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发表于 2009-3-5 00:06 |只看该作者 |倒序浏览 |打印

作者：John P. Chutea, Garrett G. Muramotoa, Holly K. Dressmanb, Gary Wolfec, Nelson J. Chaoa, Simon Lind作者单位：a Division of Cellular Therapy, Department of Internal Medicine,b Institute for Genome Sciences and Policy, Department of Molecular Genetics and Microbiology,d Department of Bioinformatics and Biostatistics, Duke University, Durham, North Carolina, USA;c Predictive Diagnostics Incorporated, Vacavill   g( z: V8 B: J; S) B$ V
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      【摘要】3 L+ _! t( L/ Y2 n
   Recent progress has been made in the identification of the osteoblastic cellular niche for hematopoietic stem cells (HSCs) within the bone marrow (BM). Attempts to identify the soluble factors that regulate HSC self-renewal have been less successful. We have demonstrated that primary human brain endothelial cells (HUBECs) support the ex vivo amplification of primitive human BM and cord blood cells capable of repopulating non-obese diabetic/severe combined immunodeficient repopulating (SCID) mice (SCID repopulating cells ). In this study, we sought to characterize the soluble hematopoietic activity produced by HUBECs and to identify the growth factors secreted by HUBECs that contribute to this HSC-supportive effect. Extended noncontact HUBEC cultures supported an eight-fold increase in SRCs when combined with thrombopoietin, stem cell factor, and Flt-3 ligand compared with input CD34  cells or cytokines alone. Gene expression analysis of HUBEC biological replicates identified 65 differentially expressed, nonredundant transcripts without annotated hematopoietic activity. Gene ontology studies of the HUBEC transcriptome revealed a high concentration of genes encoding extracellular proteins with cell-cell signaling function. Functional analyses demonstrated that adrenomedullin, a vasodilatory hormone, synergized with stem cell factor and Flt-3 ligand to induce the proliferation of primitive human CD34 CD38¨Clin¨C cells and promoted the expansion of CD34  progenitors in culture. These data demonstrate the potential of primary HUBECs as a reservoir for the discovery of novel secreted proteins that regulate human hematopoiesis.   }1 W! t8 M# p/ f* E' N+ I3 @
      【关键词】 Endothelial cell Stem cell expansion Hematopoietic stem cell
   INTRODUCTION

Hematopoietic stem cells (HSCs) possess the unique capacity to undergo self-renewal in vivo throughout the life of an individual while also providing the complete repertoire of mature hematopoietic and immune cells .
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One strategy to identify HSC growth factors involves examination of candidate niches wherein HSCs are known to reside physiologically . Therefore, ECs are a logical source of growth factors that regulate HSC growth and differentiation.) A7 U$ W4 D* P4 s6 `- V6 b4 {

Several studies have examined the capacity for stromal cell lines to support the ex vivo maintenance of HSCs . These data suggest that soluble factors elaborated by HUBECs account for the unique hematopoietic activity that we have observed.
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In this study, we have developed a molecular profile of HUBECs via comparative gene expression analysis to identify the candidate novel molecules responsible for this HSC-supportive activity. Secreted factors, extracellular proteins, and cell-cell signaling proteins are highly overrepresented within the HUBEC transcriptome. Moreover, initial functional analyses indicate that a vasoactive peptide, adrenomedullin, synergizes with other cytokines to induce human progenitor cell proliferation and expansion.3 E" R% ~$ K9 w' Q

MATERIALS AND METHODS

Noncontact Cultures of Human CB CD34  Cells with Primary HUBECs
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Primary human cord blood CD34  cells were procured from Cambrex (Cambrex, Walkersville, MD, http://www.cambrex.com). Briefly, 1 x 105 CD34  cells were placed in six-well culture plates with Iscove¡¯s modified Dulbecco¡¯s medium (IMDM) (Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com) with 10% fetal calf serum and 1% penicillin/streptomycin (pcn/strp) (Gibco-BRL) supplemented with 20 ng/ml thrombopoietin, 120 ng/ml stem cell factor, and 50 ng/ml Flt-3 ligand (TSF) (R&D Systems Inc., Minneapolis, http://www.rndsystems.com) or in noncontact cultures with primary human brain endothelial cells (RMLS-01) supplemented with TSF for 14 days. HUBECs and primary CD34  cells were separated by 0.4-µm transwell inserts (Gibco-BRL). At day 14, nonadherent cells were collected from each culture condition and washed, and cell counts were obtained. Immunophenotypic analysis using fluorescent monoclonal antibodies CD34 and CD38 and appropriate isotype controls (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) was performed at day 0 and day 14 to compare the hematopoietic content at each time point.8 [  r: V, C( d5 C: f

Transplantation of Human Hematopoietic Cells into NOD/SCID Mice+ Q( k2 s# |2 B/ h  I; p
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Six- to 8-week-old nonobese diabetic severe combined immunodeficient (NOD/SCID) mice (Jackson Laboratory, Bar Harbor, ME, http://www.jax.org) were used for all experiments .4 N$ ~  m& i4 Y2 d7 h* o) k
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Isolation of RNA from HUBECs and Human Umbilical Vein Endothelial Cells and Gene Expression Analysis

Primary human brain endothelial cells were placed in culture as previously described . Briefly, 1 x 105 HUBECs were cultured on gelatin-coated six-well plates (Corning Incorporated Life Sciences, Acton, MA, http://www.corning.com) in complete endothelial cell culture medium (5 ml per well) containing M199 (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), 10% fetal bovine serum (FBS), 100 µg/ml L-glutamine (Invitrogen), 50 µg/ml heparin, 60 µg/ml endothelial cell growth supplement (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), and 1% pcn/strp at 37¡ãC in 5% CO2 atmosphere. For analysis of HUBEC gene expression, confluent HUBECs were cultured for 72 hours, washed twice, and trypsinized, and the cells were pelleted and resuspended in TRIzol reagent (Sigma-Aldrich) for RNA preservation.& B+ e% D; c: K$ _1 f6 _( M6 R' u
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Human umbilical vein endothelial cells (HUVECs) (ATCC, Manassas, VA, http://www.atcc.org) were used as control cells and were cultured primarily as previously described . Briefly, 1 x 105 HUVECs were plated in gelatin-coated six-well plates in medium containing F12K medium (ATCC) with 2 mM L-glutamine, 0.1 mg/ml heparin, 0.05 mg/ml endothelial cell growth supplement, and 10% FBS. After 72 hours, the confluent HUVECs were trypsinized, washed twice, and resuspended in TRIzol reagent for RNA preservation.( Y6 I- _# j( O4 O! n( P' ~

