Murine hemogenic endothelial precursors display heterogeneous hematopoietic potential ex vivo
2017; Elsevier BV; Volume: 51; Linguagem: Inglês
10.1016/j.exphem.2017.04.006
ISSN1873-2399
AutoresMiguel Ganuza, Brandon Hadland, Ashley Chabot, Chen Li, Guolian Kang, Irwin D. Bernstein, Shannon McKinney‐Freeman,
Tópico(s)Angiogenesis and VEGF in Cancer
Resumo•HE constitutes a minor fraction of midgestation murine endothelium.•Midgestation HE is highly heterogeneous in its hematopoietic potential.•AKT-expressing AGM endothelial cells are a superior supportive niche for the derivation of HSPCs. Hematopoietic stem and progenitor cells (HSPCs) sustain life-long hematopoiesis and are first detected in the embryo by transplantation at embryonic day 10.5 (E10.5). HSPCs are mesodermal in origin and ultimately emerge from a subset of arterial endothelium (i.e., hemogenic endothelium [HE]), which is highly concentrated in the aorta–gonad–mesonephros region of the midgestation embryo. Here, we used clonal ex vivo assays, in which endothelial cells isolated from the midgestation aorta and vitelline and umbilical arteries are co-cultured on supportive stroma, to show that only about 0.1%, 1.3%, and 0.29% of E9.5, E10.5, and E11.5 endothelium are functional HE, respectively. We further show high phenotypic and functional variability in the hematopoietic potential of individual hemogenic endothelial precursors. Using unique niche stroma capable of providing the signals necessary for definitive hematopoietic stem cell (dHSC) induction, we demonstrate that this variability in HE includes their potential to support phenotypic dHSCs. These data suggest the presence of a continuum of maturing HE with distinct hematopoietic potential or HE representative of a heterogeneous pool of precursors that give rise to HSPCs with disparate hematopoietic potential. Hematopoietic stem and progenitor cells (HSPCs) sustain life-long hematopoiesis and are first detected in the embryo by transplantation at embryonic day 10.5 (E10.5). HSPCs are mesodermal in origin and ultimately emerge from a subset of arterial endothelium (i.e., hemogenic endothelium [HE]), which is highly concentrated in the aorta–gonad–mesonephros region of the midgestation embryo. Here, we used clonal ex vivo assays, in which endothelial cells isolated from the midgestation aorta and vitelline and umbilical arteries are co-cultured on supportive stroma, to show that only about 0.1%, 1.3%, and 0.29% of E9.5, E10.5, and E11.5 endothelium are functional HE, respectively. We further show high phenotypic and functional variability in the hematopoietic potential of individual hemogenic endothelial precursors. Using unique niche stroma capable of providing the signals necessary for definitive hematopoietic stem cell (dHSC) induction, we demonstrate that this variability in HE includes their potential to support phenotypic dHSCs. These data suggest the presence of a continuum of maturing HE with distinct hematopoietic potential or HE representative of a heterogeneous pool of precursors that give rise to HSPCs with disparate hematopoietic potential. Hematopoiesis arises in multiple waves during embryonic development, beginning with primitive hematopoietic progenitors at embryonic day 7.5 (E7.5), followed by definitive erythro-myeloid progenitors at E8.5. A third wave of hematopoiesis begins around E10 and yields definitive hematopoietic stem cells (dHSCs) that originate from arterial endothelium throughout the embryo, although their emergence is concentrated in the aorta–gonad–mesonephros (AGM) and the vitelline artery (VA) and umbilical artery (UA) [1Medvinsky A. Rybtsov S. Taoudi S. Embryonic origin of the adult hematopoietic system: Advances and questions.Development. 