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a U362 Inserm, PR1, Institut Gustave Roussy, and
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b Department of Clinical Biology, Institut Gustave Roussy, Villejuif, France;1 I( m( q% e/ M; ?4 x3 c5 C
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c U506 Inserm, H?pital Paul Brousse, Villejuif, France;1 l; g9 N. T9 O! i$ X! \; J
+ v6 z; B2 W: Ld Children’s Hospital of Pittsburgh–Rangos Research Institute, Pittsburgh, PA, USA
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$ `" C5 q% ~ e/ b% D6 ]/ LKey Words. Hematopoietic stem cell ? Erythropoiesis ? Human embryo ? Embryonic liver
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Correspondence: E. Lauret, Ph.D., U362 Inserm, Institut Gustave Roussy, PR1, 94800 Villejuif, France. Telephone: 33-1-42-11-42-33; Fax: 33-1-42-11-52-40; e-mail: elauret@igr.fr
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( S, `- B/ e. t" k5 {& D, ZABSTRACT
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- ^5 t. J. P3 r7 p4 ~! g' C; wThe hematopoietic system of higher vertebrates emerges in a series of finely controlled spatial and temporal events that occur through the sequential appearance and colonization of specific embryonic territories . Hematopoietic cells are first detected in the yolk sac, defined as the site of primitive hematopoiesis, which is responsible for the preliminary wave of circulating blood cells but does not contain long-term repopulating hematopoietic stem cells (HSCs). Definitive hematopoiesis, which implies the stem cells that give rise to the hematopoietic system in the adult, appears in the embryo, within the aorta-gonads-mesonephros (AGM) region. We previously mapped the emergence of definitive hematopoiesis in the human embryo to the truncal part of the dorsal aorta and vitelline artery within the AGM, from 27–40 days postconception (dpc) . From 31 dpc onward, hepatic hematopoiesis commences, which remains the major hematopoietic organ for the first trimester, an event which we have suggested to occur through the colonization of the liver by the AGM-derived HSCs.
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; A& O' N7 F' V; g8 U3 dNotch was first identified in Drosophila , in which it was demonstrated to specifically control wing development but was later determined to also influence the development of many other tissues . Notch-mediated signal transduction represents a highly conserved series of events with implications in asymmetric cell division , lateral inhibition , and cell fate determination in both developmental and adult processes.- }2 Y4 N$ r( h2 H- S# z; f
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As a result of the direct influence on cell fate, the hematopoietic system from both mice and humans has received extensive study to determine if activation of the Notchpathway can influence HSC fate. Although knockout studies have not shown a clear role for Notch in adult HSCs, experiments implying Notch activation show that Notch does modulate adult HSC fate. The constitutive expression of an active form of Notch1 in murine hematopoietic progenitors promotes HSC self-renewal . Notch ligands, Jagged1–2, and Delta1 inhibit differentiation of hematopoietic progenitors . Furthermore, Jagged1 and Delta1 were shown to be growth factors for hematopoietic progenitors . In contrast, we and others have documented a role for Delta4 and Jagged1 as negative regulators of the cell cycle .9 v' h7 F# f! D6 }8 T8 E
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Previous work has argued a role for Notch signaling in the development of the murine hematopoietic system. Coculture of AGM or d11 fetal liver CD34 c-kit cells with S17 stroma expressing Jagged1 increased the committed colony-forming potential of the output cells . Recent studies in mice implicate Notch1 in the generation of the earliest embryonic HSCs . Splanchnopleural explant cultures from Notch1-deficient but not Notch2-deficient mice were markedly impaired in their ability to generate hematopoietic colonies.0 _3 ^3 |4 f, B
1 X' u) j1 `0 H& D! H- u/ |& eIn the study reported here, we addressed the role of the Notch signaling pathway in human hematopoietic development. Using, in parallel, immunohistochemistry and in vitro analysis of embryonic hematopoietic progenitors cocultured with Notch ligand–expressing stroma, we have documented a role for Notch activation during the first trimester of human embryonic hematopoietic emergence. Chronologically, Notch expression in the major hematopoietic sites was only detected after the establishment of definitive hematopoiesis, while functional studies of the Notch ligands detected by immunohistochemistry showed that Notch activation through Delta4 induces fetal CD34 CD38– cells to generate erythroid progenitors, while maintaining their long-term culture-initiating cell (LTC-IC) potential, thus preventing progenitor cell exhaustion in human embryonic development.