RNA isolation from HUBECs and HUVECs was performed as follows. Briefly, 5 x 106 endothelial cells were pelleted and incubated with 1 ml of TRIzol reagent and incubated for 5 minutes. Cells were then mixed with 0.2 ml of chloroform for 3 minutes at room temperature and then centrifuged at 11,500 rpm for 15 minutes at 4o C. The upper aqueous phase of the sample was then collected into RNase-free Eppendorf tubes and mixed with 0.5 ml of isopropanol for 10 minutes. Samples were then centrifuged at 11,500 rpm for 15 minutes at 4¡ãC. The supernatant was then aspirated, and the pellet was resuspended in 75% ethanol in DEPC-H20 by vortexing. Samples were then air-dried, and RNA quantity was measured via spectrophotometry.

After RNA isolation, samples were run through an RNeasy column to eliminate potential DNA and protein contamination as previously described .

Quantitative Real-Time RT-PCR Analysis of HUBEC Gene Expression5 @# t( L* {3 o6 [& t  A/ `3 n; Y) a
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Total RNA was isolated from 1 x 106 HUBECs or HUVECs (ATCC) using the RNeasy Mini kit (Qiagen, Valencia, CA, http://www1.qiagen.com), according to the manufacturer¡¯s protocol. Total RNA was quantified using a SmartSpec 3000 spectrophotometer (Bio-Rad, Hercules, CA, http://www.bio-rad.com), and 2 µg per sample was reverse transcribed using the High Capacity cDNA Archive kit (Applied BioSystems, Foster City, CA, http://www.appliedbiosystems.com), using the recommended reaction conditions. Fifty-nanogram equivalents of cDNA were then used for quantitative real-time PCR using TaqMan Gene Expression Assays (Applied Biosystems) for decorin, insulin-like growth factor binding protein 2 (IGFBP-2), myocardin, adrenomedullin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), with an ABI Prism 7700 Sequence Detection System (Applied Biosystems). Relative gene expression between HUBECs and HUVECs was calculated using the Ct method, using GAPDH expression as a normalization reference.1 ]' t# I& G$ W$ m1 Q( o1 Q( l
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CB Processing, Lineage Depletion, and FACS Sorting
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Umbilical cord blood units were obtained from the Duke University Stem Cell Laboratory within 48 hours of collection. Volume reduction was accomplished by 10-minute incubation at room temperature with 1% Hetastarch (Abbott Laboratories, North Chicago, IL), followed by centrifugation at 700 rpm for 10 minutes without brake, to facilitate component separation. The buffy coat was collected and washed twice with Dulbecco¡¯s phosphate-buffered saline (DPBS) (Invitrogen) containing 10% heat-inactivated FBS (HyClone, Logan, UT, http://www.hyclone.com), 100 U/ml penicillin, and 100 µg/ml streptomycin (1% pcn/strp; Invitrogen). Cell pellets were thoroughly resuspended in DPB 10% FBS 1% pcn/strp and overlaid onto Lymphoprep (Axis-Shield, Olso, Norway) and centrifuged at 1,500 rpm for 30 minutes without brake to isolate the mononuclear cell (MNC) fraction. MNC monolayers were collected and washed twice before proceeding to lineage marker depletion.

Lineage depletion was conducted using the Human Progenitor Enrichment Cocktail (Stem Cell Technologies, Vancouver, BC, Canada), which contains monoclonal antibodies to human CD2, CD3, CD14, CD16, CD9, CD56, CD66b, and Glycophorin A, according to the manufacturer¡¯s suggested protocol. Briefly, CB MNCs were resuspended at 5¨C8 x 107 cells per ml in DPBS 10% FBS 1% pcn/strp, and incubated with 100 µl/ml antibody cocktail for 30 minutes on ice, followed by incubation with 60 µl/ml magnetic colloid for 30 minutes on ice. Cells were then magnetically depleted on a pump-fed negative selection column (Stem Cell Technologies), using the manufacturer¡¯s recommended procedure. Lin¨C cells were washed twice, quantified by manual hemacytometer count using trypan blue exclusion dye (Invitrogen), and cryopreserved in 90% FBS 10% dimethylsulfoxide (Sigma-Aldrich) or used for further experimentation.

Lin¨C CB cells were thawed, washed once in IMDM (Invitrogen) containing 10% FBS and 1% pcn/strp, counted, and resuspended at 5 x 106 to 1 x 107 cells per ml. Immunofluorescent staining was conducted using anti-human CD34-FITC and anti-human CD38-PE monoclonal antibodies (Becton, Dickinson and Company) for 30 minutes on ice. Stained cells were washed twice and resuspended at 1 x 107 cells per ml in IMDM 10% FBS 1% pcn/strp. Sterile cell sorting was conducted using a FACSvantage flow cytometer (Becton, Dickinson and Company) to isolate CD34 CD38¨C and CD34 CD38  subsets. For proliferation experiments, cells were automatically sorted into 60-well Terasaki plates (Nunclon, Rochester, NY), containing 5 µl per well of the appropriate growth factor media. The CD34 CD38¨C sort gate was set to collect only those CD34  events falling in the lowest 5% of PE fluorescence within the total CD34  population, as determined by staining with isotype-matched mouse IgG1 controls (BD Biosciences), to ensure acquisition of highly purified CD34 CD38¨C cells.- h( e+ d: u) e! B
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To screen for hematopoietic activity of HUBEC-secreted growth factors, we placed human CB CD34  cells in culture with 50 ng/ml thrombopoietin, 100 ng/ml stem cell factor, and 50 ng/ml Flt-3 ligand (TSF) for 7 days with and without supplementation with the following recombinant proteins that we found to be differentially overexpressed by HUBECs: IGFBP2 (R&D Systems), IGFBP3 (R&D Systems), follistatin (R&D Systems), and adrenomedullin (R&D Systems).1 U% N8 Y( q$ `
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RESULTS
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Soluble Factors Elaborated by HUBECs Support the Expansion of Human HSC

