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• W1996504403 abstract "•In vivo repopulating hematopoietic cells from serum-free differentiated ESCs•Repopulating activity arises immediately upon commitment of mesoderm•In vivo repopulating progenitors are extremely transient•In vivo repopulating progenitors are exquisitely sensitive to the cytokine milieu The generation of in vivo repopulating hematopoietic cells from in vitro differentiating embryonic stem cells has remained a long-standing challenge. To date, hematopoietic engraftment has mostly been achieved through the enforced expression of ectopic transcription factors. Here, we describe serum-free culture conditions that allow the generation of in vivo repopulating hematopoietic cells in the absence of ectopically expressed factors. We show that repopulating activity arises immediately upon the commitment of mesodermal precursors to the blood program, within the first wave of hematopoietic specification. We establish that the formation of these progenitors is extremely transient and exquisitely sensitive to the cytokine milieu. Our findings define the precise differentiating stage at which hematopoietic repopulating activity first appears in vitro, and suggest that during embryonic stem cell differentiation, all hematopoietic programs are unraveled simultaneously from the mesoderm in the absence of cues that restrict the coordinated emergence of each lineage as is normally observed during embryogenesis. The generation of in vivo repopulating hematopoietic cells from in vitro differentiating embryonic stem cells has remained a long-standing challenge. To date, hematopoietic engraftment has mostly been achieved through the enforced expression of ectopic transcription factors. Here, we describe serum-free culture conditions that allow the generation of in vivo repopulating hematopoietic cells in the absence of ectopically expressed factors. We show that repopulating activity arises immediately upon the commitment of mesodermal precursors to the blood program, within the first wave of hematopoietic specification. We establish that the formation of these progenitors is extremely transient and exquisitely sensitive to the cytokine milieu. Our findings define the precise differentiating stage at which hematopoietic repopulating activity first appears in vitro, and suggest that during embryonic stem cell differentiation, all hematopoietic programs are unraveled simultaneously from the mesoderm in the absence of cues that restrict the coordinated emergence of each lineage as is normally observed during embryogenesis. Recent advances in the generation, propagation, and differentiation of pluripotent stem cells (PSCs) offer great promise in the field of regenerative medicine. Both embryonic stem cells (ESCs) and induced PSCs (iPSCs) provide limitless sources of self-renewing cells endowed with the potential to generate tissue-specific cell populations that can be used in transplantation therapy (Grabel, 2012Grabel L. Prospects for pluripotent stem cell therapies: into the clinic and back to the bench.J. Cell. Biochem. 2012; 113: 381-387Crossref PubMed Scopus (29) Google Scholar, Keller, 2005Keller G. Embryonic stem cell differentiation: emergence of a new era in biology and medicine.Genes Dev. 2005; 19: 1129-1155Crossref PubMed Scopus (953) Google Scholar). However, one major hurdle in realizing this potential is the lack of specific and efficient protocols for differentiating these PSCs to specific populations that can be used for therapeutic applications. Although stem-cell-based regenerative medicine is still a distant goal, outstanding progress has been made in generating and engrafting ESC-derived lineages such as dopamine neurones (Kriks et al., 2011Kriks S. Shim J.W. Piao J. Ganat Y.M. Wakeman D.R. Xie Z. Carrillo-Reid L. Auyeung G. Antonacci C. Buch A. et al.Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease.Nature. 2011; 480: 547-551Crossref PubMed Scopus (1361) Google Scholar) and cardiomyocytes (Shiba et al., 2012Shiba Y. Fernandes S. Zhu W.Z. Filice D. Muskheli V. Kim J. Palpant N.J. Gantz J. Moyes K.W. Reinecke H. et al.Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts.Nature. 2012; 489: 322-325Crossref PubMed Scopus (574) Google Scholar, Yang et al., 2008Yang L. Soonpaa M.H. Adler E.D. Roepke T.K. Kattman S.J. Kennedy M. Henckaerts E. Bonham K. Abbott G.W. Linden R.M. et al.Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population.Nature. 2008; 453: 524-528Crossref PubMed Scopus (1170) Google Scholar). In contrast, since the first report of blood cell generation from ESCs 30 years ago (Doetschman et al., 1985Doetschman T.C. Eistetter H. Katz M. Schmidt W. Kemler R. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium.J. Embryol. Exp. Morphol. 1985; 87: 27-45PubMed Google Scholar), progress in deriving hematopoietic cells that are able to engraft in vivo has been rather modest. To date, the most successful in vitro derivation of hematopoietic cells capable of repopulating mouse models has relied on the ectopic expression of transcription factors such as HOXB4 (Kyba et al., 2002Kyba M. Perlingeiro R.C. Daley G.Q. HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors.Cell. 2002; 109: 29-37Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar), CDX4 (Wang et al., 2005bWang Y. Yates F. Naveiras O. Ernst P. Daley G.Q. Embryonic stem cell-derived hematopoietic stem cells.Proc. Natl. Acad. Sci. USA. 2005; 102: 19081-19086Crossref PubMed Scopus (177) Google Scholar), LHX2 (Kitajima et al., 2011Kitajima K. Minehata K. Sakimura K. Nakano T. Hara T. In vitro generation of HSC-like cells from murine ESCs/iPSCs by enforced expression of LIM-homeobox transcription factor Lhx2.Blood. 2011; 117: 3748-3758Crossref PubMed Scopus (43) Google Scholar), and RUNX1a (Ran et al., 2013Ran D. Shia W.J. Lo M.C. Fan J.B. Knorr D.A. Ferrell P.I. Ye Z. Yan M. Cheng L. Kaufman D.S. Zhang D.E. RUNX1a enhances hematopoietic lineage commitment from human embryonic stem cells and inducible pluripotent stem cells.Blood. 2013; 121: 2882-2890Crossref PubMed Scopus (100) Google Scholar). However, although HOXB4 overexpression has been shown to confer reproducible engraftment capability in differentiating mouse ESCs (Bonde et al., 2008Bonde S. Dowden A.M. Chan K.M. Tabayoyong W.B. Zavazava N. HOXB4 but not BMP4 confers self-renewal properties to ES-derived hematopoietic progenitor cells.Transplantation. 2008; 86: 1803-1809Crossref PubMed Scopus (16) Google Scholar, Kyba et al., 2002Kyba M. Perlingeiro R.C. Daley G.Q. HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors.Cell. 2002; 109: 29-37Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar, Lesinski et al., 2012Lesinski D.A. Heinz N. Pilat-Carotta S. Rudolph C. Jacobs R. Schlegelberger B. Klump H. Schiedlmeier B. Serum- and stromal cell-free hypoxic generation of embryonic stem cell-derived hematopoietic cells in vitro, capable of multilineage repopulation of immunocompetent mice.Stem Cells Transl. Med. 2012; 1: 581-591Crossref PubMed Scopus (15) Google Scholar, Matsumoto et al., 2009Matsumoto K. Isagawa T. Nishimura T. Ogaeri T. Eto K. Miyazaki S. Miyazaki J. Aburatani H. Nakauchi H. Ema H. Stepwise development of hematopoietic stem cells from embryonic stem cells.PLoS ONE. 2009; 4: e4820Crossref PubMed Scopus (41) Google Scholar), this approach has not been successfully translated to human ESCs (Wang et al., 2005aWang L. Menendez P. Shojaei F. Li L. Mazurier F. Dick J.E. Cerdan C. Levac K. Bhatia M. Generation of hematopoietic repopulating cells from human embryonic stem cells independent of ectopic HOXB4 expression.J. Exp. Med. 2005; 201: 1603-1614Crossref PubMed Scopus (266) Google Scholar). An alternative approach to the use of HOXB4 in differentiated human ESCs was recently reported by Doulatov et al., 2013Doulatov S. Vo L.T. Chou S.S. Kim P.G. Arora N. Li H. Hadland B.K. Bernstein I.D. Collins J.J. Zon L.I. Daley G.Q. Induction of multipotential hematopoietic progenitors from human pluripotent stem cells via respecification of lineage-restricted precursors.Cell Stem Cell. 2013; 13: 459-470Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, who showed that the ectopic expression of transcription factors (HOXA9, ERG, RORA, SOX4, and