Effects of Delta1 and Jagged1 on Early Human Hematopoiesis: Correlation with Exp - 《stemcells》干细胞学杂志干细胞之家



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作者：Hlia Nevesa,b, Floor Weerkampc, Andreia C. Gomesa,b, Brigitta A. E. Naberc, Paula Gameirod, Jrg D. Beckerb, Paulo Lciod, Nuno Clodee, Jacques J. M. Van Dongenc, Frank J. T. Staalc, Leonor Parreiraa,b作者单位：a Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa, Lisbon, Portugal;b Instituto Gulbenkian de Cincia, Oeiras, Portugal;c Department of Immunology, Erasmus Medical Center, University Medical Center Rotterdam, Rotterdam, The Netherlands;d Servio de Hematologia, Instituto Portugus de O
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      【摘要】
   It has been shown that Notch signaling mediated by ligands of both Jagged and Delta families expands the hematopoietic stem cell compartment while blocking or delaying terminal myeloid differentiation. Here we show that Delta1- and Jagged1-expressing stromal cells have distinct effects on the clonogenic and differentiation capacities of human CD34  CD38  cells. Jagged1 increases the number of bipotent [colony-forming unit-granulocyte macrophage (CFU-GM) and unipotent progenitors (CFU-granulocytes and CFU-macrophages), without quantitatively affecting terminal cell differentiation, whereas Delta1 reduces the number of CFU-GM and differentiated monocytic cells. Expression analysis of genes coding for Notch receptors, Notch targets, and Notch signaling modulators in supernatant CD34  cells arising upon contact with Jagged1 and Delta1 shows dynamic and differential gene expression profiles over time. At early time points, modest upregulation of Notch1, Notch3, and Hes1 was observed in Jagged1-CD34  cells, whereas those in contact with Delta1 strikingly upregulated Notch3 and Hes1. Later, myeloid progenitors with strong clonogenic potential emerging upon contact with Jagged1 upregulated Notch1 and Deltex and downregulated Notch signaling modulators, whereas T/NK progenitors originated by Delta1 strikingly upregulated Notch3 and Deltex and, to a lesser extent, Hes1, Lunatic Fringe, and Numb. Together, the data unravel previously unrecognized expression patterns of Notch signaling-related genes in CD34  CD38  cells as they develop in Jagged1- or Delta1-stromal cell environments, which appear to reflect sequential maturational stages of CD34  cells into distinct cell lineages.
      【关键词】 Delta Jagged Notch signaling Myelopoiesis Microenvironment Human cord blood Hematopoietic progenitor cells
   INTRODUCTION
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Notch receptors and their ligands are evolutionary conserved trans-membrane proteins that regulate cell-fate decisions in many developmental processes .5 l: \- `- u! K  X7 A) }1 a
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The role of Notch signaling in myeloid differentiation is still controversial. Conditional deletion of Notch1 .2 I' l! O8 v8 U/ `5 S% ?. v# w

Most in vitro experiments (but not all  corroborated these findings. Thus, both Jagged and Delta Notch ligands appear to act as growth- and self-renewal factors for hematopoietic stem cells. Whether such a redundancy of effects also occurs at later stages of hematopoiesis remains to be determined. The restricted and developmentally regulated patterns of expression of Jagged and Delta proteins (and Notch receptors) in bone marrow stromal cells and developing hematopoietic cells strongly suggest that they may have specific effects in myeloid cell development.. O! L+ `  n" a( f" M' I+ ~

To address this possibility, the effects of bone-marrow-derived stromal cells expressing human Delta1 or Jagged1  on cord blood CD34  CD38  cells were investigated. We observed that Delta1 and Jagged1 have different effects on myeloid bipotent and unipotent progenitors and differentially regulate the development of granulocytic and monocytic cell lineages. Dynamic changes in the transcriptional activity of genes coding for Notch receptors, Notch targets, and Notch signaling modulators were observed, which differed according to whether CD34  were cultured in the absence or presence of Jagged1 or Delta1.0 A+ j' x) C- q+ W" n4 Z