To extend our previous observations that primary HUBECs uniquely induce the expansion of human HSCs , respectively; p = .01, likelihood estimator model; Fig. 1C). These studies demonstrate the distinctly soluble hematopoietic activity elaborated by primary HUBECs and indicate its synergistic effect on SRC expansion when combined with thrombopoietin, stem cell factor (SCF), and Flt-3 ligand.' }  f0 p# c9 ?

Figure 1. Noncontact culture with HUBECs increases total cells, CD34  cells, and severe combined immunodeficient-repopulating cells (SRCs) compared with cytokines alone. (A): Total cell expansion is shown comparing input cord blood (CB) CD34  cells versus day 14 TSF-cultured progeny versus noncontact HUBEC culture supplemented with TSF. (B): CD34  cell expansion is shown demonstrating a significant increase in CD34  cells following HUBEC culture compared with TSF alone at day 14. (C): SRC activity of day 0 CB CD34  cells versus the progeny of CB CD34  cells following 14-day culture with TSF alone versus the progeny of noncontact HUBEC-culture plus TSF at day 14. Human CD45  cell engraftment was significantly higher in the nonobese diabetic severe combined immunodeficient (NOD/SCID) mice transplanted with the progeny of noncontact HUBEC cultures compared with either input or the progeny of TSF cultures. Abbreviations: HUBEC, human brain endothelial cell; SRC, severe combined immunodeficient-repopulating cell; TSF, thrombopoietin, stem cell factor, and Flt-3 ligand.

Gene Expression Analysis Identifies Unique HUBEC Transcripts% x2 k- D" B& w3 k7 E+ w2 M

To identify with a high degree of certainty the novel HUBEC-derived factors involved in HSC regulation, we applied a repetitive cDNA microarray analysis using Affymetrix human 133A and 133B chips, representing >47,000 annotated human genes. Highly purified RNA was isolated from biological replicates of primary HUBECs (n = 5) and HUVECs (n = 4) at 72 hours of culture and provided for array hybridization. Transcript lists generated from each sample were collected and analyzed following the MGED/MIAMI guidelines and subsequently reannotated into 26,570 nonredundant Unigene identifiers. To statistically identify the genes that were differentially expressed between HUBECs and HUVECs, a total of 4,477,203 probes in 18 hybridizations were fitted by using gene-by-gene ANOVA models. Volcano plot analysis revealed a highly consistent and nonredundant list of genes that were differentially expressed between HUBECs and HUVECs (Fig. 2).

Figure 2. Volcano plot of the nonconvergent nature of transcripts identified within human brain endothelial cells (HUBECs) and human umbilical vein endothelial cells (HUVECs). The average fold change of gene expression was calculated by comparing HUBECs (n = 5) to HUVECs (n = 4) in replicated experiments. Statistical significance was estimated by analysis of variance models. For each gene, the t score was plotted against the average fold change. Using stringent statistical cutoff values (gray line), 32 upregulated genes (red) and 33 downregulated (green) were identified as differentially expressed. The minimum fold change of these selected genes was 2, and the Bonferroni corrected p value is ' g! _; N+ ^! d( Q
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ANOVA identified 65 nonredundant transcripts that were most consistently and highly over- or underexpressed within HUBECs. The minimum fold change of these genes was >2-fold, and the p value for each gene, corrected by the Bonferroni method, was 8-fold enriched within the top 65 transcripts, and 25 of the 65 genes (38%) were annotated to have a cell communication function (Table 1). Transcripts annotated to have extracellular location, extracellular activity, cell growth activity, and collagen structure were significantly enriched (p 1.5-fold increased within HUBECs. These molecules are shown in supplemental online Table 1. The raw data from the complete HUBEC gene expression studies (Affymetrix CEL files) and all analyzed data (ratio of all genes) can be accessed directly at the Duke Bioinformatics Shared Resource web site (http://dbsr.duke.edu/pub/hubec). This web site provides accessible links to allow investigators to readily examine the complete HUBEC database.

Figure 3. Expression pattern of the top 65 differentially expressed genes within HU-BECs versus HUVECs. Each column represents one independent experiment and each row represents a distinct gene. The relative expression ratio between HUBECs versus HUVECs is represented by color (red, higher; green, lower; black, no change). Abbreviations: HUBEC, human brain endothelial cell; HUVEC, human umbilical vein endo-thelial cell.
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Table 1. Gene ontology categories of top 65 transcripts+ K( v& n" a3 w$ r
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Table 2. Genes most overrepresented and underrepresented within human brain endothelial cells  I2 h9 S1 l7 o) S4 R6 H/ s, ?( L" t) B
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The murine fetal liver stromal cell line, AFT024, has been shown to support the ex vivo maintenance of murine and human HSCs in cell-to-cell contact cultures . Autotaxin and phosphodiesterase 1a fall within the same family of phospholipases, suggesting that the action of these phospholipases on target HSCs may contribute independently to their maintenance in vitro.$ ^$ T  v" n7 e2 f& r# b