MYB) in differentiating ESCs promotes short-term erythroid and myeloid engraftment. Few reports have documented the in vitro generation of hematopoietic repopulating potential from unmanipulated ESCs (Burt et al., 2004Burt R.K. Verda L. Kim D.A. Oyama Y. Luo K. Link C. Embryonic stem cells as an alternate marrow donor source: engraftment without graft-versus-host disease.J. Exp. Med. 2004; 199: 895-904Crossref PubMed Scopus (97) Google Scholar, Hole et al., 1996Hole N. Graham G.J. Menzel U. Ansell J.D. A limited temporal window for the derivation of multilineage repopulating hematopoietic progenitors during embryonal stem cell differentiation in vitro.Blood. 1996; 88: 1266-1276PubMed Google Scholar, Müller and Dzierzak, 1993Müller A.M. Dzierzak E.A. ES cells have only a limited lymphopoietic potential after adoptive transfer into mouse recipients.Development. 1993; 118: 1343-1351PubMed Google Scholar, Potocnik et al., 1997Potocnik A.J. Kohler H. Eichmann K. Hemato-lymphoid in vivo reconstitution potential of subpopulations derived from in vitro differentiated embryonic stem cells.Proc. Natl. Acad. Sci. USA. 1997; 94: 10295-10300Crossref PubMed Scopus (68) Google Scholar). However, these approaches have not been reproduced or pursued, suggesting that they involve serum-dependent conditions that cannot be easily replicated. The use of high serum concentrations (Wang et al., 2005aWang L. Menendez P. Shojaei F. Li L. Mazurier F. Dick J.E. Cerdan C. Levac K. Bhatia M. Generation of hematopoietic repopulating cells from human embryonic stem cells independent of ectopic HOXB4 expression.J. Exp. Med. 2005; 201: 1603-1614Crossref PubMed Scopus (266) Google Scholar) and/or stroma cell lines (Ledran et al., 2008Ledran M.H. Krassowska A. Armstrong L. Dimmick I. Renström J. Lang R. Yung S. Santibanez-Coref M. Dzierzak E. Stojkovic M. et al.Efficient hematopoietic differentiation of human embryonic stem cells on stromal cells derived from hematopoietic niches.Cell Stem Cell. 2008; 3: 85-98Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar) to support the formation of repopulating hematopoietic cells derived from human ESCs has also shown promising results, but to date, no follow-up studies have further validated or extended these differentiation protocols. It is likely that the reported successes in deriving repopulating hematopoietic cells relied on specific factors present in rare batches of serum—parameters that are impossible to control for and thus are extremely difficult to reproduce. It is thought that a better understanding of the molecular and cellular mechanisms that regulate the emergence and maintenance of long-term repopulating hematopoietic stem cells (HSCs) during embryonic development would aid in the development of optimal protocols to generate such cells in vitro from PSCs. HSCs have been shown to emerge first from the aorta-gonad-mesonephros (AGM) region around embryonic day 10.5 (E10.5) in murine embryos (Medvinsky and Dzierzak, 1996Medvinsky A. Dzierzak E. Definitive hematopoiesis is autonomously initiated by the AGM region.Cell. 1996; 86: 897-906Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar). This occurs several days after the actual onset of hematopoietic activity, which is observed first in the yolk sac from E7.5 and next in the embryo proper from E9.0 (Palis et al., 1999Palis J. Robertson S. Kennedy M. Wall C. Keller G. Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse.Development. 1999; 126: 5073-5084Crossref PubMed Google Scholar). These early waves of hematopoiesis successively give rise to primitive erythroid, myeloid, definitive erythroid, and lymphoid progenitors (Costa et al., 2012Costa G. Kouskoff V. Lacaud G. Origin of blood cells and HSC production in the embryo.Trends Immunol. 