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TISSUE-SPECIFIC STEM CELLS

Isolation and Angiogenesis by Endothelial Progenitors in the Fetal Liver

^a Department of Molecular and Experimental Medicine;
^b Department of Cell Biology, The Scripps Research Institute, La Jolla, California, USA

Key Words. Endothelial progenitor cells • Angiogenesis • Murine fetal liver • CD31⁺Sca1⁺ cells

Correspondence: Daniel R. Salomon, M.D., Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. Telephone: 858-784-9381; Fax: 858-784-2121; e-mail: dsalomon{at}scripps.edu

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

 
Endothelial progenitor cells (EPCs) have significant therapeutic potential. However, the low quantity of such cells available from bone marrow and their limited capacity to proliferate in culture make their use difficult. Here, we present the first definitive demonstration of the presence of true EPCs in murine fetal liver capable of forming blood vessels in vivo connected to the host’s vasculature after transplantation. This population is particularly interesting because it can be obtained at high yield and has a high angiogenic capacity as compared with bone marrow–derived EPCs. The EPC capacity is contained within the CD31⁺Sca1⁺ cell subset. We demonstrate that these cells are dependent for survival and proliferation on a feeder cell monolayer derived from the fetal liver. In addition, we describe a novel and easy method for the isolation and ex vivo proliferation of these EPCs. Finally, we used gene expression profiling and tandem mass spectrometry proteomics to examine the fetal liver endothelial progenitors and the feeder cells to identify possible proangiogenic growth factor and endothelial differentiation-associated genes.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

 
Endothelial progenitor cells (EPCs) have the capacity to proliferate and differentiate into mature endothelial cells. An unexpected and exciting development was the discovery that endothelial progenitors, resident in peripheral blood [1–3] and bone marrow [4–7], can be recruited from the circulation and participate in angiogenesis at sites of tissue injury and/or ischemia [8, 9]. Vascular progenitors can also be isolated from skeletal muscle and can differentiate into endothelial cells during ischemic injury-induced neovascularization [10]. Similarly, embryonic lung mesenchyme contains endothelial precursors [11].

Many have suggested the therapeutic potential of progenitor-driven angiogenesis [9]. However, the differentiation of EPCs into functional blood vessels in vivo is complex and incompletely understood. For example, what is the biological significance and regulation of angiogenesis mediated by EPCs at sites of tissue injury in contrast to angiogenesis mediated by local endothelial cells? Moreover, the quantity of EPCs within the circulation, even mobilized from the bone marrow, is low, and only a small percentage of CD34⁺ hematopoietic stem cells have endothelial progenitor capacity [12, 13]. Therefore, understanding growth factor and cell signal pathways that direct EPC proliferation, survival, and differentiation is an important strategy to enrich for angiogenesis-competent cells suitable for clinical applications.

Fetal liver is a source of stem cells that can give rise to hepatocytes and biliary epithelial cells [14, 15]. It is also well established that fetal liver contains Sca1⁺ hematopoietic stem cells capable of differentiation to myeloid and lymphoid lineages [16–18]. A subset of fetal liver stem cells has been shown to express EPC markers in vitro [2, 9, 12], suggesting that they might be EPCs. However, this commonly accepted fact has never been proven by demonstration of angiogenesis in vivo mediated directly by participation of these putative EPCs. Our interests in the potential of manipulating tissue compartment-specific progenitors as a means of enhancing revascularization of cell transplants during tissue engineering led us to develop a new method to purify stem cells from murine fetal liver. We characterized a CD31⁺Sca1⁺ population of cells that contains the EPC. While it is certainly possible that some hematopoietic stem cell activity is also contained within this population, the present work is focused on their endothelial progenitor and angiogenic potential. Thus, we established that the CD31⁺Sca1⁺ cells have a high efficiency of angiogenesis in vivo. We then used biology, genomics, and proteomics to better characterize these EPCs and possible growth factors and receptors required for survival, proliferation, and maturation.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

 
Animals
Balb/c and C57BL/6 mice were obtained from The Scripps Research Institute’s animal facility. Non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice were obtained from our own colony. Transgenic Tie2-GFP (Green Fluorescent Protein) mice (strain FVB/N-TgN(Tie2GFP)287Sato) were purchased from Jackson Laboratory (Bar Harbor, ME, http://www.jax.org). Protocols were approved by the Institutional Animal Care and Use Committee. This program is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care and conforms to all guidelines of the U.S. Department of Agriculture and the Office for Protection from Research Risks.