Table 3. Common transcripts between HUBEC and AFT024

Validation of Differential Expression of HUBEC-Specific Transcripts6 X. r' h8 Y" v# i* l" o
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To validate the results of the gene array analyses, quantitative real-time reverse transcription (RT)-PCR was performed for several genes identified to be overexpressed by HUBECs compared with HUVECs. Table 4 shows the expression of each gene within HUBECs and HUVECs relative to GAPDH control. IGFBP2, myocardin, and decorin were expressed in HUBECs but were below the level of detection within HUVECs, whereas adrenomedullin was 40-fold greater within HUBECs than HUVECs. These results demonstrated a good correlation between "present" and "absent" determinations within the gene array datasets and measurements of transcription by quantitative real-time PCR.: _; H3 v0 {9 Y# l' K
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Table 4. Quantitative real-time RT-PCR analysis of representative genes
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Functional Assay of HUBEC-Derived Soluble Proteins" K* n4 d$ O1 v  ~, a$ O

To begin to define the hematopoietic capacity of the novel proteins produced by HUBECs, we first assayed the activity of four HUBEC-derived proteins against primary human CB CD34  cells based upon their fold upregulation (IGFBP2 and IGFBP3), their annotated soluble or extracellular activity (adrenomedullin and follistatin), and their collective lack of defined hematopoietic activity. As shown in Figure 4, neither IGFBP2, IGFBP3, nor follistatin demonstrated any additive hematopoietic effect with regard to total cell or CD34  cell expansion when combined with TSF. However, the addition of 50¨C100 ng/ml adrenomedullin to TSF caused a significant increase in total cell and CD34  cell expansion compared with TSF alone (p = .001 and p = .002, respectively), suggesting a potentially direct effect of adrenomedullin on human hematopoietic progenitor cells. Moreover, when we assayed HSC-enriched CD34 CD38¨Clin¨C cells alone, the addition of 100 ng/ml adrenomedullin significantly increased the proliferation of this primitive population when combined with SCF or Flt-3 ligand, as compared with either cytokine alone (p = .01 and p = .003, respectively; Fig. 5). Again, neither IGFBP2 nor follistatin supported an additive effect upon SCF, Flt-3 ligand, or TSF. However, IGFBP3 at 50 ng/ml was associated with an increase in the proliferation of CD34 CD38¨Clin¨C cells in combination with Flt-3 ligand as compared with Flt-3 ligand alone (p = .01). Taken together, these data suggested that IGFBP3 and, in particular, adrenomedullin, are candidate endothelial cell-derived growth factors with hematopoietic activity.
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Figure 4. Adrenomedullin supports an increase in CD34  progenitor cell expansion in short-term culture. Primary human cord blood CD34  cells (2.5 x 104) were placed in culture with thrombopoietin, stem cell factor, and Flt-3 ligand with and without 50¨C100 ng/ml IGFBP2, IG-FBP3, follistatin, or adrenomedullin for 7 days. (A): The addition of IGFBP2, IGFBP3, or follistatin had no effect on total progenitor cell expansion, whereas adrenomedullin caused a significant increase in total cells (p = .001). (B): The addition of adrenomedullin also caused a significant increase in the number of CD34  progenitor cells over time (p = .002). Abbreviations: ADM, adrenomedullin; Foll, follistatin; IGFBP, insulin-like growth factor binding protein; TSF, thrombopoi-etin, stem cell factor, and Flt-3 ligand.

Figure 5. Hematopoietic activity of human brain endothelial cell (HUBEC)-secreted factors on primitive human CB CD34 CD38¨Clin¨C progenitors. FACS-sorted human CB CD34 CD38¨Clin¨C cells (n = 5 cells per well) were sorted into individual Terasaki culture wells with stem cell factor (SCF), Flt-3 ligand, or TSF with and without 50¨C100 ng/ml recombinant IGFBP2, IGFBP3, follistatin, or adrenomedullin. The bar graphs indicate the mean total cell expansion under each condition at day 7. As shown, IGFBP2 failed to induce significant proliferation of human progenitors (A), whereas 50 ng/ml IGFBP3 appeared to have an additive effect with Flt-3 ligand (B) (p = .01). Follistatin had no effect on progenitor cell proliferation (C), whereas adrenomedullin demonstrated a dose-responsive additive effect on CD34 CD38¨Clin¨C progenitor cell proliferation when combined with both SCF (p = .01) and Flt-3 ligand (p = .003) (D). Abbreviations: SCF, stem cell factor; TSF, thrombopoietin, stem cell factor, and Flt-3 ligand.
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DISCUSSION
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One strategy that has been employed to characterize the biology of HSCs has involved the molecular analysis of purified cell populations enriched for HSCs compared with committed progenitors. Ivanova et al. examined the gene expression profile of murine BM Lin¨Cc-kit  Sca-1 Rholow cells (HSCs) versus murine fetal liver HSC, human fetal liver HSC, embryonic neural stem cells, and an embryonic stem cell line and found 283 transcripts enriched within all three stem cell populations , particularly due to a lack of effector cells capable of inducing HSC expansion. The approach we have taken offers the benefit of identifying human genes that have a likelihood of direct involvement in signaling the maintenance and expansion of human HSCs. Concordantly, we are pursuing studies to determine whether the conditioned medium alone from HU-BECs is capable of inducing HSC expansion, as well as the identification of HUBEC-secreted proteins via high-throughput chromatographic separation.9 {, w" I& i! K8 m
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Our analysis identified 65 genes that were significantly over- or underexpressed within HUBECs compared with HUVECs, with consistency across multiple biological replicates. Gene ontology studies demonstrated that the majority of the upregulated genes within HUBECs were extracellular and/or involved in triggering cell growth. The identification of transforming growth factor-ß-induced protein as one of the most highly overexpressed HUBEC gene products is noteworthy in light of the established function of transforming growth factor-ß in inhibiting HSC cycling and proliferation , raising the possibility that collagen moieties produced by HUBECs may contribute to the soluble hematopoietic activity we have observed.
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The upregulation of two BMP antagonists, follistatin and gremlin 1 homolog, was somewhat surprising in light of the previously demonstrated contribution of BMP signaling in embryonic hematopoiesis , it is plausible that either might inhibit differentiation of proliferating HSCs.