2012; 33: 215-223Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, Lin et al., 2014Lin Y. Yoder M.C. Yoshimoto M. Lymphoid progenitor emergence in the murine embryo and yolk sac precedes stem cell detection.Stem Cells Dev. 2014; 23: 1168-1177Crossref PubMed Scopus (49) Google Scholar). Several studies, including lineage tracing (Zovein et al., 2008Zovein A.C. Hofmann J.J. Lynch M. French W.J. Turlo K.A. Yang Y. Becker M.S. Zanetta L. Dejana E. Gasson J.C. et al.Fate tracing reveals the endothelial origin of hematopoietic stem cells.Cell Stem Cell. 2008; 3: 625-636Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar) and in vivo imaging (Boisset et al., 2010Boisset J.C. van Cappellen W. Andrieu-Soler C. Galjart N. Dzierzak E. Robin C. In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium.Nature. 2010; 464: 116-120Crossref PubMed Scopus (672) Google Scholar) studies, have revealed the endothelial origin of HSCs emerging from a hemogenic endothelium (HE) population within the AGM region. Similarly, earlier waves of hematopoietic progenitors were also shown to derive from the HE (Ema et al., 2006Ema M. Yokomizo T. Wakamatsu A. Terunuma T. Yamamoto M. Takahashi S. Primitive erythropoiesis from mesodermal precursors expressing VE-cadherin, PECAM-1, Tie2, endoglin, and CD34 in the mouse embryo.Blood. 2006; 108: 4018-4024Crossref PubMed Scopus (88) Google Scholar, Lancrin et al., 2010Lancrin C. Sroczynska P. Serrano A.G. Gandillet A. Ferreras C. Kouskoff V. Lacaud G. Blood cell generation from the hemangioblast.J. Mol. Med. 2010; 88: 167-172Crossref PubMed Scopus (57) Google Scholar, Nishikawa et al., 1998Nishikawa 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). The in vitro differentiation of ESCs has been widely used as a model system to dissect and understand the early events of hematopoietic specification in terms of both molecular mechanisms and cellular steps. The careful dissection of this in vitro program has demonstrated that, similarly to in vivo development, blood cells are generated from mesodermal hemangioblast precursors through an HE intermediate (Choi et al., 1998Choi K. Kennedy M. Kazarov A. Papadimitriou J.C. Keller G. A common precursor for hematopoietic and endothelial cells.Development. 1998; 125: 725-732Crossref PubMed Google Scholar, Choi et al., 2012Choi K.D. Vodyanik M.A. Togarrati P.P. Suknuntha K. Kumar A. Samarjeet F. Probasco M.D. Tian S. Stewart R. Thomson J.A. Slukvin I.I. Identification of the hemogenic endothelial progenitor and its direct precursor in human pluripotent stem cell differentiation cultures.Cell Rep. 2012; 2: 553-567Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, Eilken et al., 2009Eilken H.M. Nishikawa S. Schroeder T. Continuous single-cell imaging of blood generation from haemogenic endothelium.Nature. 2009; 457: 896-900Crossref PubMed Scopus (469) Google Scholar, Fehling et al., 2003Fehling H.J. Lacaud G. Kubo A. Kennedy M. Robertson S. Keller G. Kouskoff V. Tracking mesoderm induction and its specification to the hemangioblast during embryonic stem cell differentiation.Development. 2003; 130: 4217-4227Crossref PubMed Scopus (399) Google Scholar, Huber et al., 2004Huber T.L. Kouskoff V. Fehling H.J. Palis J. Keller G. Haemangioblast commitment is initiated in the primitive streak of the mouse embryo.Nature. 2004; 432: 625-630Crossref PubMed Scopus (550) Google Scholar, Kennedy et al., 2007Kennedy M. D’Souza S.L. 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 (363) Google Scholar, Lancrin et al., 2009Lancrin 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 (501) Google Scholar, Wang et al., 2004Wang L. Li L. Shojaei F. Levac K. Cerdan C. Menendez P. Martin T. Rouleau A. Bhatia M. Endothelial and hematopoietic cell fate of human embryonic stem cells originates from primitive endothelium with hemangioblastic properties.Immunity. 2004; 21: 31-41Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar) and that the same network of transcription factors orchestrates both in vivo and in vitro processes (Moignard et al., 2013Moignard V. Woodhouse S. Fisher J. Göttgens B. Transcriptional hierarchies regulating early blood cell development.Blood Cells Mol. Dis. 2013; 51: 239-247Crossref PubMed Scopus (16) Google Scholar). Detailed studies of the generation of primitive erythroid, myeloid, and lymphoid progenitors have suggested a temporal emergence of these blood lineages in vitro, reflecting their sequential emergence in vivo during embryonic development (Irion et al., 2010Irion S. Clarke R.L. Luche H. Kim I. Morrison S.J. Fehling H.J. Keller G.M. Temporal specification of blood progenitors from mouse embryonic stem cells and induced pluripotent stem cells.Development. 