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! ~7 }/ H" c/ Z; g6 `MATERIALS AND METHODS' A+ P/ [' K2 p" U' Z- ?, s
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Detection After Commencement of Definitive Hematopoiesis in the Embryonic Liver8 v, ]& _7 v( }% T) ]
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We first focused on in situ analysis of Notch and Notch ligand protein expression (Notch1, Notch2, and Notch4; Delta1 and Delta4; Jagged1) to the preliminary sites of hematopoietic development. Extraembryonic blood islands, which appear in the human yolk sac from 16–17 dpc and give rise to the first wave of blood cell circulation, did not express any of the Notch or Notch ligands tested (data not shown). We analyzed extraembryonic tissues from 20–25 dpc (n = 6), representing Carnegie stages 9 to 11 (Table 1), and never detected expression in the blood islands. Furthermore, no expression of Notch or Notch ligands was detected anywhere within the embryos analyzed.) \4 r8 c4 Y( D/ C5 N
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From 27–40 dpc, hematopoiesis switches to the embryo proper on the ventral wall of the dorsal aorta in the AGM region. The CD34 CD45 cell clusters found here also did not express any of the Notch and Notch ligands tested, although a weak Jagged1 expression encircling the aorta was observed, while rare cells expressing Notch1 were found both anterior and posterior to the aorta (data not shown).2 D3 N6 H9 p" D ], F
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We next switched our focus to the embryonic liver, which represents the major hematopoietic organ within the developing embryo and fetus in the first trimester of development. The onset of CD34 cell-mediated hematopoiesis commences here at approximately 30 dpc, and we were only able to detect Notch1, Notch2, and Delta4 expression from 34 dpc onward, and then only at a low frequency. No Notch or Notch ligand expression was detected in the liver prior to this time. We continued to observe the rare expression of these proteins for the remainder of the gestational period analyzed (Table 1). In one embryo at 36 dpc, in addition to Notch1, Notch2, and Delta4, we observed the rare appearance of cells expressing Delta1 (Fig. 1). In all cases, the major proteins expressed were Notch1, Notch2, and Delta4, all at low frequency, which prevented us from comparing sequential tissue sections and from clearly determining whether the positive cells were also expressing CD34 or CD45 or were part of the liver vasculature. It is noteworthy that at no time in any of the tested hematopoietic tissues did we observe expression of the endothelium-associated Notch4 protein or Jagged1. However, we could rarely detect Notch4 in other vascular tissues, while Jagged1 was highly expressed in the neural tube, mesonephros, and hepatic ductal plate and rarely in the vasculature (data not shown).
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Figure 1. Notch and Notch ligand expression in the embryonic human liver. The 36-day liver sections are shown, stained with antibodies to (A) CD34, (B) CD45, (C) Notch1, (D) Notch2, (E) Notch4, (F) Jagged1, (G) Delta1, (H) Delta4. Scale bar: 25 μm.# i( {" s( s+ c5 G
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We next wished to more directly determine if the Notch expression observed in situ was present on sorted CD34 CD38– hematopoietic progenitors. Following purification, analysis of the 6.5- to 9.5-week-old CD34 CD38– mononuclear cells revealed that a mean of 26.7% and 32.3% of these cells expressed both Notch1 and Notch2 proteins (Fig. 2A), with a mean intensity of fluorescence of 37.2 and 36.48, respectively. We did not find any apparent correlation with gestational stage and protein expression (Table 2). Similar to in situ analysis, no Notch4 expression could be observed on these cells (data not shown).
