• Endothelial cell;
  • Stem cell expansion;
  • Hematopoietic stem cell


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

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 [SRCs]). 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+CD38lin 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. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

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 [13]. Currently, transplantation of human HSCs from adult bone marrow (BM), mobilized peripheral blood, and umbilical cord blood (CB) is applied in the curative treatment of both malignant and nonmalignant diseases [46]. More recently, the potential contribution of transplanted HSCs toward immune tolerance induction [7], vascular repair [8], and in vivo tissue regeneration [9] has been suggested. Since HSCs comprise <0.1% of the CD34+ population within the bone marrow of adults [10], numerous studies have focused on the development of methods to expand HSC numbers in vitro with a goal of generating larger numbers of transplantable repopulating cells [1114]. Concordantly, strategies have been applied to identify novel growth factors that stimulate HSC self-renewal in vivo [1518]. Despite these efforts, few hematopoietic growth factors have achieved clinical application [1921].

One strategy to identify HSC growth factors involves examination of candidate niches wherein HSCs are known to reside physiologically [2224]. Two recent studies have demonstrated that HSCs reside in contact with osteoblasts in the BM niche [23, 24] and these cells provide signaling through Notch ligand and cadherin interactions to maintain quiescent HSCs in vivo. A vascular niche within the marrow has also been postulated, comprised of sinusoidal endothelial cells, in which HSC proliferation and differentiation are thought to occur [22]. The role of endothelial cells (ECs) as regulators of hematopoiesis is supported by evidence from embryogenesis, in which development of blood islands is critically dependent upon the presence of flk-1-positive vascular precursor cells [25, 26]. Gene marking studies have also suggested a common precursor cell, the hemangioblast, which appears to give rise to both HSCs and endothelial precursor cells [27]. Yolk sac ECs support hematopoietic progenitor cell growth ex vivo [28], and adult BM ECs support the in vitro proliferation of erythroid, myeloid, and megakaryocytic progenitors [29, 30]. Anatomically, human HSCs embed within the intimal layer of the aorta at day 35 of embryogenesis [31] and reside in association with ECs in the fetal liver [26] and, ultimately, in the adult BM [22, 32]. Therefore, ECs are a logical source of growth factors that regulate HSC growth and differentiation.

Several studies have examined the capacity for stromal cell lines to support the ex vivo maintenance of HSCs [14, 33, 34]. Whereas studies of primary human BM stroma have been disappointing [33], a murine fetal liver stromal cell line, AFT024, has been shown to support the maintenance of human CB severe combined immunodeficient-repopulating cell (SRCs) in vitro [34, 35]. Conversely, our laboratory has shown that coculture with brain-derived porcine microvascular endothelial cells supports the expansion of human BM CD34+ and CD34+CD38 cells during short-term culture [36]. We subsequently showed that coculture with porcine brain ECs augmented the genetic modification of human HSCs [37] and, remarkably, induced the functional repair and expansion of lethally irradiated murine HSCs [38]. In collaboration with Brandt et al. [39], we also showed that the progeny of BM cells cultured with porcine brain ECs were capable of providing long term repopulation in lethally irradiated baboons. Our recent studies indicate that this HSC-supportive activity is conserved within primary human brain ECs (HUBECs) as well, whereas nonbrain ECs fail to maintain human CD34+CD38 cells in culture [13]. Coculture with HUBECs supports a four fold expansion of both human BM SRCs [13] and CB SRCs [40] in 7-day cultures, and in contrast to comparative stromal cell lines [34], cell-to-cell contact does not appear to be required for HUBECs to stimulate the expansion of human HSCs [40, 41]. These data suggest that soluble factors elaborated by HUBECs account for the unique hematopoietic activity that we have observed.

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.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Noncontact Cultures of Human CB CD34+ Cells with Primary HUBECs

Primary human cord blood CD34+ cells were procured from Cambrex (Cambrex, Walkersville, MD, Briefly, 1 × 105 CD34+ cells were placed in six-well culture plates with Iscove's modified Dulbecco's medium (IMDM) (Gibco-BRL, Gaithersburg, MD, 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, 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, was performed at day 0 and day 14 to compare the hematopoietic content at each time point.