2010; 137: 2829-2839Crossref PubMed Scopus (64) Google Scholar). This led to the concept that repopulating activity might emerge at late stages of the hematopoietic program during ESC differentiation (Kardel and Eaves, 2012Kardel M.D. Eaves C.J. Modeling human hematopoietic cell development from pluripotent stem cells.Exp. Hematol. 2012; 40: 601-611Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, Lis et al., 2013Lis R. Rafii S. James D. Wading through the waves of human embryonic hemogenesis.Cell Cycle. 2013; 12: 859-860Crossref PubMed Scopus (2) Google Scholar, Sturgeon et al., 2013Sturgeon C.M. Ditadi A. Clarke R.L. Keller G. Defining the path to hematopoietic stem cells.Nat. Biotechnol. 2013; 31: 416-418Crossref PubMed Scopus (37) Google Scholar) and that the emergence of lymphoid potential marks the establishment of the definitive program (Kennedy et al., 2012Kennedy M. Awong G. Sturgeon C.M. Ditadi A. LaMotte-Mohs R. Zúñiga-Pflücker J.C. Keller G. T lymphocyte potential marks the emergence of definitive hematopoietic progenitors in human pluripotent stem cell differentiation cultures.Cell Rep. 2012; 2: 1722-1735Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, Slukvin, 2013Slukvin I.I. Hematopoietic specification from human pluripotent stem cells: current advances and challenges toward de novo generation of hematopoietic stem cells.Blood. 2013; 122: 4035-4046Crossref PubMed Scopus (94) Google Scholar). To date, however, attempts to derive in vivo repopulating hematopoietic cells from late stages of ESC differentiation have been largely unsuccessful. To revisit this long-standing challenge, we took an alternative approach and explored the very first step of hematopoietic specification from the mesoderm. We hypothesized that multilineage progenitors with in vivo repopulating ability might be specified very early upon commitment of mesoderm to the blood program, and might be difficult to maintain as such in the presence of serum or hematopoietic cytokines. We first evaluated the growth factor requirement for optimal specification of hemangioblast to HE. Next, defining the full hematopoietic potential of this emerging population, we observed the concomitant emergence of erythroid, myeloid, and lymphoid progenitors. Interestingly, this early population was also endowed with the capability to engraft immunocompromised mice and to confer multilineage, long-term engraftment. Further studies allowed us to define the temporal emergence of this repopulating ability and to determine the growth factor requirement and immunophenotypic characteristic of this population. Collectively, our findings demonstrate that in vitro repopulating cells emerge very rapidly from mesoderm precursors, are extremely transient, and are exquisitely sensitive to the growth factors present in the differentiating conditions. We previously showed that a combination of BMP4, Activin A, FGF, and VEGF was sufficient to efficiently drive the formation of blood precursors from differentiating ESCs in serum-free culture conditions (Pearson et al., 2008Pearson S. Sroczynska P. Lacaud G. Kouskoff V. The stepwise specification of embryonic stem cells to hematopoietic fate is driven by sequential exposure to Bmp4, activin A, bFGF and VEGF.Development. 2008; 135: 1525-1535Crossref PubMed Scopus (136) Google Scholar). However, optimal specification to each differentiation stage is likely to require precise temporal exposure to cytokine stimuli. Therefore, we set out to define which cytokines were specifically required for the transition from hemangioblast to HE, and then from HE to hematopoietic progenitors, with a particular emphasis on HE, from which repopulating cells are known to emerge in vivo (Bertrand et al., 2010Bertrand J.Y. Chi N.C. Santoso B. Teng S. Stainier D.Y. Traver D. Haematopoietic stem cells derive directly from aortic endothelium during development.Nature. 