Liver Endothelial Progenitor Isolation
Livers obtained from five to seven fetal embryos (15–21 days postimplantation) were dissected, minced, and washed. After spinning 30 seconds at 99g, the tissue was digested with 3 mg/ml collagenase P (Sigma, St. Louis, http://www.sigmaaldrich.com) in 4 ml of Hanks’ balanced salt solution (HBSS) with 1 M CaCl₂ and hand-agitated at 37°C for 3.15 minutes. Digestion was stopped by adding 20 ml of HBSS with bovine serum albumin (BSA) 0.35% and cooling in ice 10 minutes. After discarding 20 ml of supernatant and adding 5 ml of HBSS+BSA, the pieces were triturated six times using a 14-gauge needle attached to a 10-ml syringe. The mixture was then pelleted by centrifugation (99g for 30 seconds), 5 ml of buffer was replaced, and this entire procedure was repeated twice. The digested tissue pellet was then resuspended in 30 ml of complete RPMI-1640 medium (Cambrex, East Rutherford, NJ, http://www.cambrex.com), 10% fetal calf serum, 4 mM glutamine, 1 mM sodium pyruvate, and 100 U penicillin/streptomycin (In-vitrogen Corporation, Carlsbad, CA, http://www.invitrogen.com), split in a six-well plate with 5 ml per well, and cultured at 37°C and 7% CO₂. The media was changed every 2 days. We performed all our studies with cells harvested at 8 days.

Bone Marrow Stem Cell Isolation
Bone marrow cells were extracted by flushing from the tibias and femurs of C57Bl/6 mice at 6–10 weeks of age.

Sorting of Sca1⁺ Stem Cells
Sorting of both fetal liver and adult bone marrow cells was performed using anti-Sca1 antibody conjugated to mini-magnetic beads (Miltenyi Biotec, Inc., Auburn, CA, http://www.miltenyibiotec.com) according to the manufacturer’s instructions. Sca1⁺ cells were eluted with a purity of better than 90% by flow cytometry and greater than 99% viability by vital dye exclusion.

Proliferation and Survival Assay
A hundred thousand cells per well were seeded in triplicate in 96-well plates and cultured for 3 days in medium or supplemented with 10%, 20%, and 40% of 48 hours conditioned supernatants harvested from a feeder cell monolayer. The cells were harvested with trypsin-EDTA and counted. Apoptosis and cell viability were measured using annexin V and propidium iodide according to the manufacturer’s protocols (Molecular Probes, Inc., Eugene, OR, http://probes.invitrogen.com). Proliferation was measured with [³H]thymidine in round-bottom 96-well plates added at 48 hours (1 µCi/ml), and 16 hours later the cells were harvested onto glass fiber filters and counted by liquid scintillation (Wallac MicroBeta, PerkinElmer Life and Analytical Sciences, Inc., Boston, http://las.perkinelmer.com).

Matrigel Vascular-Like Tube-Forming Assay
Matrigel (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com) was added to the wells of a 24-well plate in a volume of 300 µl and allowed to solidify at 37°C for 30 minutes. After the Matrigel solidified, 1 x 10⁶ liver endothelial progenitor (LEP) cells were added in 1 ml of media: Endothelial Cell Basal Medium (EBM)-2 supplemented with FCS 2%, hydrocortisone, human Fibroblast Growth Factor basic (hFGFb), vascular endothelial growth factor (VEGF), insulin-like growth factor–1 (IGF1), human Epidermal Growth Factor (hEGF), ascorbic acid, and heparin Endothelial Cell Medium (EGM-2 Bulletkit; Cambrex). The cells were incubated at 37°C, 7% CO₂ for 7 days and then photographed.