Two of the most highly overexpressed transcripts within HUBECs, stanniocalcin 1 and 2, regulate calcium/phosphorus homeostasis in fish and humans and induce proliferation and differentiation of osteoblasts in vitro  also suggests that brain-derived factors may have hematopoietic activity.
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Of additional interest was the examination of transcripts that were downregulated in HUBECs compared with HUVECs. Of note, protocadherin and VE-cadherin were markedly underexpressed in HUBECs compared with HUVECs. Since cadherin-based interactions have recently been implicated in the contact-dependent maintenance of quiescent HSCs in vivo within the osteoblastic marrow niche , this implies that such interactions might be important for ex vivo maintenance of HSCs in culture. Despite this, noncontact HUBEC cultures and the results of this gene expression analysis indicate that the cadherin-based contact interactions are not important for expansion of HSCs in the HUBEC culture system. Taken together, these data implicate a novel soluble factor or factors elaborated by HU-BECs that promote the expansion of human repopulating cells. Moreover, these data suggest that the interaction of HSCs with cadherin moities may inhibit the proliferation of HSCs, thereby maintaining quiescence. Further studies will be important to delineate differences in the cell cycle status and SCID-repopulating capacity of HSCs cultured with osteoblasts and those cultured under noncontact conditions with HUBECs.

The fetal liver murine stromal cell line AFT024 has been shown, in contact cultures, to support the ex vivo maintenance of murine and human HSCs . When we queried the most upregulated HUBEC transcripts against the AFT024 database, we identified seven transcripts in common between HUBECs and AFT024, including collagen type I and VI, IGFBP3, URB, and autotaxin. We have prioritized functional assay of these genes via loss of function small inhibitory RNA studies, since these molecules should have a high probability of participation in HSC signaling. We also anticipate that other extracellular HUBEC transcripts unique from the AFT024 transcriptome will prove to be functionally important in HSC regulation in light of the distinctly soluble nature of the HUBEC hematopoietic activity that we have observed.; d, b. H5 G0 d. i4 P7 b

As an initial strategy to screen for the hematopoietic activity of novel growth factors expressed by HUBECs, we have analyzed a group of proteins that are available in recombinant form and have established extracellular function: IGFBP2, IGFBP3, follistatin, and adrenomedullin. Interestingly, one of these proteins, adrenomedullin, augments the expansion of human CD34  cell when combined with thrombopoietin, SCF, and Flt-3 ligand, while also enhancing the individual activities of SCF and Flt-3 ligand on HSC-enriched CD34 CD38¨Clin¨C cells in vitro. These data indicate that further studies are merited to define the effects of adrenomedullin on HSC fate and hematopoiesis in general, in addition to our planned siRNA gene silencing studies to determine the precise contribution of adrenomedullin to HUBEC-mediated HSC expansion. Although it has been established that adrenomedullin is required for normal cardiovascular development . We plan to recombinantly produce and functionally assay each of the genes with secretory or extracellular domains that are overexpressed by HUBECs and anticipate that the reproduction of HUBEC stem cell-supportive activity may require the combination of several proteins identified thus far./ N% `8 f* |/ b; Q6 Q

In summary, we have presented a molecular profile of novel endothelial cells that support the ex vivo expansion of human HSCs. Since HUBECs are unlike other established stromal cell lines (e.g., AFT024) in the soluble nature of their HSC-supportive activity, it is plausible that novel soluble proteins produced by HUBECs can be identified and characterized. The identities of these factors may overlap with secreted factors produced within the BM microenvironment that support in vivo HSC maintenance and proliferation . The HUBEC molecular profile is a template for the identification of soluble factors that mediate hematopoietic stem cell fate.
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ACKNOWLEDGMENTS* h) ]' n# Z7 I) J( O9 A

We thank Dr. David Venzon from the National Cancer Institute for critical assistance with the biostatistical analysis. Primary HUBECs were kindly provided by the Naval Medical Research Center (Silver Spring, MD) for a portion of the studies.
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DISCLOSURES. D7 ]& @! Q( i# |. i, a- Q% B. {

The authors indicate no potential conflicts of interest.
      【参考文献】: W- C+ P+ ]$ i0 J5 Y% C

Sorrentino P. Clinical strategies for expansion of haematopoietic stem cells. Nat Rev Immunol 2004;4:878¨C888.

Osawa M, Hanada K, Hamada H, Nakauchi H. Long-term lymphohematopoietic reconstitution from a single CD34-/low hematopoietic stem cell. Science 1996;273:242¨C245.

Cashman J et al. Kinetic evidence of the regeneration of multilineage hematopoiesis from primitive cells in normal human bone marrow transplanted into immune deficient mice. Blood 1997;80:4307¨C4316.

Rocha V, Cornish J, Sievers E et al. Comparison of outcomes of unrelated bone marrow and umbilical cord blood transplants in children with acute leukemia. Blood 2001;97:2962¨C2971.7 z6 C8 j' T/ A

Storb R, Blume K, O¡¯Donnell M et al. Cyclophosphamide and anti-thymocyte globulin to condition patients with aplastic anemia for allogeneic marrow transplantations: The experience of 4 centers. Biol Blood Marrow Transplant 2001;7:39¨C44.
8 f% K' d; [3 H# v; G# Q0 _! d
Kiehl M, Kraut L, Schwerdtfeger T et al. Outcome in hematopoietic stem cell transplantation in adult patients with acute lymphoblastic leukemia: No difference in related compared with unrelated transplant in first complete remission. J Clin Oncol 2004;15:2816¨C2825.6 T) B6 B# D8 J; v2 b7 k& E, j
3 o: L3 p- s! p; I
Gur H, Krauthgamer R, Berrebi A et al. Tolerance induction by megadose hematopoietic progenitor cells: Expansion of veto cells by short term culture of purified human CD34  cells. Blood 2002;99:4174¨C 4181.

Cogle C, Wainman D, Jorgensen M et al. Adult human hematopoietic cells provide functional hemangioblast activity. Blood 2004;103:133¨C135.

Rafii S, Lyden D. Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration. Nat Med 2003;9:702¨C712.