2010; 464: 108-111Crossref PubMed Scopus (753) Google Scholar, Boisset et al., 2010Boisset J.C. van Cappellen W. Andrieu-Soler C. Galjart N. Dzierzak E. Robin C. In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium.Nature. 2010; 464: 116-120Crossref PubMed Scopus (672) Google Scholar, Kissa and Herbomel, 2010Kissa K. Herbomel P. Blood stem cells emerge from aortic endothelium by a novel type of cell transition.Nature. 2010; 464: 112-115Crossref PubMed Scopus (701) Google Scholar). As depicted in Figure 1A, ESCs were differentiated via embryoid body (EB) for 3 days in serum-free culture with the successive addition of BMP4 at day 0, and then Activin A and FGF at day 2.5. This sequential exposure to growth factors was previously shown to promote hemangioblast specification efficiently in developing mesoderm (Pearson et al., 2008Pearson S. Sroczynska P. Lacaud G. Kouskoff V. The stepwise specification of embryonic stem cells to hematopoietic fate is driven by sequential exposure to Bmp4, activin A, bFGF and VEGF.Development. 2008; 135: 1525-1535Crossref PubMed Scopus (136) Google Scholar). At day 3 of the EB culture, FLK1+ cells enriched for hemangioblast were isolated and then further cultured with no added factors, a combination of four factors (BMP4 [B], Activin A [A], FGF [F], and VEGF [V]), or various combinations of these factors. The successful differentiation of FLK1+ cells into HE was measured at day 2 of the culture by the coexpression of TIE2 and cKIT, as previously described (Lancrin et al., 2009Lancrin 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 (501) Google Scholar). The efficient generation of hematopoietic progenitors was assessed at day 3 by CD41 expression, which is known to mark emerging progenitors (Ferkowicz et al., 2003Ferkowicz M.J. Starr M. Xie X. Li W. Johnson S.A. Shelley W.C. Morrison P.R. Yoder M.C. CD41 expression defines the onset of primitive and definitive hematopoiesis in the murine embryo.Development. 2003; 130: 4393-4403Crossref PubMed Scopus (267) Google Scholar, Mikkola et al., 2003Mikkola H.K. Fujiwara Y. Schlaeger T.M. Traver D. Orkin S.H. Expression of CD41 marks the initiation of definitive hematopoiesis in the mouse embryo.Blood. 2003; 101: 508-516Crossref PubMed Scopus (308) Google Scholar). In the absence of added factors, few cells coexpressed TIE2 and cKIT (Figure 1B), and the generation of CD41+ cells was limited (Figure 1C). In contrast, the addition of all factors (BAFV) led to the detection of a substantial TIE2+cKIT+ population and the enhanced generation of CD41+ cells. Dissecting the role of each factor individually or in combination revealed that individual factors on their own and most combinations were not able to generate or maintain an HE population (Figure 1B) and/or to produce a substantial frequency of CD41+ cells (Figure 1C). Both Activin A and VEGF appeared to be critically required for the generation and maintenance of HE cells, since only culture conditions containing both factors led to the formation of a clear TIE2+cKIT+ population (AV, AFV, and BAV). As observed in V, BV, and BFV culture conditions, the absence of Activin A in the culture led to CD41 cell production associated with a decrease in TIE2+cKIT+ frequency that was already observed at day 2 (Figures 1B and 1C). In contrast, the absence of BMP4 in the culture led to a dramatic decrease in CD41+ cell production, as observed in A, AV, and AFV culture conditions, suggesting that while this factor is dispensable for the generation or maintenance of a TIE2+cKIT+ population, BMP4 is required for the emergence of CD41+ cells. To address this issue, we supplemented AV culture with BMP4 at day 2 and assayed for CD41 expression at day 3 (Figures S1A and S1B). However, the delayed addition of BMP4 did not enhance the generation of CD41+ cells, suggesting that although it does not impact the generation of a TIE2+cKIT+ immunophenotypic population from FLK1+ cells, BMP4 exposure is nonetheless critical for shaping the hematopoietic potential of this population at the onset of FLK1 differentiation. The expression of a panel of endothelial markers, such as ICAM2, FLK1, and