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Figure 2. Notch1–2 protein expression on embryonic liver CD34 CD38– cells. Mononuclear cells from first-trimester embryonic liver were stained for CD34, CD38, and Notch molecules. Following gating on the CD34 CD38– population, cells were analyzed for coexpression of CD34 and Notch1 or Notch2.; p% `8 C; t9 B/ y
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Table 2. Notch1–2 protein expression on embryonic liver CD34 CD38– cells
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Delta4 Enhances BFU-E Generation, |: |; z; B$ [( z* j& G
& ~. j$ j& |! C8 p- ?Because Delta4 appeared to be the highest expressed Notch ligand in the embryonic liver during the first trimester, we wanted to determine if any effect, and what effect, was elicited on embryonic HSCs following activation through this Notch ligand. For this purpose, we used 6.5- to 9.5-week-old embryonic liver CD34 CD38– mononuclear cells as HSCs. Preliminary characterization revealed that liver-derived first-trimester CD34 CD38– mononuclear cells represented 2.2% ± 1.1% of the total mono-nuclear population, with no detectable CD3, CD15, CD19, or CD33 expression, although low levels of CD56 were observed. Clonogenically, 77 ± 11 hematopoietic colonies (including 18 ± 5 BFU-E) per 1,000 CD34 CD38– cells were generated in standard colony-forming unit-culture (CFU-C) assays, while the LTC-IC frequency was approximately 0.014 ± 0.003 (n = 5), in which each LTC-IC gave rise to 6.1 ± 4.4 clonogenic progenitors.7 w) A, R7 G6 U9 c
4 ?: Y" ^" ~* U0 ~+ r0 ]0 CThe role of Delta4 on HSCs was analyzed as follows. CD34 CD38– mononuclear cells obtained from human embryonic liver at 6.5–9.5 weeks of gestation were cultured with either S17 stroma stably transfected with an empty vector (C/S17) or S17 stroma stably transfected with a construct coding for the membrane-bound form of Delta4 (mb4/S17), in a cytokine-rich milieu known to support the proliferation of primitive HSCs, for 7 days. Output CD34 mononuclear cells were then purified by cell sorting, analyzed for hematopoietic characteristics, or replated onto fresh stromal layers for a further 7 days.
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Following culture of 1,000 CD34 CD38– cells with either the C/S17 or mb4/S17 stroma (n = 12), no significant differences were observed in the number of output CD34 mononuclear cells at weeks 1 and 2 (28 ± 23 x 103 versus 12 ± 7 x 103 for C/S17 and 11 ± 7 x 103 versus 8 ± 4 x 103 for mb4/S17), as well as the percentage of CD34 cells at weeks 1 and 2 (16% ± 12% versus 15% ± 11% for C/S17 and 10% ± 5% versus 9% ± 6% for mb4/S17). In contrast, clonogenicity of the output CD34 cells revealed that mb4/S17 significantly augmented the total colony number per 1,000 output CD34 cells at week 1 (104 ± 68 colonies for mb4/S17 versus 68 ± 31 for C/S17; p = .03) (Fig. 3A). Dissection of the colony type generated revealed that the C/S17 and mb4/S17 stromas generated equivalent numbers of nonerythroid myeloid colonies (67 ± 18 colonies for mb4/S17 versus 59 ± 17 for C/S17) (Fig. 3B). More striking was the effect of mb4/S17 on the erythroid cell–forming capacity of CD34 cells. After 7 days of culture with mb4/S17, output CD34 cells gave rise to 35% of BFU-E (36 ± 10 colonies for mb4/S17 versus 14 ± 12 for C/S17 per 1,000 output CD34 cells; p = .001) (Fig. 3C). A similar significantly higher number of BFU-E was observed with mb4/S17 at week 2 (16 ± 7 colonies for mb4/ S17 versus 4 ± 3 for C/S17 per 1,000 output CD34 cells; p = .001) (Fig. 3C). No differences in myeloid colony type were detected at week 2 of culture between mb4/S17 and C/S17 (Fig. 3B).' V [0 W& d" r; g! M, y( {
# Z7 |4 S% K7 l0 F# w& t. fFigure 3. Effect of Delta4 on hematopoietic progenitors. Following coculture of CD34 CD38– mononuclear cells with either control (white) or Delta4 (gray) stroma, output CD34 cells were assessed for their direct colony-forming capacity. (A): Frequency of total colonies. (B): Frequency of nonerythroid colonies. (C): Frequency of erythroid colonies, all at weeks 1 and 2. (D): Following coculture of CD34 CD38– cells with either control (white) or Delta4 (gray) stroma, in the presence of 10 μM DAPT (a secretase inhibitor: N--(S)-phenylglycine t-butyl ester) or dimethyl sulfoxide (DMSO) (as control) for 1 week, output nucleated cells were assessed for their BFU-E content. Asterisks indicate statistical significance: *p ) b' o, E4 f; I, p. G% z
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To assess the implication of Notch signaling in the enhanced production of BFU-E observed following culture of CD34 CD38– cells with mbD4/S17 stroma, we performed the 7-day culture in the presence of a -secretase inhibitor that is capable of blocking Notch cleavage. Addition of the -secretase inhibitor significantly (p = .001) reduced the erythroid-enhancing activity of mbD4 stroma (Fig. 3D). This strongly supports the involvement of Notch signaling in the enhanced BFU-E production by Delta4.