Bhatia M, Wang J, Kapp U et al. Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice. Proc Natl Acad Sci U S A 1997;94:5320¨C5325.
' c( z6 X6 i( J& y, G! J) A
Krosl J, Austin P, Beslu N et al. In vitro expansion of hematopoietic stem cells by recombinant TAT-HOXB4 protein. Nat Med 2003;9:1428¨C1432.
3 A4 N, ?1 f* @* }  Z
Piacibello W, Bruno S, Sanavio F et al. Lentiviral gene transfer and ex vivo expansion of human primitive stem cells capable of primary, secondary, and tertiary multilineage repopulation in NOD/SCID mice. Blood 2002;15:4391¨C4400.8 D( U. ^& @1 ?; @

Chute J, Saini AA, Chute DJ et al. Co-culture with human brain endothelial cells increases the SCID repopulating capacity of adult human bone marrow. Blood 2002;100:4433¨C4439.
) s/ X% @; T( ^# N
Kawano Y, Kobune M, Yamaguchi M et al. Ex vivo expansion of human umbilical cord hematopoietic progenitor cells using a coculture system with human telomerase catalytic subunit (hTERT) transfected human stromal cells. Blood 2003;101:532¨C540.

Reya T, Duncan A, Ailles L et al. A role for Wnt signalling in the self renewal of hematopoietic stem cells. Nature 2003;423:409¨C414.
) W+ }4 S' N$ J8 t
Varnum-Finney B, Xu L, Brashem-Stein C et al. Pluripotent, cytokine-dependent, hematopoietic stem cells are immortalized by constitutive Notch1 signaling. Nat Med 2000;6:1278¨C1281.( q" w, [( m. `

Bhardwaj G, Murdoch B, Wu D et al. Sonic hedgehog induces the proliferation of primitive human hematopoietic cells via BMP regulation. Nat Immunol 2001;2:172¨C180.
4 J# c( t0 X. ]3 M5 H
Amsellem S, Pflumio F, Bardinet D et al. Ex vivo expansion of human hematopoietic stem cells by direct delivery of the HOXB4 homeoprotein. Nat Med 2003;9:1423¨C1427.# I  Q1 x* K( y
$ w$ R( R  Y" J& t
Basser R, To L, Begley C et al. Rapid hematopoietic recovery after multicycle high dose chemotherapy: Enhancement of filgrastim-induced progenitor cell mobilization by recombinant human stem cell factor. J Clin Oncol 1998;16:1899¨C1908.0 W% V* s+ }9 ~0 |9 ]" P$ c
- W% M* W% M+ J/ k" G9 d
Rizzo J, Lichtin A, Woolf S et al. Use of epoietin in patients with cancer: Evidenced based clinical practice guidelines of the American Society of Clinical Oncology and the American Society of Hematology. J Clin Oncol 2002;20:4083¨C4107.7 m, G+ K; ^9 ]" ~3 z
8 d( C  ^' s. R+ \' m$ }# W' G# {
Bennett C, Smith T, Weeks J et al. Use of hematopoietic colony stimulating factors: The American Society of Clinical Oncology survey. The Health Services Research Committee of the American Society of Clinical Oncology. J Clin Oncol 1996;14:2511¨C2520.
" ^+ S% Z2 l$ n  [! j  b
Avecilla S, Hattori K, Heissig B et al. Chemokine mediated interaction of hematopoietic progenitors with the bone marrow vascular niche is required for thrombopoiesis. Nat Med 2004;10:64¨C71.
' z' P3 [/ i8 X% Y2 O5 r
Calvi L, Adams G, Weibrecht K et al. Osteoblastic cells regulated the haematopoietic stem cell niche. Nature 2003;425:841¨C846.5 W: J5 i% g, J' b+ B4 e9 U, r
6 g2 t- w  |) R; q. k# y' a/ p; K
Zhang J, Niu C, Ye L et al. Identification of the haematopoietic stem cell niche and control of the niche size. Nature 2003;425:836¨C841.
7 N! M5 ?# x- I0 {" h
Shalaby F, Rossant J, Yamaguchi TP et al. Failure of blood island formation and vasculogenesis in flk-1 deficient mice. Nature 1995;376:62¨C66.
) ~( Q2 T0 B# g) d* c
Oberlin E, Tavian M, Blazsek I et al. Blood-forming potential of vascular endothelium in the human embryo. Development 2002;129:4147¨C 4157.6 p, }# h+ L* O$ h
2 V6 r* f% s! `  ], R# j3 y  u
Choi K, Kennedy M, Kazarov A et al. A common precursor for hematopoietic and endothelial cells. Development 1998;125:725¨C732.

Lu L, Wang S, Auerbach R. In vitro and in vivo differentiation into B cells, T cells, and myeloid cells of primitive yolk sac hematopoietic progenitor cells expanded >100 fold by a clonal yolk sac endothelial cell line. Proc Natl Acad Sci U S A 1996;93:14782¨C14787.

Rafii S, Shapiro F, Rimarachin J et al. Isolation and characterization of human bone marrow microvascular endothelial cells: Hematopoietic progenitor cell adhesion. Blood 1994;84:10¨C19.. Q4 j6 M9 i* D$ _
; b; x  P3 T6 c& Y
Rafii S, Shapiro F, Pettengell R et al. Human bone marrow micro-vascular endothelial cells support long term proliferation and differentiation of myeloid and megakaryocytic progenitors. Blood 1995; 86:3353¨C3363.# P8 u1 ~+ D& a" s; _$ O6 P
' n3 |# z0 G2 ?" F3 u% r
Tavian M, Coulombel L, Luton D et al. Aorta associated CD34  hematopoietic cells in the early human embryo. Blood 1996;87:67¨C72.  O! c1 l: B$ o3 G3 g8 u1 s