MATERIALS AND METHODS9 K; |$ \1 u/ [! u: H; s

CD34  Cells

Umbilical cord blood (CB) was collected according to the guidelines approved by the Ethical Committee of the Lisbon Medical School, mononuclear cells were isolated by density gradient centrifugation (Ficoll-Paque; Plus, Amersham Biosciences, Lisbon, Portugal, http://www.amersham.com), and CD34  cells were obtained using the VarioMACS separation column (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com). Expression of CD34  on collected cells was 96.5% ¡À 1.5% (n = 10). Cells were immediately processed for coculture experiments. CD34  cells differentiating upon contact with Delta1 or Jagged1 were purified using the same procedure (purity >97%). Sorting of Delta1-derived CD7¨C and CD7  subpopulations was further performed with purity higher than 98%.3 h! g* f% Y$ h1 p$ c' S9 K( {3 F

Retroviruses and Producer Cell Lines
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Full-length cDNAs encoding either the human Delta1 or Jagged1 (provided by G. Artavanis-Tsakonas, Harvard Medical School, Charlestown, MA) were cloned into the LZRS linker internal ribosome entry site (IRES)-enhanced green fluorescent protein (GFP) retrovirus (provided by H. Spits, The Netherlands Cancer Institute, Amsterdam, Holland; Garry Nolan, Stanford University, Stanford, CA), as previously described in detail .; S3 |2 S8 B" A. l5 J! X) O/ T+ g
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Transduction of S17 Cells with Retroviruses Containing Delta1 or Jagged1 c-DNA" d+ S5 u4 T+ D2 x9 Z: B
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S17 bone marrow-derived stromal cells (provided by K. Dorshkind, University of California, Los Angeles, CA) were transduced and functionally assayed as previously described in detail .

Functional Assays for Jagged1 and Delta1 Proteins

Efficiency of transduced Delta1 and Jagged1 proteins in the activation of the Notch pathway was assessed as described . Myotube formation was assessed by phase contrast analysis and expression of Myosin (clone My-32; Sigma-Aldrich, Sintra, Portugal, http://www.sigmaaldrich.com) (immunocytochemical detection).' J' R4 s7 n, @) {. _

Coculture Assays) R' B, s) _3 l
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Twenty-four hours before their use in coculture, 2 x 104 S17 cells in 1 ml of medium were plated in 24-well flat-bottomed plates. Cocultures were initiated by seeding 4 x 104 CD34  cells to the wells precoated with parental or transduced S17 stroma and maintained at 37oC, 5% CO2. Half of the culture medium (RPMI ) was replaced by fresh medium once a week. No exogenous cytokines were added to the cultures in any time point. After 48 hours, 1 week, 2 weeks, and 4 weeks of incubation, cells were harvested, counted, stained, and used for subsequent analyses. The expression of Notch signaling-related genes was analyzed in purified CD34  cells by quantitative reverse transcription polymerase chain reaction (qRT-PCR) at the different time points. CD34  cells persisting at 4 weeks of culture were further used for clonogenic assays.
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Flow Cytometry and Cell Sorting

Flow cytometry analysis was performed using a FACSCalibur (BD Biosciences, Madrid, Spain, http://www.bdbiosciences.com). Fresh CB progenitors or supernatant cells harvested at different time points were stained with the following monoclonal antibodies as described : CD10-FITC, CD15-FITC, CD14-PE, CD19-PE, CD34-PE, CD38-PE, CD3-PerCP, CD10-PE-Cy5, CD34-PerCP-Cy5.5, CD45-APC (all from BD Biosciences), and CD7-PE (Beckman Coulter, Buckinghamshire, U.K., http://www.beckmancoulter.com). To sort CD34 CD7  and CD34 CD7¨C cells from Delta1-derived CD34  cells, cells were stained with PE-conjugated anti-CD7 (BD Biosciences) and sorted using triple laser (488 nm argon laser, 599 nm dye laser, and UV laser) fluorescence-activated cell sorting (FACS DiVa flow cytometer and cell sorter; BD Biosciences).1 A: O3 V: U+ q0 V7 Z
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Methylcellulose Assays6 [: }( d/ s+ ?
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At day 0 (control for clonogenic assays) and 4 weeks of coculture (pool of cells from 2¨C3 different donors per sample), supernatant-purified CD34  cells were plated in quadruplicate of two dilutions in methylcellulose medium (Methocult GF  H4435; Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) according to the manufacturer¡¯s recommendations. Cultures were maintained at 37¡ãC in 5% CO2, and colonies were evaluated by phase microscopy, stained by May-Gr¨¹nwald Giemsa, and classified according to morphologic criteria after 14¨C16 days.( o8 K- {: T3 d% V& Q