Transplantation of Human Hematopoietic Cells into NOD/SCID Mice

Six- to 8-week-old nonobese diabetic severe combined immunodeficient (NOD/SCID) mice (Jackson Laboratory, Bar Harbor, ME, were used for all experiments [42]. All animal studies were performed under protocols approved by the Duke University Institutional Animal Care and Use Committee. Briefly, mice were irradiated with 300 cGy from a Cs137 source. Four hours postirradiation, mice were transplanted via tail vein injection with either 2 × 104 day 0 CB CD34+ cells or their progeny following 14-day culture with either TSF alone or HUBEC transwell cultures supplemented with TSF. Eight weeks post-transplantation, all mice were sacrificed, bilateral femurs were harvested, and BM cells were collected. Immunophenotypic analysis of human cell engraftment and lineage repopulation within the murine marrow was performed using antibodies against human CD45PerCP, anti-murine CD45 fluorescein isothiocyanate (FITC), anti-huCD34 phycoerythrin (PE), anti-huCD38FITC, anti-huCD33FITC, anti-huCD13PE, anti-huCD19PE, anti-huCD3FITC, anti-huCD56FITC, and anti-huCD71PE, along with isotype controls (Becton, Dickinson and Company). Estimation of SRC frequency in each cell source was calculated using the maximum likelihood estimator as described previously by Taswell [43] for the single-hit Poisson model [43, 44].

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 [13]. Briefly, 1 × 105 HUBECs were cultured on gelatin-coated six-well plates (Corning Incorporated Life Sciences, Acton, MA, in complete endothelial cell culture medium (5 ml per well) containing M199 (Invitrogen, Carlsbad, CA,, 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,, and 1% pcn/strp at 37°C 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.

Human umbilical vein endothelial cells (HUVECs) (ATCC, Manassas, VA, were used as control cells and were cultured primarily as previously described [45]. Briefly, 1 × 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.

RNA isolation from HUBECs and HUVECs was performed as follows. Briefly, 5 × 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 4°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°C. 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 [46]. The samples were then precipitated with ethanol. Following ethanol precipitation, samples were analyzed via spectrophotometry and TBE ethidium bromide gel electrophoresis to verify the presence of highly pure RNA. Total RNA was used to develop the targets for Affymetrix microarray analysis and probes were prepared according to the manufacturer's instructions. Briefly, biotin-labeled cRNA was produced by in vitro transcription, fragmented, and hybridized to the Human 133A and 133B arrays (Affymetrix, Santa Clara, CA, containing >47,000 representative human gene sequences, as previously described [47]. Arrays were hybridized at 45°C for 16 hours and then washed and stained using the GeneChip Fluidics and scanned on the Affymetrix scanner. The hybridization signals from each array were normalized against the signals from human maintenance genes, which show consistent levels of expression across a variety of tissues prior to comparisons with other array results [48, 49]. To verify the consistency of gene expression within the endothelial cell samples, multiple biological replicates were subjected to microarray hybridization in an identical manner. Unified gene lists for each endothelial cell group, representing only those genes consistently up- or downregulated, were then generated. Collection of probe list data and analysis followed the Microarray Gene Expression Database Group/Minimum Information About a Microarray Experiment (MGED/MIAMI) guidelines [50]. All genes on U133A and B chips were reannotated into 26,570 nonredundant Unigene identifiers. An unsupervised cluster analysis using all of these genes suggested a difference between the transcriptional programs of HUBECs and HUVECs. To statistically identify these differentially expressed genes, a total of 4,477,203 probes in 18 hybridizations were fitted using gene-by-gene analysis of variance (ANOVA) linear models. All calculations were conducted using the R/Bio-conductor package [51]. The top 65 candidate genes were analyzed by gene ontology using the EASE algorithm [52].

Quantitative Real-Time RT-PCR Analysis of HUBEC Gene Expression

Total RNA was isolated from 1 × 106 HUBECs or HUVECs (ATCC) using the RNeasy Mini kit (Qiagen, Valencia, CA,, according to the manufacturer's protocol. Total RNA was quantified using a SmartSpec 3000 spectrophotometer (Bio-Rad, Hercules, CA,, and 2 μg per sample was reverse transcribed using the High Capacity cDNA Archive kit (Applied BioSystems, Foster City, CA,, 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 ΔΔCt method, using GAPDH expression as a normalization reference.

CB Processing, Lineage Depletion, and FACS Sorting

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,, 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 5–8 × 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 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 CB cells were thawed, washed once in IMDM (Invitrogen) containing 10% FBS and 1% pcn/strp, counted, and resuspended at 5 × 106 to 1 × 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 × 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 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 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 cells.

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. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

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 [13, 40, 41], we compared the in vitro expansion and in vivo repopulating capacity of human CB CD34+ cells following extended (14-day) culture with an optimal cytokine combination, thrombopoietin, stem cell factor, and Flt-3 ligand (TSF), versus transwell (noncontact) HUBEC cultures supplemented with TSF. As shown in Figure 1A and 1B, total hematopoietic cell and CD34+ cell expansion at day 14 was significantly increased in the noncontact HUBEC cultures compared with TSF alone (p = .008 and p = .001, respectively; t test). More importantly, the progeny of 14-day noncontact cultures with HUBECs + TSF contained approximately eight fold increased numbers of SRCs compared with both input CD34+ cells and TSF-cultured cells (1 SRC in 8,200 cells [confidence interval: 1/3,800 to 1/19,000] versus 1 in 64,000 cells [1/14,000 to 1/1,140,000], 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.