Endothelial Progenitor Colony-Forming Unit Assay
Five million LEPs per well were resuspended in 2 ml of Endocult Liquid Medium (StemCell Technologies, Vancouver, British Columbia, Canada, http://www.stemcell.com), plated on fibronectin-coated six-well culture dishes (BD Biosciences), and incubated for 2 days at 37°C, 7% CO₂. The nonadherent cells were then collected and plated at 5 x 10⁵ cells per well on fibronectin-coated 24-well culture dishes in 1 ml of Endocult Liquid Medium. After 3 days, the colony-forming units (CFU) were counted and photographed.

Transplantation of Matrigel Templates
One million cells were mixed in 500 µl of iced Matrigel Basement Membrane Matrix to prevent gelification and were injected subcutaneously into the flank of mice, using a 23-gauge needle.

Estimation of Blood Vessels
Mouse tissues or Matrigel templates explanted 2 weeks after transplant, were minced and digested in collagenase P (1.6 mg/ml; Sigma) and DNaseI (10 U/ml; Roche Molecular Biochemicals, Indianapolis, http://www.rochemb.com) for 2 hours at 37°C and resuspended by pipetting every 30 minutes. After filtering through a 70 µm filter (BD Biosciences), cells were collected and stained with blood vessel–specific antibody: phycoerythrin (PE) anti-CD31 antibody, a PE-conjugated rat IgG2a was used as the isotype control (Caltag Laboratories, Burlingame, CA, http://www.caltag.com). A PE-labeled anti-IIbß3 antibody (EMFRET Analytics, Würzbug, Germany, http://www.emfret-analytics.com) and the corresponding PE-Rat IgG2b isotype control were used to determine the quantity of platelets in the CD31⁺ subset. PE-streptavidin was used to reveal binding of biotinylated Griffonia simplicifolia lectin I isolectin B4 (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com). The lectin (25 µg) was injected in the tail vein of NOD/SCID mice 20 minutes before harvesting the transplanted Matrigel templates.

Antibody Phenotyping of LEPs
Anti-mouse F_cR CD16/CD32 (BD Biosciences) at 1 µg per 10⁶ cells incubated 10 minutes on ice was used as a blocking step. The following directly conjugated anti-mouse antibodies were used at 1 µg per 10⁶ cells and incubated 1 hour on ice: fluoroscein isothiocyanate (FITC)–anti-Sca1 (D7 Ly-6A/E; eBioscience, San Diego, http://www.ebioscience.com), FITC-anti-CD45R/B220 (BD Biosciences), and the isotype control, FITC-Rat IgG2a. Similarly, we used PE–anti-CD31 (Caltag Laboratories) with PE-Rat IgG2a as isotype control and FITC–anti-F4/80 (Caltag Laboratories) with FITC-Rat IgG2b as isotype control.

Histology
Explanted Matrigel templates were fixed in 4% paraformaldehyde 4 hours at room temperature and incubated overnight in 10% sucrose at 4°C. Specimens were frozen in Tissue-Tek OCT (optimal cutting temperature) embedding medium (Electron Microscopy Sciences, Hatfield, PA, http://www.emsdiasum.com) at –80°C, and 8 µm–thick frozen sections were made. After blocking with 1% BSA, 10% donkey serum in PBS for 1 hour at room temperature, sections were stained with rabbit anti-human von Willebrand Factor (vWF) (DakoCytomation, Carpinteria, CA, http://www.dakocytomation.us) at 1:200 dilution for 1 hour at room temperature, followed by donkey anti-rabbit IgG conjugated with Cy5 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, http://www.jacksonimmuno.com) at 1:200 dilution for 1 hour at room temperature. Cy5-streptavidin (1:100 dilution) identified biotinylated G. simplicifolia isolectin. Sections were visualized using an MRC1024 laser scanning confocal microscope (Bio-Rad Laboratories, Hercules, CA, http://www.bio-rad.com).