2011; 138: 1017-1031Crossref PubMed Scopus (276) Google Scholar]. dHSCs are defined by their ability to reconstitute hematopoiesis when transplanted into conditioned recipients. dHSCs that can reconstitute adult recipients are first detected at E10.5 in the embryo [2Müller A.M. Medvinsky A. Strouboulis J. Grosveld F. Dzierzak E. Development of hematopoietic stem cell activity in the mouse embryo.Immunity. 1994; 1: 291-301Abstract Full Text PDF PubMed Scopus (677) Google Scholar, 3Medvinsky A. Dzierzak E. Definitive hematopoiesis is autonomously initiated by the AGM region.Cell. 1996; 86: 897-906Abstract Full Text Full Text PDF PubMed Scopus (1146) Google Scholar, 4Kumaravelu P. Hook L. Morrison A.M. et al.Quantitative developmental anatomy of definitive haematopoietic stem cells/long-term repopulating units (HSC/RUs): Role of the aorta-gonad-mesonephros (AGM) region and the yolk sac in colonisation of the mouse embryonic liver.Development. 2002; 129: 4891-4899Crossref PubMed Google Scholar], whereas cells capable of reconstituting conditioned newborns can be detected earlier, between E9 and E10 [5Yoder M.C. Hiatt K. Dutt P. Mukherjee P. Bodine D.M. Orlic D. Characterization of definitive lymphohematopoietic stem cells in the day 9 murine yolk sac.Immunity. 1997; 7: 335-344Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 6Yoder M.C. Hiatt K. Mukherjee P. In vivo repopulating hematopoietic stem cells are present in the murine yolk sac at day 9.0 postcoitus.Proc Natl Acad Sci U S A. 1997; 94: 6776-6780Crossref PubMed Scopus (212) Google Scholar, 7Fraser S.T. Ogawa M. Yu R.T. Nishikawa S. Yoder M.C. Nishikawa S. Definitive hematopoietic commitment within the embryonic vascular endothelial-cadherin(+) population.Exp Hematol. 2002; 30: 1070-1078Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar]. dHSCs emerge during development from a subset of “blood-forming” endothelium known as “hemogenic endothelium” (HE), a concept first proposed in the 19th century when embryologists reported that the “blood islands” of the yolk sac appeared to bud from developing vascular tissue [1Medvinsky A. Rybtsov S. Taoudi S. 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Aorta-associated CD34+ hematopoietic cells in the early human embryo.Blood. 1996; 87: 67-72Crossref PubMed Google Scholar]. This model has been supported by studies demonstrating that endothelial cells purified from murine embryos and embryonic stem cells (ESCs) possess lymphopoietic potential [12Nishikawa S.I. Nishikawa S. Hirashima M. Matsuyoshi N. Kodama H. Progressive lineage analysis by cell sorting and culture identifies FLK1+VE-cadherin+ cells at a diverging point of endothelial and hemopoietic lineages.Development. 1998; 125: 1747-1757Crossref PubMed Google Scholar, 13Nishikawa S.I. Nishikawa S. Kawamoto H. et al.In vitro generation of lymphohematopoietic cells from endothelial cells purified from murine embryos.Immunity. 1998; 8: 761-769Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar] and labeling vascular endothelium before the onset of hematopoiesis results in labeled hematopoietic clusters [14Jaffredo T. Gautier R. Eichmann A. Dieterlen-Lièvre F. 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As mentioned, functional dHSCs cannot be detected via transplantation into adult recipients before E10.5 [2Müller A.M. Medvinsky A. Strouboulis J. Grosveld F. Dzierzak E. Development of hematopoietic stem cell activity in the mouse embryo.Immunity. 