Real-Time qRT-PCR' r% {9 f+ m( N* ]- i% @* O

The expression of selected Notch signaling-related genes was analyzed by qRT-PCR of purified CD34  cells freshly isolated and after 48 hours, 1 week, 2 weeks, and 4 weeks of contact with control, Jagged1, and Delta1 stromas (further sorted for CD7¨C and CD7  subpopulations) (a pool of cells from 2¨C3 different donors was used for each sample). Three different samples were analyzed in triplicate for each culture condition. All procedures were done as described  ). Expression of each target gene was normalized for the endogenous gene huGAPDH (TaqMan PDAR; Applied BioSystems). PCRs were performed in a TaqMan 7900 machine (Applied BioSystems), according to the manufacturer¡¯s instructions. The Universal Human Reference RNA (Stratagene, La Jolla, CA, http://www.stratagene.com) was used as standard to allow relative quantification and expression values of control cells used as calibrators. All tests were performed in triplicate, and the mean cycle threshold (Ct) value was taken as the final result (mean Ct ¡À SD, if SD / d4 y2 N0 x7 N- B: n) a* l

Statistical Analysis

Statistical analysis was performed using the nonpaired Student¡¯s t test." r/ G* [0 y2 ?& a( z) B. b( z7 Z4 j; W1 c
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RESULTS

Stromal Cells Expressing Delta1 or Jagged1 Differentially Affect the Expansion of CD34  and CD14  Cells
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The experimental design of the present study is depicted in the diagram of Figure 1A. Figure 1B shows that S17 stromal cells transduced with Jagged1 and Delta1 are efficient in inhibiting the fusion of C2 myoblasts into multinucleated myotubes, as previously described ./ w8 Y$ h6 t. Z; E, F, j8 q

Figure 1. Experimental design and functional assay of S17 cells transduced with Delta1 or Jagged1. (A): Diagram depicting the sequential steps of the experimental approach used in the present study. (B): Differentiation of C2 myoblasts cells after 4 days of coculture on S17-vector, S17-Delta1, or S17-Jagged1 in the presence of carrier alone (0.5% dimethyl sulfoxide -S-phenylglycine t-butylester (DAPT). Original magnification x20. Abbreviations: CFU, colony-forming unit; DAPI, 4,6-diamidino-2-phenylindole; GFP, green fluorescent protein; qRT-PCR, quantitative reverse transcription polymerase chain reaction; S17-Delta1, S17 cells transduced with Delta1 cDNA; S17-Jagged1, S17 cells transduced with Jagged1 cDNA; S17-vector, S17 cells transduced with GFP-vector; wk, week.+ q3 K% K& J! j; v! B9 A% A6 Q8 U

As CD34  CD38  cells derived from cord blood, when cultured with stromal cells, do not generate colony-forming units (CFU) beyond day 40 (reaching the maximum of expansion and clonogenic potential around day 30) , coculture experiments were ended at 4 weeks. The phenotypic analysis of supernatant cells obtained by culturing freshly collected CD34  cells with parental (nontransduced) S17 cells over time is shown in Figure 2A. After an initial decline, a progressive expansion of CD34  cells, together with expansion of early B and myeloid cells, was observed.8 I$ F# C- u* [2 F0 x