Heissig B, Hattori K, Dias S et al. Recruitment of stem and progenitor cells from the bone marrow niche requires MMP-9 mediated release of kit ligand. Cell 2002;109:625¨C637.
; Y- t! Q1 Q3 j! h
Gan O, Murdoch B, Larochelle A et al. Differential maintenance of primitive SCID-repopulating cells, clonogenic progenitors, and long-term culture initiating cells after culture on human bone marrow stromal cells. Blood 1997;90:641¨C650.
1 s, @/ z3 X; c7 W6 l$ ]9 _" w& I3 i
Punzel M, Liu D, Zhang T et al. The symmetry of initial divisions of human hematopoietic progenitors is altered only by the cellular microenvironment. Exp Hematol 2003;31:339¨C347.
9 u, O& U+ P6 t5 S$ z
Lewis I, Almeida-Porada G, Du J et al. Umbilical cord blood cells capable of engrafting in primary, secondary, and tertiary xenogeneic hosts are preserved after ex vivo culture in a non-contact system. Blood 2001;7:3441¨C3449.$ `2 g: M4 R2 W# F* a
& I3 o$ n/ d! h* v
Chute JP, Kampen R, Wells R et al. A comparative study of the cell cycle status and primitive cell adhesion molecule profile of human CD34  cells cultured in stroma-free versus porcine microvascular endothelial cell cultures. Exp Hematol 1999;27:370¨C379.
+ k3 C7 Q; ~7 n
Chute JP, Saini A, Wells M et al. Pre-incubation with endothelial cell monolayers increases gene transfer efficiency into human bone marrow CD34 CD38¨C progenitor cells. Hu Gene Ther 2000;11:2515¨C2528.6 y$ y. ^( t4 E1 n4 J' x

Chute J, Fung J, Muramoto G et al. Ex vivo culture rescues hematopoietic stem cells with long term repopulating capacity following harvest from lethally irradiated mice. Exp Hematol 2004;32:308¨C317.
! Z  \$ x5 z* T1 f* u* S
Brandt JE, Bartholemew AM, Fortman JD et al. Ex vivo expansion of autologous bone marrow stem cells with porcine microvascular endothelial cells results in a graft capable of rescuing lethally irradiated baboons. Blood 1999;94:106¨C113.6 _  u- r7 W8 A/ k. n4 s( F+ R8 \

Chute JP, Fung J, Muramoto G, Oxford C. Quantitative analysis demonstrates expansion of SCID-repopulating cells (SRC) and increased engraftment capacity within human cord blood cells following culture with human brain endothelial cells. STEM CELLS 2004;22:202¨C215.

Chute JP, Muramoto G, Fung J et al. Soluble factors elaborated by human brain endothelial cell induce the concomitant expansion of purified BM CD34 CD38¨C cells and SCID repopulating cells. Blood 2005; 105:576¨C583.7 T" u  f7 m9 x/ t5 g; W+ O  v
: n; M0 ~9 Q2 d% ?
Schulz L, Schweitzer P, Christianson S et al. Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice. J Immunol 1995;154:180¨C191.& p  s( g# }; _( U! f
  J( T1 Z( ?/ a6 U/ G" j
Taswell C. Limiting dilution assays for the determination of immuno-competent cell frequencies, I: Data analysis. J Immunol 1981;126:1614¨C1619.& y. n& M6 e  j( S/ K0 m
+ b/ B* P. D3 t3 ^6 B* u
Wang J, Doedens M, Dick J. Primitive human hematopoietic cells are enriched in cord blood compared with adult bone marrow or mobilized peripheral blood as measured by the quantitative in vivo SCID-repopulating cell assay. Blood 1997;89:3919¨C3924." W+ o# P# V- k  B5 a9 b/ t2 N

Jazwiec B, Solanilla A, Grosset C et al. Endothelial cell support of hematopoiesis is differentially altered by IL-1 and glucocorticoids. Leukemia 1998;8:1210¨C1220.1 R' |2 b# T- v* \7 g

Coombes B, Mahoney J. cDNA array analysis of altered gene expression in human endothelial cells in response to Chlamydia pneumoniae infection. Infect Immun 2001;69:1420¨C1427.
, u" d1 _, V" I6 O" D5 v: t6 p8 S+ C
Gerritsen M, Soriano R, Yang S et al. Branching out: A molecular fingerprint of endothelial differentiation into tube-like structures generated by Affymetrix oligonucleotide arrays. Microcirculation 2003;10:63¨C81.2 t3 Z3 H, Z( n
3 z" g4 u# g* }# n3 F  z
Soon L, Yie T, Shvarts A et al. Overexpression of WISP-1 downregulated motility and invasion of lung cancer cells through inhibition of Rac activation. J Biol Chem 1003:278:11465¨C11470.

Cousins R, Blanchard R, Popp M et al. A global view of the selectivity of Zinc deprivation and excess on genes expressed in human THP-1 mononuclear cells. Proc Natl Acad Sci U S A 2003;100:6952¨C6957.: t) O( I4 g5 M; D: [
" u9 ~& N" q0 C$ Q( _9 D+ b4 {. l
Brazma A, Hingamp P, Quackenbush J et al. Minimum information about a microarray experiment (MIAME)-toward standards for microarray data. Nat Genet 2001;29:365¨C371.' H) o4 M( J  |4 i

Dudoit S, Gentleman R, Quackenbush J. Open source software for the analysis of microarray data. Biotechniques 2003;suppl:45¨C51.
" u; X" V  D. N9 ?
Hosack D, Dennis G, Sherman B et al. Identifying biological themes within lists of genes with EASE. Genome Biol 2003;4:R70.

Klein N, Battaglia D, Woodruff T et al. Ovarian follicular concentrations of activin, follistatin, inhibin, insulin-like growth factor I (IGF-1), IGF-II, IGF binding protein 2 (IGFBP2), IGFBP3, and vascular endothelial growth factor in spontaneous menstrual cycles of normal women of advanced reproductive age. J Clin Endocrinol Metab 2000;85:4520¨C4525.