1994; 1: 291-301Abstract Full Text PDF PubMed Scopus (677) Google Scholar, 3Medvinsky A. Dzierzak E. Definitive hematopoiesis is autonomously initiated by the AGM region.Cell. 1996; 86: 897-906Abstract Full Text Full Text PDF PubMed Scopus (1146) Google Scholar, 4Kumaravelu P. Hook L. Morrison A.M. et al.Quantitative developmental anatomy of definitive haematopoietic stem cells/long-term repopulating units (HSC/RUs): Role of the aorta-gonad-mesonephros (AGM) region and the yolk sac in colonisation of the mouse embryonic liver.Development. 2002; 129: 4891-4899Crossref PubMed Google Scholar, 6Yoder M.C. Hiatt K. Mukherjee P. In vivo repopulating hematopoietic stem cells are present in the murine yolk sac at day 9.0 postcoitus.Proc Natl Acad Sci U S A. 1997; 94: 6776-6780Crossref PubMed Scopus (212) Google Scholar, 7Fraser S.T. Ogawa M. Yu R.T. Nishikawa S. Yoder M.C. Nishikawa S. Definitive hematopoietic commitment within the embryonic vascular endothelial-cadherin(+) population.Exp Hematol. 2002; 30: 1070-1078Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar], making identification of earlier developing hematopoietic stem and progenitor cells (HSPCs) and their precursors technically difficult. To overcome this, several experimental platforms have been developed in which E11, E10, or E9 AGM-derived cells can be cultured ex vivo in a manner that ongoing specification and expansion of dHSCs are preserved [21Taoudi S. Gonneau C. 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Therefore, a series of precursors at E11, E10, and E9 have been identified using these assays. However, the exact frequency of the HE precursors within these populations and their precise hematopoietic potential has not yet been defined. We recently developed a novel system that also supports the ongoing specification and expansion of HSPCs from HE ex vivo [10Smith R.A. Glomski C.A. “Hemogenic endothelium” of the embryonic aorta: Does it exist?.Dev Comp Immunol. 1982; 6: 359-368Crossref PubMed Scopus (56) Google Scholar]. In this system, E11 AGM-derived endothelial cells are engineered to express Myr-AKT (AGM AKT-endothelial cells or AA-ECs). These cells constitute an endothelial niche that is sufficient to support the specification and maturation of dHSCs ex vivo, including the maturation of E9 VE-Cadherin+c-Kit+ cells to transplantable dHSCs [25Hadland B.K. Varnum-Finney B. Poulos M.G. et al.Endothelium and NOTCH specify and amplify aorta-gonad-mesonephros-derived hematopoietic stem cells.J Clin Invest. 2015; 125: 2032-2045Crossref PubMed Scopus (52) Google Scholar]. Here, we employ this system to demonstrate at the clonal level that the hematopoietic output of HE is highly heterogeneous with respect to cell surface phenotype and function. To our knowledge, our study is the first to interrogate clonally the phenotype and function of HSPCs emerging from HE ex vivo. In parallel, we also employed OP9 stromal cells, a widely used hematopoietic supportive cell line, to assess the frequency and heterogeneity of HE in E9.5, E10.5, and E11.5 embryos [12Nishikawa S.I. Nishikawa S. Hirashima M. Matsuyoshi N. Kodama H. Progressive lineage analysis by cell sorting and culture identifies FLK1+VE-cadherin+ cells at a diverging point of endothelial and hemopoietic lineages.Development. 1998; 125: 1747-1757Crossref PubMed Google Scholar, 13Nishikawa S.I. Nishikawa S. Kawamoto H. et al.In vitro generation of lymphohematopoietic cells from endothelial cells purified from murine embryos.Immunity. 1998; 8: 761-769Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 25Hadland B.K. Varnum-Finney B. Poulos M.G. et al.Endothelium and NOTCH specify and amplify aorta-gonad-mesonephros-derived hematopoietic stem cells.J Clin Invest. 2015; 125: 2032-2045Crossref PubMed Scopus (52) Google Scholar, 26Nakano T. Kodama H. Honjo T. Generation of lymphohematopoietic cells from embryonic stem cells in culture.Science. 