoiesis Hedgehog signaling in Gene 2010; PubMed Scopus Google Scholar]. of the SANT-1 or the agonist purmorphamine reduced colony formation to (Figure at SANT-1 did not hematopoietic or endothelial surface but reduced cells at high colony of (Figure and at The in colony formation with SANT-1 was with of a in and differentiation by the K differentiation in is by an of PubMed Scopus Google Scholar]. Notch signaling is required for and for development but is not to required for yolk sac hematopoietic colony formation Notch signaling and the emergence of hematopoietic stem 2020; PubMed Scopus Google Scholar, A et signaling hemogenic endothelium from human pluripotent stem PubMed Scopus Google Scholar, K S A et but not is for hematopoietic stem cells from endothelial PubMed Scopus Google Scholar, J et al.A for 2 of hematopoiesis during 2004; PubMed Scopus Google Scholar]. To the role of Notch signaling in human blast colony we Notch signaling using the in a of The of hematopoietic and endothelial lineages was the and the supplemented cultures (Figure at but colony frequency was in the of (Figure These are with mouse data a in after the of a Keller G A quantitative analysis of hemogenic endothelium of hematopoiesis by Cell PubMed Scopus Google Scholar]. This that the blast colony assay an cell during hPSC differentiation that a of to Notch signals [4Bruveris FF Ng ES Leitoguinho AR et al.Human yolk sac-like haematopoiesis generates RUNX1-, GFI1- and/or GFI 1B-dependent blood and SOX17-positive endothelium.Development. 2020; 147dev193037Crossref PubMed Scopus (8) Google Scholar]. The of the small molecule DMH-1 to the methylcellulose culture selected for SOX17+ endothelial differentiation at the of hematopoiesis (Figure at DMH-1 blast were by SOX17+ endothelial and colonies, of in blast colony assays of RUNX1 cells [4Bruveris FF Ng ES Leitoguinho AR et al.Human yolk sac-like haematopoiesis generates RUNX1-, GFI1- and/or GFI 1B-dependent blood and SOX17-positive endothelium.Development. 2020; 147dev193037Crossref PubMed Scopus (8) Google Scholar] (Figure DMH-1 and cultures were by of the hematopoietic CD43, and and of endothelial and and of gene expression revealed of RUNX1 in and DMH-1 in to the of RUNX1 expression in the RUNX1 samples (Figure of RNA sequencing data revealed that mesoderm cells the blast colony assay the and BMP4 and that RUNX1 was not at the endothelial day RUNX1 was but expression at These data that hemogenic endothelium by RUNX1 in the of exogenous BMP4, but that the of in endothelium were to the endothelial-to-hematopoietic we have a for VEGF and in human blast colony differentiation during the transition of mesoderm to endothelium (Figure BMP4 was needed for cells to a subsequent of endothelial-to-hematopoietic transition (Figure Activin A and high concentrations of SOX17+ endothelial and cells (Figure These studies help to define distinct roles for prerequisite growth factors that commit mesoderm to hemogenic endothelium and subsequently allocate cells to blood lineages. The This was by the and Research of through to and and to AG Elefanty and EG Stanley by the Research Research in Stem and by the Research to the Murdoch Children's Research Institute was provided by the and Research Research Institute and the

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