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5 K& q4 e3 U i/ C; ?2 |4 T2 vThe CD34 CD38– cell population, though representing purified primitive cells, possesses significant amounts of functional diversity. It therefore became important to determine whether Delta4 was expanding progenitors already committed toward the erythroid lineage or favoring differentiation toward erythropoiesis. To address this question, CD34 CD38– cells were seeded at 1 cell per well on either the C/S17 or mb4/S17 stroma in the presence of cytokines. After 1 week, the total cells were assessed for their erythroid colony-forming potential. Both the C/S17 and mb4/S17 stroma conditions yielded equivalent frequencies of wells that could generate colonies (16.2% ± 2.8% and 17.8% ± 1.1%, respectively; n = 4) (Table 3), while the frequency of wells containing erythroid colonies was augmented in the mb4/S17 condition (42% ± 7% for mb4/S17 compared with 31% ± 5% for C/S17). No differences were observed in the frequency of wells giving rise to myeloid colonies. Additionally, the fact that no well gave rise to exclusive erythroid colonies and that the number of erythroid colonies generated per single cell was almost identical
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w8 L/ O2 M. ~2 I+ C: Y: j0 t% \Table 3. Clonal analysis of Delta4 influence on BFU-E generation from embryonic liver hematopoietic cells (n = 4)6 o. Z' k- Z3 z8 j' Y$ O
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To confirm that the effect of Delta4 on BFU-E generation was specific to this Delta isoform, similar experiments were performed using S17 expressing the membrane-bound form of Delta1 (mb1/S17). This stroma has been previously demonstrated to maintain a high proportion of LTC-ICs in cord blood CD34 cells in culture, when compared with culture grown on control stroma (data not shown). In neither the bulk culture nor the single-cell cloning experiments did Delta1 elicit an effect on erythropoiesis (data not shown), implying that the observed effects were indeed specific for Delta4.
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# k- i5 o+ c3 T& z9 H" _Delta4 and LTC-IC Frequency' F9 o" j. E @% r7 O- j% S
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In parallel to the study on committed progenitors, we also examined the effect of Delta4 on more primitive progenitors, as measured using the in vitro LTC-IC assay. Following the initial 7-day coculture period with either the control or the Delta4 stroma, the output CD34 cells were plated on MS-5 for 5 weeks and then cultured in methylcellulose to analyze the LTC-IC–derived CFCs. The frequency of LTC-ICs in output CD34 cells was maintained following mb4/S17 coculture (Fig. 4) at a level similar to the one of input CD34 CD38– cells (frequency of 0.04 versus 0.01, respectively). In contrast, coculture with the C/S17 stroma led to a rapid and significant decrease in the LTC-IC frequency (0.0043), which was 10-fold lower than the LTC-IC frequency of output CD34 cells exposed to mb4/S17 (p = .03) and threefold lower than the LTC-IC frequency of input CD34 CD38– cells (p = .0003). No differences in the number of clonogenic progenitors, either total or BFU-E, generated per LTC-IC were observed between the mb4/S17 and C/S17 conditions. The decrease in the LTC-IC frequency of output CD34 cells continued into the second week of coculture, dropping below the sensitivity limit of the assay following C/S17 coculture (p/ |( t% h" U5 F
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Figure 4. Effect of Delta4 on long-term culture-initiating cell (LTC-IC) potential. Following coculture of CD34 CD38– mononuclear cells with either control (white) or Delta4 (gray) stroma, output CD34 cells were assessed for their primitive LTC-IC characteristics: LTC-IC frequency of input CD34 CD38– cells and LTC-IC frequency of output CD34 cells at week 1 or week 2. Results are shown as the mean ± SE from six embryonic livers. Asterisks indicate statistical significance: *p # R+ w$ O% e, s& |; b( _