CD144 (VE-cadherin), further revealed that the presence of both BMP4 and Activin A was critical to maintain the endothelial identity of the cKIT+ population at day 2 of the culture (Figure S1C). Only a fraction of cKIT+ cells maintained the expression of these endothelial markers when cultured in the presence of AFV or BFV. Altogether, these data revealed that the combination of BMP4, Activin A, and VEGF is critical for the generation of both HE and CD41+ cells. Interestingly, early exposure to BMP4 appears to confer hematopoietic potential to the TIE2+cKIT+ population. We next compared the emergence and frequency of HE when FLK1+ cells were cultured with BAV and BAFV, as these two conditions were the most effective for generating HE (Figure 1B). In both cases, a low frequency of TIE2+cKIT+ cells was observed at day 1 of the culture; the frequency of this population peaked at day 2 and decreased thereafter (Figure 2A). No noticeable differences were observed in the temporal emergence and frequency of this population regardless of whether FGF was added to the culture or not (Figures 2A and 3D ). In contrast, the formation of CD41+ cells was negatively affected by the presence of FGF in the culture, with on average a 2-fold increase in the frequency of CD41+ cells produced in the absence of FGF from day 2 onward (Figures 2B and 3E). Both cultures gave rise to primitive erythroid and definitive colonies upon replating in clonogenic assays; however, FLK1+ cells cultured in the BAV condition resulted in the production of higher frequencies of hematopoietic precursors (Figure 2C), in agreement with the CD41 flow cytometry data. Altogether, these data suggest that although FGF does not affect the temporal emergence and frequency of HE, this growth factor negatively impacts the formation of hematopoietic progenitors.Figure 3Activin A Impairs the Maintenance of HEShow full caption(A) Schematic representation of the experimental strategy. FLK1+ cells sorted from day 3 EBs were seeded on gelatinized plates in serum-free media supplemented with BAV for the first day and then with BV from day 1 onward (B, BMP4; A, Activin A; V, VEGF).(B) Flow cytometry analysis of TIE2 and cKIT coexpression at day 1 and 2 of FLK1+ cell culture grown in a BAV or BAV-BV cytokine combination.(C) Flow cytometry analysis of CD41 expression at days 2 and 3 of the same cultures.(D and E) Graph of data obtained from BAFV, BAV, and BAV-BV cultures, showing the frequencies of TIE2+cKIT+ cells at day 2 (D) and CD41+ cells at day 3 (E). Each point represents an independent experiment.(F and G) Representative time-lapse imaging of FLK1+ sorted cells cultured in serum-free media supplemented with a BAV-BV (F) or BAV (G) cytokine combination. Data shown are representative of at least three independent experiments (n.s., nonsignificant).See also Figure S2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Schematic representation of the experimental strategy. FLK1+ cells sorted from day 3 EBs were seeded on gelatinized plates in serum-free media supplemented with BAV for the first day and then with BV from day 1 onward (B, BMP4; A, Activin A; V, VEGF). (B) Flow cytometry analysis of TIE2 and cKIT coexpression at day 1 and 2 of FLK1+ cell culture grown in a BAV or BAV-BV cytokine combination. (C) Flow cytometry analysis of CD41 expression at days 2 and 3 of the same cultures. (D and E) Graph of data obtained from BAFV, BAV, and BAV-BV cultures, showing the frequencies of TIE2+cKIT+ cells at day 2 (D) and CD41+ cells at day 3 (E). Each point represents an independent experiment. (F and G) Representative time-lapse imaging of FLK1+ sorted cells cultured in serum-free media supplemented with a BAV-BV (F) or BAV (G) cytoki" @default.
• W1996504403 created "2016-06-24" @default.
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• W1996504403 date "2015-03-01" @default.
• W1996504403 modified "2023-10-04" @default.
• W1996504403 title "In Vivo Repopulating Activity Emerges at the Onset of Hematopoietic Specification during Embryonic Stem Cell Differentiation" @default.
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