1994; 265: 1098-1101Crossref PubMed Scopus (690) Google Scholar]. We found that only 0.1%, 1.3%, and 0.29% of the E9.5, E10.5, and E11.5 VE-Cadherin+CD45− endothelium, respectively, generates hematopoietic colonies after OP9 co-culture. Further, only about 9% of these colonies contain phenotypic dHSCs ex vivo, detection of which is critically dependent upon exposure to relevant niche signals, because AA-ECs display superior support of phenotypic dHSC and multilineage hematopoietic progenitors compared with OP9 stroma. C57BL/6J mice were acquired from The Jackson Laboratory (Bar Harbor, ME) and housed in a pathogen-free facility. All animal experiments were performed according to procedures approved by the St. Jude Children's Research Hospital's Institutional Animal Care and Use Committee. Timed pregnancies were set up overnight, and vaginal plugs were assessed before 8:00 the next morning (designated as E0.5). Embryos were staged based on somite pair (sp) counts: E9.5 (19–23 sp), E10.5 (32–36 sp), and E11.5 (46–47 sp). E9.5 caudal halves and E10.5/E11.5 AGMs were dissociated with collagenase (0.0012 g/mL, Sigma-Aldrich, St. Louis, MO) in PBS (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FCS (Omega Scientific, Tarzana, CA) and then stained with CD144-PE (11D4.1, BD Biosciences, San Diego, CA) and CD45-FITC (30-F11, BD Biosciences). VE-Cadherin+CD45− cells were then sorted by fluorescence-activated cell sorting (FACS) via a FACSAria III (BD Biosciences) into 384-well or 96-well cell culture–treated plates (Sigma-Aldrich) at 1, 2, 5, 10, or 20 cells/well. Each 384-well plate was pre-plated with 1,000 AA-ECs overnight after collagen coating (STEMCELL Technologies, Vancouver, Canada), and each 96-well plate was pre-plated with 3,000 OP9 cells or 3,000 AA-ECs to ensure equivalent cell densities in 96- and 384-well plates. Each 96- and 384-well plate contained 150 or 75 μL of X-vivo 20 serum-free medium (Lonza, Walkersville, MD) supplemented with 100 ng/mL of recombinant murine stem cell factor (SCF), recombinant murine FLT3L, recombinant murine interleukin-3 (IL-3), and 20 ng/mL of recombinant murine thrombopoietin (Peprotech, Rocky Hill, NJ). Sorted cells were co-cultured with either AA-ECs or OP9 cells for 7 days before each well was inspected for the presence of hematopoietic colonies. Individual colonies were then either replated in M3434 (STEMCELL Technologies) or stained with the following antibodies and analyzed by flow cytometry on an LSRFortessa (BD Biosciences): c-Kit-APC (2B8), CD4-e605 (RM4-5), CD8-e605 (53-6.7), Gr1-e605 (RB6-8C5), B220-e605 (RA3-6B2), Ter119-e605 (Ter119), CD4-Brilliant Violet 605™ (RM4-5), CD8- Brilliant Violet 605™ (53-6.7), Gr1- Brilliant Violet 605™ (RB6-8C5), B220- Brilliant Violet 605™ (RA3-6B2), Ter119- Brilliant Violet 605™ (Ter119), Sca-1-PerCPCy5.5 (E13-161.7), CD150-PE (TC15-12F12.2), and CD48-FITC (HM48-1). All antibodies were obtained from BioLegend (San Diego, CA) except c-Kit-APC (2B8), which was obtained from eBiosciences (San Diego, CA). Colony-forming units (CFUs) on M3434 were scored 10 days after plating. OP9 stromal cells (CRL2749, ATCC, Manassas VA) were maintained in MEM-20 medium (MEM Alpha 1x media 12561, Invitrogen), fetal bovine serum 20% (Omega), 1% penicillin/streptomycin (Hyclone, Thermo Scientific), and kept in culture at medium density to avoid cell differentiation. AA-ECs were derived and cultured as previously described [25Hadland B.K. Varnum-Finney B. Poulos M.G. et al.Endothelium and NOTCH specify and amplify aorta-gonad-mesonephros-derived hematopoietic stem cells.J Clin Invest. 