Reverse Transcription–Polymerase Chain Reaction
Total RNA was prepared in 1 ml of Trizol (Invitrogen Corporation), purified using RNeasy columns (Qiagen, Inc., Valencia, CA, http://www.qiagen.com) and quality confirmed on an Agilent 2100 BioAnalyzer (Agilent Technologies, Inc., Palo Alto, CA, http://www.agilent.com). Five micrograms of total RNA, treated with DNase (DNA-free; Ambion, Inc., Austin, TX, http://www.ambion.com), was reverse-transcribed using the SuperScript First-Strand Synthesis System with oligo dT primers (Invitrogen Corporation). First-strand DNA was treated with DNase-free RNase (Invitrogen Corporation). Polymerase chain reaction (PCR) was performed as follows: 94°C, 30 seconds; 55–60°C, 1 minute; and 72°C, 1 minute for 40 cycles. Primer sequences for the following genes are published: Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2), Tie-1, Tie-2, Flk-1 and Flt-1 [5], Flt-4 [19], Cd34 [20], Endoglin [21], c-kit [22], Aa4 [23], and Vcam-1 [24]. We also designed primers for the following: Hhex, 5'-ATCTCAGAGGATTCCGACCAGG-3' forward, 5'-ATTCCCCAATGTTGCCCCCAC-3' reverse (513 bp); Cd133, 5'-GGAAAAGTTGCTCTGCGAACC-3' forward, 5'-TGCTTGTTTGCTGGAGGGTC-3' reverse (608 bp); Tal1, 5'-GCCCAAAGATTTCCCCAATG-3' forward, 5'-AAACCCAGTGCCCCAAACAC-3' reverse (543 bp); VE-Cadherin 5'-CAGCCAGCATCTTGAACCTG-3' forward, 5'-GAGATTCACGAGCAGTTGGT-3' reverse (506 bp) and vWF 5'-TGTTTTGTGGCGTGTATGTGAGG-3' forward, 5'-GTGTTCTGGGTTTTCTGGAGTTTG-3' reverse (584 bp).

DNA Microarrays
Affymetrix GeneChip (Santa Clara, CA, http://www.affymetrix.com) protocols were used for all hybridizations. Samples were hybridized to MOE430A GeneChip arrays. Data were analyzed using GeneChip Operating Software (GCOS) Version 1.0 (Affymetrix), which computes signal intensity and p values for each probe set (Wilcoxon Rank Sum test) and generates Present/Absent calls. We used RMA Express for signal normalization [25], BRB ArrayTools for class comparisons (http://linus.nci.nih.gov/BRB-ArrayTools.html), and Cluster/TreeView for creation of heat map displays [26]. Microarray data for all the GeneChips are available at the Gene Expression Omnibus (GEO) Website (www.ncbi.nlm.nih.gov/geo) under the series ID GSE1727 [NCBI GEO] .

Protein Extraction
Proteins were extracted with isopropyl alcohol from phenol-ethanol supernatants of Trizol extracts after RNA was removed. Samples were allowed to precipitate for 10 minutes (25°C) and sedimented at 12,000g (10 minutes, 4°C). The protein pellet was washed three times in two volumes of 0.3 M guanidine hydrochloride in 95% ethanol for 20 minutes at 25°C and then centrifuged at 7,500g (5 minutes, 4°C). The pellet was then vortexed in 2 ml of 80% ethanol and centrifuged at 7,500g (5 minutes, 4°C). The protein pellet was dried at room temperature for 10 minutes and stored at –20°C.