Gene Expression Analysis Identifies Unique HUBEC Transcripts

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).

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 <.01. Figure 3 is a colorimetric plot demonstrating the differential expression of these genes within the HUBEC and HUVEC sample sets. As anticipated, subtraction of the HUBEC transcriptome against that of HUVECs eliminated many housekeeping endothelial cell genes that we hypothesized were unlikely to play a role in HSC regeneration. In addition, this analysis revealed that primary HUBECs do not differentially express many established hematopoietic growth factors, including granulocyte colony stimulating factor, Flt-3 ligand, stem cell factor, thrombopoietin, interleukin (IL)-1, and IL-3. Table 1 shows the fold enrichment for various gene ontology categories within the top 65 transcripts. Fold enrichment was calculated by comparing each gene ontology category in the 65-gene set against all the genes on the chip. When organized by biological process, molecules involved in cell growth or the regulation of cell growth were >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 < .001) within the genes upregulated in HUBECs. Conversely, transcripts annotated for function in cell adhesion and proteins integral to the membrane were significantly enriched (p < .001) within the most downregulated genes within HUBECs. The Unigene identifiers and fold changes for each of the top 65 up- or downregulated HUBEC transcripts, along with their common gene names, are shown in Table 2. Fifteen of the 32 (47%) upregulated transcripts have extracellular activity or secreted protein properties, consistent with the soluble hematopoietic activity detected in our functional studies. As shown in Table 2, certain gene families were overrepresented within HUBECs, including insulin-like growth factor binding proteins (2 and 3), collagens (type I α2, IV α1, and VI α3), bone morphogenetic protein (BMP) antagonists (gremlin I homolog and follistatin), and stanniocalcins (1 and 2). The established interactions of these genes in other biological systems, such as folliculogenesis (IGFBPs and follistatin) [53], suggest that these overrepresented genes within HUBECs may participate in a coordinated process. Interestingly, none of the most upregulated transcripts within HUBECs are known to have definitive function in hematopoiesis or HSC self-renewal. Conversely, cell adhesion molecules, including ICAM 2, protocadherin, VE cadherin, and PECAM (CD31), were significantly downregulated within HUBECs compared with HUVECs (Table 2). Using less stringent fold change-only criteria, we extended our analysis to include all transcripts with cell-cell signaling activity, hormone activity, and extracellular location that were >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 ( This web site provides accessible links to allow investigators to readily examine the complete HUBEC database.

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 [34, 54] The molecular profile of the AFT024 cell line has recently been published [54]. We hypothesized that common transcripts between HUBECs and AFT024 might represent an informatically validated list of HSC regulatory molecules. We interrogated the public StroCDB database ( [54] against the full-length sequences of all overrepresented transcripts within HUBECs, as shown in Table 2. Whereas the majority of the upregulated HUBEC transcripts failed to match with genes within the AFT024 transcriptome, 7 of the 32 sequences (22%) were found to be exact sequence matches (Table 3), including the soluble proteins IGFBP3, pregnancy-associated plasma protein A, autotaxin, and phosphodiesterase Ia. Autotaxin is of particular interest since this is a secreted phosphodiesterase that inhibits the cell adhesion of normal and malignant cells and promotes their motility [55]. 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.

Validation of Differential Expression of HUBEC-Specific Transcripts

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.

Functional Assay of HUBEC-Derived Soluble Proteins

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 50–100 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+CD38lin 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+CD38lin 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.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

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 Linc-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 [56]. Ramahlo-Santos et al. [57] similarly identified 216 transcripts that were enriched within murine HSC, neural stem cells, and embryonic stem cells. Interestingly, comparison of the lists of stem cell-associated genes from the two studies revealed only six genes in common between the two [58]. An additional analysis by Georgantas et al. comparing the unique genes within the transcriptome of human CD34+CD38lin cells versus the reported findings of overrepresented genes within three other data sets of purified murine and human HSC populations determined that only one gene, the GATA3 transcription factor, was common to all data sets [58]. One explanation for the lack of commonalities between these studies may be the inherent limitations in methods to isolate pure HSC populations in the absence of contaminating cells, as well as the different methods used to isolate stem cell populations across these studies. In this study, we chose to examine the transcriptional profile of a homogeneous population of primary human endothelial cells that support the ex vivo expansion of human HSCs [40, 41]. Molecular analyses of effector cells that support the maintenance or expansion of HSCs have been much less frequently reported [54], 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.