2015; 125: 2032-2045Crossref PubMed Scopus (52) Google Scholar]. The Shapiro–Wilk test was performed to test for normality of the data. Exact Chi-squared tests and two-sample t tests or exact Wilcoxon rank–sum tests, depending on the normality, were used to compare two groups for categorical and continuous variables, respectively. Box plots, bar plots with error bars, and bar charts were used to present the data. All analyses were performed in SAS version 9.4 (SAS Institute). For limiting dilution analysis (LDA), parameters were estimated using a generalized linear model with a complementary log–log link. The Chi-squared test (Pearson and deviance) was used to assess the goodness-of-fit to the LDA model. Differences in frequencies between groups were assessed by relying on the asymptotic normality of the maximum likelihood estimator. LDA was performed using L-Calc (STEMCELL Technologies). OP9 co-culture is used widely in the maintenance of HSPCs from adults, embryos, and differentiating ESCs [12Nishikawa S.I. Nishikawa S. Hirashima M. Matsuyoshi N. Kodama H. Progressive lineage analysis by cell sorting and culture identifies FLK1+VE-cadherin+ cells at a diverging point of endothelial and hemopoietic lineages.Development. 1998; 125: 1747-1757Crossref PubMed Google Scholar, 13Nishikawa S.I. Nishikawa S. Kawamoto H. et al.In vitro generation of lymphohematopoietic cells from endothelial cells purified from murine embryos.Immunity. 1998; 8: 761-769Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 25Hadland B.K. Varnum-Finney B. Poulos M.G. et al.Endothelium and NOTCH specify and amplify aorta-gonad-mesonephros-derived hematopoietic stem cells.J Clin Invest. 2015; 125: 2032-2045Crossref PubMed Scopus (52) Google Scholar, 26Nakano T. Kodama H. Honjo T. Generation of lymphohematopoietic cells from embryonic stem cells in culture.Science. 1994; 265: 1098-1101Crossref PubMed Scopus (690) Google Scholar, 27McKinney-Freeman S.L. Naveiras O. Yates F. et al.Surface antigen phenotypes of hematopoietic stem cells from embryos and murine embryonic stem cells.Blood. 2009; 114: 268-278Crossref PubMed Scopus (87) Google Scholar]. Here, we employed OP9 co-culture to investigate the hematopoietic output of midgestation endothelium from E9.5, E10.5, and E11.5 embryos (Fig. 1A). VE-Cadherin+CD45− (VE+CD45−) cells were isolated by FACS from E9.5 caudal halves and from E10.5 or E11.5 AGM, UA, and VA and co-cultured at limiting dilution with OP9 cells in 96-well plates (Fig. 1 and Supplemental Fig. E1, online only, available at www.exphem.org). This population represents a mixture of HE and non-HE [28Kondo M. Weissman I.L. Akashi K. Identification of clonogenic common lymphoid progenitors in mouse bone marrow.Cell. 1997; 91: 661-672Abstract Full Text Full Text PDF PubMed Scopus (1646) Google Scholar], but should capture all the embryonic endothelium [12Nishikawa S.I. Nishikawa S. Hirashima M. Matsuyoshi N. Kodama H. Progressive lineage analysis by cell sorting and culture identifies FLK1+VE-cadherin+ cells at a diverging point of endothelial and hemopoietic lineages.Development. 1998; 125: 1747-1757Crossref PubMed Google Scholar, 13Nishikawa S.I. Nishikawa S. Kawamoto H. et al.In vitro generation of lymphohematopoietic cells from endothelial cells purified from murine embryos.Immunity. 1998; 8: 761-769Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 29Ayalon O. Sabanai H. Lampugnani M.G. Dejana E. Geiger B. Spatial and temporal relationships between cadherins and PECAM-1 in cell-cell junctions of human endothelial cells.J Cell Biol. 1994; 126: 247-258Crossref PubMed Scopus (175) Google Scholar, 30Matsuyoshi N. Toda K. Horiguchi Y. et al.In vivo evidence of the critical role of cadherin-5 in murine vascular integrity.Proc Assoc Am Physicians. 