Multidimensional Protein Identification Technology
We used multidimensional protein identification technology (MudPIT) for this analysis as described previously [27]. Protein samples were analyzed using two different techniques for cleavage. One replicate from each sample was enzymatically cleaved using Endoproteinase Lys-C (Roche Biochemicals) followed by digestion with sequencing grade modified Trypsin (Promega Corporation, Madison, WI, http://www.promega.com). The other replicate was chemically cleaved with Cyanogen Bromide (CnBr) in addition to enzymatic cleavage with Trypsin and Endoproteinase Lys-C.

Analysis of Tandem Mass Spectra
MS/MS spectra were analyzed using the SEQUEST software analysis protocol as described [28]. A filter, called 2to3 [29], determined the charge state (+2 or +3) of multiple peptide spectra, and poor-quality spectra were deleted. Each MS/MS spectrum after analysis and filtering was searched against the SwissProt database (Release 42.0) and EntrezProtein (July 24, 2004) using SEQUEST [28]. DTASelect was used to filter peptide identifications. Filter criteria were set to cross-correlation (Xcorr) values >2.2 for 1+ spectra, >2.5 for 2+ spectra, and >3.5 for 3+ spectra with Cn of 0.1. For the proteins hits of mRNA microarray data, moderate stringency was applied with Xcorrs of >0.8 and Cn of 0.01 followed by manual validation of each peptide spectrum based on two main criteria: (a) more than three of the most intense fragment ions must show a match, and (b) the b and y ion series must show continuity for at least three fragment ions above background noise.

Statistical Analysis
Data are expressed as mean ± SE of at least three independent experiments. ANOVA (analysis of variance) was used to detect differences in cells survival and proliferation. A p value of <.05 was considered statistically significant.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

 
Phenotypic Characterization of the Isolated LEPs
We developed a novel way to easily isolate a population of cells containing EPCs from mouse fetal livers in large numbers as detailed in Materials and Methods. Between 6 and 12 days after isolation, weakly adherent and highly light-refractile round cells grow out in clusters on a firmly adherent cell monolayer (Fig. 1A). We call these round cells LEPs. We can obtain these cells in large quantity, 5.5 ± 0.5 x 10⁶ cells per five to seven murine fetal embryos harvested 15–18 days postimplantation. Flow cytometric analysis of LEPs after 8 days in the cultures demonstrated populations of cells expressing the progenitor-associated markers, CD31 (46.3% ± 1.6%) and Sca1 (Ly6a, 26.0% ± 7.0%). On the other hand, LEPs were essentially hematopoietic lineage–negative. Flow cytometry analysis demonstrated that the cells are not macrophages (F4/80, 1.8% ± 0.6% vs. isotype control, 1.2% ± 0.2%). Staining for CD45R/B220 marking mature T, B, and natural killer cells as well as lymphocyte and macrophage progenitors in fetal liver was 3.8% ± 0.3% versus the isotype control of 1.2% ± 0.2%. LEPs are found only in fetal, not in adult, liver preparations.

View larger version (44K): [in this window] [in a new window]   Figure 1. Growth of liver endothelial progenitor (LEP) cells on feeder cell monolayers. (A): Light microscopy picture of LEP growing out of the collagenase digestions of fetal mouse liver. The LEPs are weakly or nonadherent and highly refractile round cells that grow out in clusters (arrow) on a firmly adherent cell monolayer. (B): To determine the effect of feeder cell–derived growth factors on LEP proliferation and survival, we harvested the LEPs from the feeder cell monolayer and cultured them in 0%, 10%, 20%, and 40% concentrations of conditioned feeder cell supernatants for 72 hours. The cells were then counted (upper panel) or stained with annexin V and propidium iodide (lower panel) to determine the quantity of dead cells.

LEPs Can Form Blood Vessels In Vitro
A well-established assay for the angiogenic potential of endothelial progenitors is the development of vascular-like tubes in a Matrigel in vitro culture [30]. LEPs isolated from the feeder cell monolayers after 8 days were plated in Matrigel and cultured for 5 days in an endothelial cell growth media. Figure 2 is a photomicrograph of a representative culture showing the characteristic vascular-like tubes developing from the LEPs.