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-β-induced protein as one of the most highly overexpressed HUBEC gene products is noteworthy in light of the established function of transforming growth factor-β in inhibiting HSC cycling and proliferation [59]. Many of the overexpressed transcripts include families of genes, such as IGFBPs, which regulate mesodermal cell fate decisions. IGFBP2 has been shown to inhibit embryonic fibroblast proliferation and can induce growth arrest of type II alveolar epithelial stem cells [60, 61]. IGFBP3 inhibits the proliferation of mesenchymal progenitors and fibroblasts in an IGF-1-independent manner [62]. IGFBP3 levels have also been positively associated with effective erythropoiesis in children, suggesting a potential physiologic role for this secreted protein in hematopoiesis [63]. Collagen family subtypes, specifically collagen type I α2 and collagen type IV α1, were also significantly overrepresented within HUBECs. Although the adhesion of HSCs to extracellular matrix molecules, such as fibronectin and collagen type I [64], has been associated with short-term maintenance of repopulating cells, the soluble hematopoietic activity of collagen moieties has not been demonstrated. Interestingly, adiponectin, which is a member of the family of soluble defense collagens, has recently been shown to inhibit colony-forming cell activity in suspension cultures [65], raising the possibility that collagen moieties produced by HUBECs may contribute to the soluble hematopoietic activity we have observed.

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 [66]. Follistatin is an inhibitor of follicle-stimulating hormone and activin [67] and causes lethality in knockout mice via failure of brain, lung, and soft tissue development at day 15.5 [68]. A potential role for the activin/follistatin pathway in hematopoiesis has been implied, but not confirmed, by studies indicating that activin exposure promoted red blood cell differentiation in mice [69]. Gremlin 1 homolog, like follistatin, also inhibits the activity of BMPs, specifically BMP2, BMP4, and BMP 7. Interestingly, a gremlin 1 null mutation in the mouse induces neonatal lethality secondary to failure of nephric and lung organ development, and exogenous gremlin 1 has been shown to have antiapoptotic effects on mesodermal cells in vitro [70]. The direct role for gremlin 1 and follistatin in hematopoiesis has yet to be demonstrated, but given that these molecules inhibit progenitor cell differentiation in other organ systems [71], 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 [72]. URB (steroid-sensitive gene 1) is a 150-kDa secreted protein and was recently characterized in the mouse to have a role in skeletogenesis [73]. In light of the recent demonstration of the osteoblastic niche for HSCs in the bone marrow, these data suggest the possibility that hormones elaborated by endothelial cells, possibly brain endothelial cells, may regulate osteoblast activity in the BM. A neuroendocrine-hematopoietic axis has been postulated previously [74], and the enrichment for osteoblast-regulatory factors within HUBECs further suggests this possibility. The recent demonstration of overlapping genetic programs between neural and hematopoietic stem cells [75] also suggests that brain-derived factors may have hematopoietic activity.

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 [24], 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 [34, 35]. In contrast to HUBECs, which support HSC expansion equally under contact or noncontact conditions [43, 44], AFT024 support of LTC-IC has been shown to decline under noncontact conditions [76]. 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.

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+CD38lin 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 [77], the hematopoietic activity of adrenomedullin has not been well characterized. However, human CD34+ cells express the calcitonin receptor-like receptor, the receptor for adrenomedullin [78], and stromal cells expressing adrenomedullin as well as other growth factors support human colony-forming cell growth in vitro [79]. 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.

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 [1, 2224]. The HUBEC molecular profile is a template for the identification of soluble factors that mediate hematopoietic stem cell fate.

Table Table 1.. Gene ontology categories of top 65 transcripts
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Table Table 2.. Genes most overrepresented and underrepresented within human brain endothelial cells
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Table Table 3.. Common transcripts between HUBEC and AFT024
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Table Table 4.. Quantitative real-time RT-PCR analysis of representative genes
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Figure 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.

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Figure 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 <.01.

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Figure 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 endothelial cell.

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Figure Figure 4.. Adrenomedullin supports an increase in CD34+ progenitor cell expansion in short-term culture. Primary human cord blood CD34+ cells (2.5 × 104) were placed in culture with thrombopoietin, stem cell factor, and Flt-3 ligand with and without 50–100 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.

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Figure Figure 5.. Hematopoietic activity of human brain endothelial cell (HUBEC)-secreted factors on primitive human CB CD34+CD38lin progenitors. FACS-sorted human CB CD34+CD38lin 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 50–100 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+CD38lin 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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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

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.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
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