Figure 2. CD34  cells cultured in the presence of parental and transduced S17 stromal cells for 4 weeks. (A): Expansion of supernatant cells grown on parental stroma. (B): Phenotypic analysis of supernatant cells grown on transduced and parental S17 stromal cells. Cells were analyzed for CD45 (hematopoietic cells), CD34 (progenitor cells), CD14/CD15 (myeloid cells), CD10/CD19 (pre-B cells) and CD7, within gated CD34  cells, for detection of T/NK cell precursors. (C): Expansion of supernatant cells grown in the presence of Jagged1 and Delta1 for 4 weeks (normalized to vector). Absolute cell numbers were as follows. At 2 weeks: (9.6 ¡À 4.1) x 104 (Vector), (1.1 ¡À 0.5) x 105 (Jagged1), (7.8 ¡À 5.1) x 104 (Delta1); at 4 weeks: (4.0 ¡À 1.8) x 105 (Vector), (4.0 ¡À 1.5) x 105 (Jagged1), (1.7 ¡À 0.6) x 105 (Delta1). Abbreviations: Delta1, S17 cells transduced with Delta1 cDNA; Jagged1, S17 cells transduced with Jagged1 cDNA; Vector, S17 cells transduced with green fluorescent protein vector; Wk, week; WT, parental S17 stromal cells.: l8 h$ ]5 ~7 v: R4 }1 @
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Phenotypic analysis of supernatant cells grown on transduced S17 cells (Fig. 2B and Table 1) was then performed. The following was observed: no significant differences could be detected, either in cell numbers (not shown) or expression of differentiation markers, in cells grown with S17 cells transduced with the vector containing only IRES-GFP sequences (Vector), compared with the parental conditions. Significant proportions of CD34  cells were detected at 4 weeks in all conditions; Jagged1 did not influence the percentages of CD15  (granulocytic) and CD14  (monocytic) cells; Delta1 increased the percentage of CD15  cells, whereas CD14  cells were maintained at percentages similar to control. As expected , an increase in CD10 CD19  cells was observed during the 4 weeks of culture both in control and Jagged1 stromas, and no CD10 CD19  cells were detected at any time point in the presence of Delta1; in the latter condition, almost half of the supernatant CD34  cells co-expressed CD7  at 4 weeks (not observed in the other conditions). No TdT-positive, CytCD3-positive, or CytCµ-positive cells were detected in any condition.

Table 1. CD34  cells cultured in the presence of Delta1 or Jagged1: phenotype of supernatant cells at different time points

The absolute numbers of the different cell subpopulations at 4 weeks, normalized for the control values, show that the increase in total cell numbers was significantly lower in Delta1 than in Jagged1 and control conditions (Fig. 2C). CD15  cell numbers were similar in Jagged1 and Delta1, CD14  and CD34  cells were lower in Delta1 conditions, and CD10  CD19  cell numbers were similar in Jagged1 and control conditions (Fig. 2C).. K! I5 s* z6 ^. {! \
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Thus, after 4 weeks of culture of CD34  cells with Delta1-or Jagged1-S17 cells, Delta1 caused, in addition to a block of B-cell development and increase in CD7  cells, a reduction in the absolute number of monocytic cells (CD14  cells), whereas the absolute number of CD10 CD19  and CD14  cells in Jagged1 conditions did not differ from control. In addition, Delta1 induces a lower expansion of CD34  cells at 4 weeks, compared with Jagged1 and control stromas.
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Stromal Cells Expressing Delta1 or Jagged1 Differentially Affect the Myeloid Potential of CD34  Cells0 P' |7 n6 M& d, H/ n
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CD34  cells purified after 4 weeks were used for CFU assays (Table 2). The total number of CFU originated by fresh CD34  cells and CD34  cells grown with the control stroma (Vector) were similar for the two dilutions (results from three independent experiments are shown in Table 2). In contrast, the total number of CFU originating from Jagged1-CD34  cells was higher than in control (p : V; P! F- h9 }* c% z- {