View larger version (91K): [in this window] [in a new window]   Figure 2. Matrigel tube-formation assay. Light microscopy picture of liver endothelial progenitor forming tube-like structures (arrow) in Matrigel after 7 days in vitro. Magnification x200.

Next, we tested the use of Matrigel templates as a platform technology to verify the endothelial capacity of LEPs isolated from transgenic Tie2-GFP mice. LEP cells were seeded in Matrigel (10⁶ LEPs per 500 µl) and injected subcutaneously into NOD/SCID mice. After 2 weeks, the Matrigel templates were explanted and analyzed by histology and the collagenase-based flow cytometry assay described above. By flow cytometry, GFP⁺ cells (i.e., Tie2⁺) are observed in templates containing LEPs (Fig. 3A), and these cells are also CD31⁺ representing the mature endothelial cells derived from the LEPs. CD31⁺ endothelial cells comprise 35.5% ± 1.6% (corresponding to approximately 1.3 x 10⁶ cells) of all the cells in the explanted Matrigel templates. In turn, the GFP⁺CD31⁺ cells derived from the LEPs represent 9.3% ± 0.4% (corresponding to approximately 3.4 x 10⁵ cells) of the total cells in the template or 26% of the endothelial cells. Note that the presence of host-derived endothelial (GFP^–CD31⁺) cells in both the LEP and control templates is consistent with the fact that Matrigel is a proangiogenic material.

View larger version (38K): [in this window] [in a new window]   Figure 3. Demonstration that LEPs differentiate into endothelial cells in vivo. (A): Analysis of LEPs isolated from Tie2-GFP mice collected from Matrigel templates 2 weeks after transplant. The two left panels marked ratIgG2a represent the isotype controls for the anti-CD31 staining shown in the two right panels. GFP+ endothelial cells (R1) are present in the Matrigel with LEPs (lower) but not in the Matrigel control (upper). The two GFP/CD31 plots show that all the GFP+ (Tie2+) cells in the Matrigel+LEP are also CD31+ (R2). Based on R2, the number of endothelial cells generated from LEPs in the Matrigel is 8.8%. (B–G): Demonstration that LEPs differentiate into endothelial cells in vivo by confocal microscopy. LEPs were transplanted in Matrigel templates and harvested 2 weeks after transplantation. GFP+ marking of the LEPs is seen in the green channel (B), and anti-vWF staining of blood vessels is shown in blue (C). The merged image shows colocalization of GFP+ LEP cells with anti-vWF+ blood vessels (D). To demonstrate that LEP-formed vessels are connected to the host vasculature, we harvested the Matrigel templates after injection of the biotinylated, endothelium-specific isolectin B4. GFP+ LEPs are seen in the green channel (E), and isolectin staining is shown in blue (F). The merged image (G) shows the colocalization of GFP+ LEPs and isolectin staining, proving that LEP-derived vessels are connected to the blood stream of the host. Scale bars = 21 microns. Abbreviations: GFP, Green Fluorescent Protein; LEP, liver endothelial progenitor; PE, phycoerythrin; vWF, von Willebrand Factor.

That LEPs form blood vessels in vivo in the Matrigel after transplant was confirmed by frozen sections of explanted templates stained with the blood vessel–specific anti-vWF antibody (Figs. 3B–3D). The key point is a colocalization between vWF staining and the LEP-derived GFP⁺ cells lining the vascular lumens, which have differentiated into a mature endothelial cell phenotype (Fig. 3D).

To test whether these new vessels were connected to the host vasculature, we injected the biotinylated endothelium-specific isolectin B4 in the tail vein of NOD/SCID mice 2 weeks after the transplantation of LEP-containing Matrigel templates. The explanted templates were stained with PE-streptavidin and we showed by flow cytometry that GFP⁺ cells (Tie2⁺) are also isolectin B4⁺ (data not shown). We obtained the same result by confocal microscopy by staining with streptavidin-Cy5 to identify the biotinylated lectin bound to endothelial cells. Colocalization between the GFP⁺ cells and the isolectin B4⁺ cells is clearly seen in Figure 2G. In conclusion, the LEPs are EPCs, that mature into endothelium and incorporate into blood vessels connected with the host vasculature after transplantation.