1997; 109: 362-371PubMed Google Scholar, 31Vittet D. Buchou T. Schweitzer A. Dejana E. Huber P. Targeted null-mutation in the vascular endothelial-cadherin gene impairs the organization of vascular-like structures in embryoid bodies.Proc Natl Acad Sci U S A. 1997; 94: 6273-6278Crossref PubMed Scopus (184) Google Scholar]. Individual wells were scrutinized 7 days later for hematopoietic colonies (Fig. 1A and B). Only 0.1%, 1.3%, and 0.29% of E9.5, E10.5, and E11.5 VE+CD45− cells generated hematopoietic colonies, respectively (Fig. 1B, Table 1, and Supplemental Table E1, online only, available at www.exphem.org). Therefore, hemogenic activity in this cell population peaks at E10.5. Consistent with this finding, the frequency of HE-derived c-Kit+ hematopoietic clusters also peaks at E10.5 in the mouse [32Yokomizo T. Dzierzak E. Three-dimensional cartography of hematopoietic clusters in the vasculature of whole mouse embryos.Development. 2010; 137: 3651-3661Crossref PubMed Scopus (178) Google Scholar].Table 1Limiting dilution analysis of hemogenic potential in E9.5, E10.5, and E11.5 mouse endotheliumExperimentStageHE frequencyHE frequency, % of total (95% CI)Chi-squared (Pearson)p valueChi-squared (deviance)p value1E9.51 in 5730.17 (0.056–0.54)0.19aLDA assumption holds well (p > 0.05).0.40aLDA assumption holds well (p > 0.05).E10.51 in 701.4 (1.1–1.9)0.03bLDA assumption does not hold (p < 0.05).0.013bLDA assumption does not hold (p < 0.05).E11.51 in 2670.4 (0.2–0.6)0.06aLDA assumption holds well (p > 0.05).0.09aLDA assumption holds well (p > 0.05).2E9.51 in 9750.1 (0.05–0.2)0.97aLDA assumption holds well (p > 0.05).0.91aLDA assumption holds well (p > 0.05).E11.51 in 4860.2 (0.08–0.55)0.60aLDA assumption holds well (p > 0.05).0.39aLDA assumption holds well (p > 0.05).3E9.51 in 1,0940.09 (0.02–0.36)0.62aLDA assumption holds well (p > 0.05).0.50aLDA assumption holds well (p > 0.05).E10.51 in 641.6 (1.2–2.1)0.68aLDA assumption holds well (p > 0.05).0.58aLDA assumption holds well (p > 0.05).E11.51 in 4360.2 (0.1–0.5)0.90aLDA assumption holds well (p > 0.05).0.80aLDA assumption holds well (p > 0.05).4E10.51 in 1050.95 (0.57–1.58)0.93aLDA assumption holds well (p > 0.05).0.93aLDA assumption holds well (p > 0.05).E11.51 in 2820.35 (0.27–0.46)0.35aLDA assumption holds well (p > 0.05).0.33aLDA assumption holds well (p > 0.05).a LDA assumption holds well (p > 0.05).b LDA assumption does not hold (p < 0.05). Open table in a new tab We next used flow cytometry to analyze and quantify the phenotype of the emerged hematopoietic colonies with respect to different hematopoietic markers (Fig. 1C–K). Specifically, we assessed quantitatively the output of individual HE cells regarding the following phenotypically defined mature and immature hematopoietic populations: Lineage+ (Lin+), Lin−, Lin−Sca-1lowc-Kitlow (enriched for common lymphoid progenitors, CLPs [28Kondo M. Weissman I.L. Akashi K. Identification of clonogenic common lymphoid progenitors in mouse bone marrow.Cell. 1997; 91: 661-672Abstract Full Text Full Text PDF PubMed Scopus (1646) Google Scholar]), Lin− Sca-1+c-Kit+ (LSK), Lin− Sca-1+c-Kitlow (this populations displays an intermediate CLP and LSK phenotype), LSK CD150−CD48− (multipotent progenitors, MPPs), LSK CD150−CD48+ (HPC-1), LSK CD150+CD48+ (HPC-2), and LSK CD150+CD48− (HSC) [33Kiel M.J. Yilmaz O.H. Iwashita T. Yilmaz O.H. Terhorst C. Morrison S.J. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.Cell. 2005; 121: 1109-1121Abstract Full Text Full Text PDF PubMed Scopus (2399) Google Scholar, 34Kiel M.J. Yilmaz O.H. Morrison S.J. CD150- cells are transiently reconstituting multipotent progenitors with little or no stem cell activity.Blood. 