Table 2. Myeloid colonies originated by CD34  cells grown in contact with Delta1 or Jagged1 for 4 weeks
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In summary, Jagged1, as opposed to Delta1, increases the overall myeloid clonogenic potential of CD34  cells.
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Stromal Cells Expressing Delta1 or Jagged1 Have Different Effects on the Number, Size, and Cell Morphology of Different Colony Types Generated by CD34  Cells9 ~6 f$ J' [+ Z
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Inspection of colonies emerging in the various conditions revealed that, compared with control conditions, all types of colonies (CFU-GM, CFU-G, and CFU-M) increased in Jagged1 conditions, whereas Delta1-CD34  cells (of which only 60% had myeloid potential) originated less CFU-GM and CFU-M and more CFU-G (see Table 2, last column); that all types of Jagged1 colonies were usually larger (more cells) than in control (Fig. 3A, 3B), the opposite being observed for all types of Delta1 colonies (not shown); and that all colonies included all expected cell differentiation stages, with the exception of Delta1-CFU-G, in which most cells were morphologically promyelocytes (Fig. 3B). In some of the largest Jagged1-derived CFU-GM, some erythroid cells could also be observed./ L1 V3 E4 @! o/ Q- f
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Figure 3. Myeloid clonogenic assays. Phase contrast images and May-Grunwald-Giemsa staining of cytospins obtained of colonies from vector-derived CD34  cells (A) or ligand-derived CD34  cells (B). Bar = 500 µm. Abbreviations: CFU-GM, colony-forming unit-granulocyte macrophage; CFU-G, colony-forming unit-granulocyte; CFU-M, colony-forming unit macrophage.$ w: y4 @; G# w( F1 b$ S
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Therefore, S17 cells expressing Delta1 and Jagged1 differentially affect the differentiation of CD34  progenitors into the myeloid lineages. Jagged1 increases the overall clonogenicity of CD34  cells; Jagged1 and Delta1 have opposite effects on the emergence of CFU-GM, the former increasing the latter decreasing their number. A consistent, yet nonsignificant, reduction of CFU-M and increase of CFU-G were observed in Delta1 conditions.

CD34  Cells Arising in Contact with Stromal Cells Expressing Delta1 or Jagged1 Have Different Transcription Profiles of Notch Signaling-Related Genes

In an attempt to investigate the expression dynamics, over time, of genes related to Notch signaling, qRT-PCR was performed at 48 hours, 1 week, 2 weeks, and 4 weeks in purified CD34  cells emerging in the different stromal conditions. In the case of Delta1-CD34  cells, gene expression analysis at 1, 2, and 4 weeks was done in sorted CD7¨C and CD7  subpopulations (the latter appearing at significant numbers in the supernatant at 2 weeks). Three groups of gene-transcripts were studied: 1) Notch receptors (Notch1, Notch2, and Notch3); 2) Notch targets (Hes1 and Deltex, all time points); and 3) modulators of Notch signaling (Lunatic Fringe at all time points; Manic Fringe, Radical Fringe, and Numb at 4 weeks).
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An overview of the expression dynamics of these transcripts over time is shown in Figure 4. Figure 4A shows the expression levels of all genes (relative to those in fresh CD34  cells) after 48 hours of cell contact with parental- and vector-S17 cells. No upregulation of any transcript was observed. This is in contrast with, at the same time point, Jagged1- and Delta1-CD34  cells: Jagged1-cells upregulated Notch1, Notch3, and Hes1 at modest levels (two- to threefold) (Fig. 4B), whereas Delta1-CD34  cells (whose phenotype is identical to that of control and Jagged1 CD34  cells at this point) strikingly upregulated Notch3 (36-fold) and Hes1 (13-fold) (Fig. 4C)., b7 t8 B4 M: Q- {& A  m

Figure 4. Expression of Notch signaling-related genes in CD34  cells emerging at different time points of culture (quantitative reverse transcription polymerase chain reaction). (A): CD34  cells after 48 hours of contact with parental (WT) or vector-transduced (Vector) stromal cells. (B): CD34  cells emerging, at different time points, in contact with Jagged1. Phenotype of CD34  cells at 2 and 4 weeks is shown in boxes (mean values; see also Table 1). (C, D): CD34  cells emerging, at different time points, in contact with Delta1. (C): CD7¨C cells. (D): CD7¨C cells. Expression of each transcript was measured as a ratio with the glyceraldehyde-3-phosphate dehydrogenase transcript and expressed in arbitrary units normalized to control (fresh CD34  cells ¨C 1 ). Three different samples were analyzed in triplicate for each culture condition. Abbreviations: F, Fringe; Vector, vector-transduced stromal cells; Wk, week; WT, parental stromal cells.