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Erythropoietin and Delta4 Expression in Embryonic Liver–Adherent Cells
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The rare and scattered expression of Delta4 found during the establishment of definitive hematopoiesis in the liver from 34 days on, and the clear push toward erythrogenesis of fetal liver CD34 CD38– cells cultured with Delta4, suggested that this ligand has a role in oxygen sensitivity responsiveness〞in particular, hypoxia. To address whether Delta4 expression was involved in hypoxia, we generated an adherent cell layer from the CD34 CD45– mononuclear cell fraction of 6.5- to 9.5-week-old embryonic livers (n = 5). The preliminary series of experiments, exposing these adherent cells to standard tissue culture (21%) or reduced (7%) oxygen culture conditions, revealed that neither Delta4 nor Delta1 expression was modified following reduction of oxygen (data not shown). This prompted us to investigate whether either Delta1 or Delta4 expression was influenced by the gene products of hypoxia. The two major gene products associated with hypoxia-inducible factor-1 alpha activation during hypoxia are EPO and VEGF , and endothelial and stromal cells are known to express the receptor for EPO . Therefore the embryonic liver–adherent cell layers were exposed to either EPO- or VEGF-containing media at increasing concentrations for approximately 2 hours, and expression of both Delta1 and Delta4 protein was measured by flow cytometry. Exposure of embryonic liver–derived adherent cells to either 10 or 30 U/ml of EPO resulted in a rapid and significant increase in Delta4 expression, almost doubling that observed in nontreated cells (Fig. 5A). This effect was rapidly reversible as replacement of the EPO-containing medium with standard culture medium resulted in Delta4 expression returning to levels equivalent to untreated cells after only 1 hour (data not shown). In striking contrast, VEGF did not alter the expression profile of Delta4 (Fig. 5B, part c). In the same conditions, Delta1 expression proved to be insensitive to both EPO and VEGF, even at high concentrations (Fig. 5B, parts b and d).
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Figure 5. Effect of hypoxia-associated growth factors on Delta expression on embryonic liver–adherent cells. Embryonic liver–adherent cells were exposed to increasing concentrations of either erythropoietin (EPO) or vascular endothelial growth factor (VEGF). Delta expression was measured after a continuous 2.5-hour growth factor exposure. (A): Delta4 expression of embryonic liver–adherent cells exposed to increasing concentrations of EPO in one representative experiment. (B): (a) Delta4 expression following EPO exposure; (b) Delta1 expression following EPO exposure; (c) Delta4 expression following VEGF exposure; (d) Delta1 expression following VEGF exposure. The mean result from five independent liver samples is shown ± SE.! F' r$ W3 J! m9 E/ }! a( A0 `
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DISCUSSION3 {# V- h( i% s" |- i3 ?
9 ]) y1 [$ O7 ^, p* s" `7 p. HWe are indebted to Amgen for providing us with rhu-SCF and Flt3-ligand; Kirin for providing us with rhu-PEG-MDGF; Novartis for providing us with rhu-IL-3; Cilag AG (Schaffhausen, Switzerland, http://www.cilag.ch) for providing us with rHuEPO; and Dr. K. Mori (Niigata University, Niigata, Japan) for providing us with the MS-5 cell line. We thank Y. L谷cluse and F. Larbret for performing the cell sorting. This work was supported by Inserm ("Poste Vert") and grants from CRC (Contrat de Recherche Clinique, no. 2000.10, Institut Gustave Roussy), ARC (Association de Recherche contre le Cancer, no. 4300), and HFSP (Human Frontier Science Program, no. RG0345/1999-M).
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