2008; 111 (author reply 4414-4415.): 4413-4414Crossref PubMed Scopus (45) Google Scholar, 35Oguro H. Ding L. Morrison S.J. SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors.Cell Stem Cell. 2013; 13: 102-116Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar] (Fig. 1). LSK cells are highly enriched for a heterogeneous mixture of HSPCs compared with whole bone marrow, including HPC-1, HPC-2, MPPs, and HSCs. HPC-1 and HPC-2 represent heterogeneous restricted progenitors with limited in vivo repopulating potential [33Kiel M.J. Yilmaz O.H. Iwashita T. Yilmaz O.H. Terhorst C. Morrison S.J. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.Cell. 2005; 121: 1109-1121Abstract Full Text Full Text PDF PubMed Scopus (2399) Google Scholar, 34Kiel M.J. Yilmaz O.H. Morrison S.J. CD150- cells are transiently reconstituting multipotent progenitors with little or no stem cell activity.Blood. 2008; 111 (author reply 4414-4415.): 4413-4414Crossref PubMed Scopus (45) Google Scholar, 35Oguro H. Ding L. Morrison S.J. SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors.Cell Stem Cell. 2013; 13: 102-116Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar]. MPPs are also heterogeneous with respect to lineage potential, but are generally considered to be the immediate downstream progeny of dHSCs and display multilineage short term in vivo repopulating activity [33Kiel M.J. Yilmaz O.H. Iwashita T. Yilmaz O.H. Terhorst C. Morrison S.J. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.Cell. 2005; 121: 1109-1121Abstract Full Text Full Text PDF PubMed Scopus (2399) Google Scholar, 34Kiel M.J. Yilmaz O.H. Morrison S.J. CD150- cells are transiently reconstituting multipotent progenitors with little or no stem cell activity.Blood. 2008; 111 (author reply 4414-4415.): 4413-4414Crossref PubMed Scopus (45) Google Scholar, 35Oguro H. Ding L. Morrison S.J. SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors.Cell Stem Cell. 2013; 13: 102-116Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar]. Long-term repopulating dHSCs are LSK CD150+CD48− [33Kiel M.J. Yilmaz O.H. Iwashita T. Yilmaz O.H. Terhorst C. Morrison S.J. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.Cell. 2005; 121: 1109-1121Abstract Full Text Full Text PDF PubMed Scopus (2399) Google Scholar, 34Kiel M.J. Yilmaz O.H. Morrison S.J. CD150- cells are transiently reconstituting multipotent progenitors with little or no stem cell activity.Blood. 2008; 111 (author reply 4414-4415.): 4413-4414Crossref PubMed Scopus (45) Google Scholar, 35Oguro H. Ding L. Morrison S.J. SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors.Cell Stem Cell. 2013; 13: 102-116Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar]. Remarkably, although we detected less functional HE at E11.5 than E10.5 (Fig. 1B), E11.5 HE trended toward an increased ability to produce large numbers of hematopoietic cells, including phenotypic dHSCs (Fig. 1C–K), although our previous studies indicated that the phenotypic dHSCs that emerge during OP9 co-culture cannot be transplanted [25Hadland B.K. Varnum-Finney B. Poulos M.G. et al.Endothelium and NOTCH specify and amplify aorta-gonad-mesonephros-derived hematopoietic stem cells.J Clin Invest. 2015; 125: 2032-2045Crossref PubMed Scopus (52) Google Scholar]. Because the frequency of functional HE in the VE+CD45− compartment peaked at E10.5 (Fig. 1B), we chose this developmental time point for further study. Although OP9 cells support the emergence of hematopoietic colonies they fail to promote the specification of bona fide dHSC from E9–E11 embryos with robust transplantation activity [25Hadland B.K. Varnum-Finney B. Poulos M.G. et al.Endothelium and NOTCH specify and amplify aorta-gonad-mesonephros-derived hematopoietic stem cells.J Clin Invest. 2015; 125: 2032-2045Crossref PubMed Scopus (52) Google Scholar]. In contrast, AA-ECs robustly support the m
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