At 1 and 2 weeks, levels of Notch1, Notch2, and Hes1 were maintained, with mild fluctuations, in both Jagged1- (Fig. 4B) and Delta1-CD34  cells (Fig. 4C), with the exception of Notch3 and Deltex, which increased sharply from 1¨C2 weeks in Delta1-CD34  cells (Fig. 4C, 4D). A sustained upregulation (fourfold) of Lunatic Fringe, starting at week 1, was also observed in Delta1-CD34  (Fig. 4C).
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At 4 weeks, the quantitative and qualitative differences between Jagged1- and Delta1-CD34  cells were as follows: most genes were transcribed at much higher levels in Delta1-than in Jagged1-CD34  cells, and within Delta1-CD34  cells, the same genes were transcribed at higher levels in CD7  than in CD7¨C cells. One further observation was that genes of each functional group were expressed in a differential manner within each subset. Jagged1-CD34  cells upregulated Notch1 and Notch3 genes (the former at higher levels than the latter) and Deltex and Hes1 (the former at much higher levels than the latter), and all modulators of Notch signaling were downregulated (Fig. 4B). As to Delta1-CD34  cells, Notch3 was the most upregulated gene, followed by Notch1 and Notch2, a pattern that was common to both Delta1-CD34  cell subpopulations (Fig. 4C, 4D). Hes1 was markedly upregulated in Delta1-cells, especially in CD7¨C cells (Fig. 4C), whereas Deltex transcripts predominated in CD7  cells (Fig. 4D). All Notch signaling modulators (with the exception of Radical Fringe) were up regulated in Delta1-CD34  cells, more so in CD7  than in CD7¨C cells (Fig. 4C, 4D).( ^1 d( d# E( r7 a

Therefore, dynamic changes in the transcriptional activity of genes coding for Notch receptors, Notch targets, and Notch signaling modulators were observed over time, which differed according to whether CD34  were cultured in the absence or presence of Jagged1 or Delta1.
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DISCUSSION0 {$ M* j9 \# ^% p2 f
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Here we show that stromal cell environments expressing Jagged1 or Delta1, to which no exogenous cytokines were added, have differential effects on the phenotype, clonogenic potential and expression of Notch signaling-related genes in CD34 CD38  hematopoietic progenitors.4 c9 q' u4 l! r/ E! ~0 V$ B
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Both Jagged1 and Delta1 stromas affected myeloid progenitors, not by forcing them to undergo cell fate decision processes, as is the case with Delta1 effects on lymphopoiesis , indicates that the negative effects of this ligand on the monocytic lineage must start beyond the stem cell stage, possibly at the level of the pluripotent myeloid progenitor.
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In contrast, Jagged1-expressing stromal cells increase the proliferation of bipotent and unipotent myeloid progenitors. Jagged1 was shown to induce proliferative and antidifferentiative effects in hematopoietic stem cells . Here we show that when acting in more advanced progenitors, Jagged1 maintains its proliferative effect in all cell progenies, but the block of terminal differentiation is no longer present.# o; b( g: N/ q/ D6 e6 ?  J

In an attempt to investigate whether these phenotypic and functional differences were related to changes in transcription levels of Notch signaling-related genes, qRT-PCR analysis of supernatant CD34  cells, emerging in the different stromal conditions, was done at different time points., b* S% z. G# }+ c+ U9 U' M- Q& m
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Despite the functional heterogeneity of the cell subpopulations under analysis, a few insights emerge when gene expression data are interpreted together with phenotypic changes occurring in supernatant cells over time. At the earliest time point investigated (48 hours), at which no evidence for cell differentiation was detectable, the data indicate that Delta1 and Jagged1 do indeed induce distinct responses in CD34 CD38  progenitors. In fact, whereas upregulation of Hes1 was present in both culture conditions, indicating that both ligands were activating Notch signaling through this CBF1-dependent pathway , an impressive differential regulation of Notch genes themselves was observed. Jagged1-CD34  cells had a very modest, yet similar, upregulation of Notch1 and Notch3, whereas Delta1-CD34  cells exhibited a striking upregulation of Notch3 in relation to Notch1 and Notch2.