Hackney J, Charboard P, Brunk B et al. A molecular profile of a hematopoietic stem cell niche. Proc Natl Acad Sci U S A 2002;99:13061¨C13066.
$ X' h* r$ [) r4 W& f% k# J" e
Fox M, Colello R, Macklin W et al. Phosphodiesterase-1 alpha/autotaxin: A counteradhesive protein expressed by oligodendrocytes during onset of myelination. Mol Cell Neurosci 2003;23:507¨C519.* ~. k1 n% H2 _9 w" s+ _4 f

Ivanova N, Dimos J, Schaniel C et al. A stem cell molecular signature. Science 2002;298:601¨C604.0 H7 G& ]# E! L0 @0 c( [' g3 ]

Ramalho-Santos M, Yoon S, Matsuzaki Y et al. "Stemness": Transcriptional profiling of embryonic and adult stem cells. Science 2002;298:597¨C600.: l" ~1 V( u  E1 D

Georgantas R, Tanavde V, Malehorn M et al. Microarray and serial analysis of gene expression analyses identify known and novel transcripts overexpressed in hematopoietic stem cells. Cancer Res 2004;64:4434¨C4441.% _4 f! k0 C$ h( a0 _/ Y

Sitnicka E, Ruscetti F, Priestley G et al. Transforming growth factor beta 1 directly and reversibly inhibits the initial cell divisions of long term repopulating hematopoietic stem cells. Blood 1996;88:82¨C88.
! j% I9 d  I# {
Hofflich A, Lahm H, Blum W et al. Insulin like binding protein 2 inhibits proliferation of human embryonic kidney fibroblasts and of IGF responsive colon carcinoma cell lines. FEBS Lett 1998;434:329¨C334.

Mouhieddine O, Cazals V, Kuto E et al. Glucocorticoid growth arrest of lung alveolar epithelial cells is associated with increased production of insulin like growth factor binding protein 2. Endocrinology 1996;137:287¨C295." E+ J% P0 z& j3 B2 \2 V
# [) G/ i/ l! Y# s
Longobardi L, Torello M, Buckway C et al. A novel insulin-like growth factor I independent role for insulin like growth factor binding protein 3 in mesenchymal chondroprogenitor cell apoptosis. Endocrinology 2003; 144:1695¨C1702.+ D1 i7 D$ ]  c$ l/ i
' u( m( s7 z8 m- P) c; }8 }; B+ I& I) W
Vihervouri E, Virtanen M, Koistinen H et al. Hemoglobin level is linked to growth hormone dependent proteins in short children. Blood 1996;87:2075¨C2081.
6 r: O; C" C  p8 U
Bhatia R, Williams A, Munthe H. Contact with fibronectin enhances maintenance of normal but not chronic myelogenous leukemia primitive hematopoietic progenitors. Exp Hematol 2002;30:324¨C332.9 K) C& W2 |  P! I
' U1 b* \3 r, n* g7 f
Yokota T, Oritani K, Takahashi I et al. Adiponectin, a new member of the family of soluble defense collagens, negatively regulates the growth of myelomonocytic progenitors and the functions of macrophages. Blood 2000;96:1723¨C1732.
% g2 U! o1 l8 T7 j1 w
Zon L. Self renewal versus differentiation, a job for the mighty morphogens. Nat Immunol 2001;2:142¨C143.

Meehan T, Schlatt S, O¡¯Bryan M et al. Development of germ cell and Sertoli cell development by activin A, follistatin, and FSH. Dev Biol 2000;220:225¨C237.

Matzuk M, Lu N, Vogel H et al. Multiple defects and perinatal death in mice deficient in follistatin. 1995;374:360¨C363.+ K  k) M- _9 M: d$ S
8 S' J  D0 u2 ]" q! k' d! ]
Yamashita T, Takahashi S, Ogata E. Expression of activin/erythroid differentiation factor in murine bone marrow stromal cells. Blood 1992; 79:304¨C307.

Michos O, Panman L, Vintersten K et al. Gremlin-mediated BMP antagonism induces the epithelial-mesenchymal feedback signaling controlling metanephric kidney and limb organogenesis. Development 2004; 131:3401¨C3410.: X# F; P6 Q5 |' u4 W

Zhang Y, Cleary M, Si Y et al. Inhibition of activin signaling induces pancreatic epithelial cell expansion and diminishes terminal differentiation of pancreatic beta cells. Diabetes 2004;53:2024¨C2033.

Yoshiko Y, Aubin J. Stanniocalcin 1 as a pleiotropic factor in mammals. Peptides 2004;25:1663¨C1669.3 ~+ ?6 `- E2 s  j' z: @; V( A8 J$ u

Liu Y, Monticone M, Tonachini L et al. URB expression in human bone marrow stromal cells and during mouse development. Biochem Biophys Res Commun 2004;322:497¨C507.: p, o8 Z, t6 L& B4 C9 I
/ L4 j* k" q; t2 z
Murphy W, Durum S, Anver M, Longo D. Immunologic and hematologic effects of neuroendocrine hormones: Studies on DW/J dwarf mice. J Immunol 1992;148:3799¨C3805.
1 k4 p4 b5 @7 M) t; g
Terskikh A, Easterday M, Li L et al. From hematopoiesis to neuropoiesis: Evidence of overlapping genetic programs. Proc Natl Acad Sci U S A 2001;98:7934¨C7939.

Punzel M, Gupta P, Roodell M et al. Factor(s) secreted by AFT024 fetal liver cells following stimulation with human cytokines are important for human LTC-IC growth. Leukemia 1999;13:1079¨C1084.

Caron K, Smithies O. Extreme hydrops fetalis and cardiovascular abnormalities in mice lacking the adrenomedullin gene. Proc Natl Acad Sci U S A 2001;98:615¨C 619.& ]& C( {0 u9 @+ X
- `* D1 d8 c% |2 ]! L8 U4 g
Harzenetter M, Keller U, Beer S et al. Regulation and function of the CGRP receptor complex in human granulopoiesis. Exp Hematol 2002; 30:306¨C312.
1 T- U$ \4 t' P7 V
De Angeli S, Di Liddo R, Buoro S et al. New immortalized human stromal cell lines enhancing in vitro expansion of cord blood hematopoietic stem cells. Int J Mol Med 2004;13:363¨C371.

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