When looking at subsequent time points, abrupt changes were observed at 2 weeks in Delta1-CD34 CD7¨C cells, consisting of further increase in Notch3 levels, modest upregulation of Notch1, downregulation of Hes1, and upregulation of Deltex. This time point coincides with the appearance of substantial numbers of CD34 CD7  cells in the supernatant (40% of all CD34  cells expressed CD7 at this point). Indeed, a similar, but quantitatively more robust, expression pattern was observed in CD34 CD7  cells (pure T/NK progenitors) purified at 2 weeks. Therefore, the composite expression pattern of Notch3, Notch1, Hes1, and Deltex in Delta1-CD34  cells at 2 weeks appears to reflect a transcription profile of T/NK progenitors. If so, the presence of the same profile, albeit at much lower quantitative levels in the CD7¨C subpopulation, suggests that very early T/NK progenitors might co-exist with myeloid progenitors in Delta1-CD34 CD7¨C cells.! w" C4 ^+ O% V2 I2 m; o

By week 4, when CD34  cells have reached their maximal myeloid clonogenic potential .

As to changes in gene transcription observed at 48 hours (striking upregulation of Notch3 and, to a much lesser extent, of Notch1), at least two possibilities should be considered. One is that they could be a direct consequence of Delta1-mediated Notch stimulation on CD34  cells. In fact, when the latter were switched, after 48 hours of contact with Delta1, to control stroma, no T-cell development (or block of B-cell differentiation) was observed (data not shown), in agreement with recently reported findings for mouse fetal liver precursors . The possibility cannot, therefore, be formally excluded that the observed early expression patterns reflect the effect of Delta1 on this very rare, yet already lineage-committed, cell population.
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In contrast with Delta1-CD34  cells, no substantial changes in gene expression were observed in Jagged1-CD34  cells until week 4, when a marked increase in Notch1 and Deltex transcripts occurred. Although no phenotypic differences, in relation to control cells, could be observed at any time point, Jagged1-CD34  cells were, by this time, more immature and proliferative than those in other stromal conditions, as shown by colony assays. How these differences relate to the concomitant Notch1 and Deltex transcription profiles is uncertain, as no clonogenic assays were performed at earlier time points. However, the absence in Jagged1 cells of the above-described T/NK transcription profile, together with the knowledge that murine immature B-cells (immature B-cells were also present in Jagged1 cultures; Table 1) express very low levels of Notch1 and Deltex , makes it conceivable that the upregulation of these genes at 4 weeks might be myeloid-specific and related to the increased myeloid clonogenic potential of Jagged1-CD34  cells.
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As to the expression of genes coding for Notch signaling modulators, Lunatic Fringe, Manic Fringe, and Numb were upregulated in Delta1-CD34  cells (CD7  cells at 4 weeks showing the highest levels for all transcripts) and downregulated in Jagged1-CD34  cells. Given that Lunatic Fringe suppresses Jagged1 signaling while enhancing that mediated by Delta1 , these changes are likely to be related to the regulation of Notch signaling intensity mediated by the two ligands.

In summary, the present study shows that CD34 CD38  progenitors arising on Delta1- or Jagged1-expressing stromal cells differ as to their phenotype and myeloid clonogenic capacities. The observed differences correlate with distinct and dynamic gene transcription patterns for Notch signaling-related genes. As to the latter, the results reveal that myeloid progenitors with strong clonogenic potential emerging in contact with Jagged1 upregulate Notch1 and Deltex and downregulate Notch signaling modulators, whereas the T/NK progenitors originated by Delta1  are characterized by a striking upregulation of Notch3 and Deltex and, to a lesser extent, of Hes1, Lunatic Fringe, and Numb. The quest for the mechanisms underlying these responses and their putative physiological role is certainly worth pursuing.

ACKNOWLEDGMENTS
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We are grateful to Prof. Ant¨®nio Coutinho (Instituto Gulbenkian de Ci¨ºncia, Oeiras, Portugal) for helpful discussions, to Dr. Domingos Henrique (Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa) for cDNA clones, and to Dr. Isabel Alcobia for helping with culture experiments. This work was supported by grants from Fundação para a Ci¨ºncia e Tecnologia (POCTI/37953/2001) and Fundo de Investigação Oncol¨®gica.2 ]2 {& T, U" J8 u6 L+ K

DISCLOSURES' z+ |1 P* O+ t/ O7 o

The authors indicate no potential conflicts of interest./ g2 K2 Z% Q) e% P- c
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