Finally, for the sake of a functional comparison, the same experimental design was performed with bone marrow– derived Sca1⁺ cells from adult Tie2-GFP mice. Flow cytometric analysis of the explanted Matrigel templates demonstrated that only 1.2% ± 0.03% of the total cells were GFP⁺/CD31⁺ endothelial cells. These results demonstrate the high angiogenic efficiency of the fetal liver– derived LEPs as compared with the adult bone marrow– derived Sca1⁺ cells to form blood vessels in vivo.

LEP Sca1⁺ Cells Contain the Endothelial Progenitor Cells
Fetal livers were harvested, collagenase digested, and the resulting cell mixtures were placed in culture. After 8 days, the LEPs were collected from the underlying feeder layer and magnetically sorted using anti-Sca1 antibody. The presence of EPCs was tested in LEP, LEP Sca1⁺, and LEP Sca1^– populations using the endothelial progenitor colony-forming unit (EP-CFU) assay. We counted the number of colonies characterized by a central cluster of rounded cells surrounded by radiating, thin, flat cells (Fig. 4): LEP 23.5 ± 0.5, LEP Sca1⁺ 56.5 ± 5.5, and LEP Sca1^– 3.5 x 0.5. The quantity of CFU is significantly enriched in the Sca1^– subset (p = .01) and lower in Sca1^– (p = .001).

View larger version (52K): [in this window] [in a new window]   Figure 4. Endothelial progenitor cell (EPC) colony-forming assay comparing liver endothelial progenitor (LEP), LEP Sca1+, and LEP Sca1–. Phase-contrast micrographs of EPC colonies characterized by a central cluster of round cells surrounded by radiating flat cells. Many colonies were observed in the wells seeded with LEP Sca1+ (56.5 ± 5.5) (B), fewer were observed in the unseparated LEPs (23.5 ± 0.5) (A), and almost none in LEP Sca1– (3.5 ± 0.5) (C). Magnification x200.

Gene Expression Profiling of LEP Sca1⁺ and Sca1^– Cells
To investigate potential factors that drive EPC differentiation, we compared the genes expressed by these two populations. We first performed reverse transcription (RT)–PCR on LEP Sca1⁺ and Sca1^– cells, testing 17 genes associated in the literature with hematopoietic and endothelial progenitors (Fig. 5). The genes uniquely expressed by LEP Sca1⁺ are the VEGF receptors, Kdr, Flt1, and Flt4, and also Cd34, Vcam-1, Cd133, and VE-cadherin. In contrast, vWF, Endoglin, AA4, Hhex, and Tal1 are expressed by both Sca1⁺ and Sca1^– populations. All the primers were verified using the feeder cell monolayer, a negative control of NIH 3T3 fibroblasts, and a positive control of a mouse endothelial cell line, MS1 [34] (data not shown).

View larger version (61K): [in this window] [in a new window]   Figure 5. Comparisons of liver endothelial progenitor (LEP) Sca1+ and LEP Sca1–. Reverse transcription–polymerase chain reaction analysis of hematopoietic and vascular-specific gene expression.

View larger version (39K): [in this window] [in a new window]   Figure 6. Heat map for differential gene expression of 62 angiogenesis-associated genes comparing liver endothelial progenitor (LEP) Sca1+ and LEP Sca1–. We identified 445 differentially expressed genes: 228 genes were upregulated in the Sca1+ subset, and 217 genes were upregulated in the Sca1– subset. We filtered this set by all genes associated with angiogenesis in the literature and public databases. These 62 genes were then clustered; relative expression levels are displayed in the heat map for the LEP Sca1+ versus Sca1– subsets as a color gradation from high (magenta) to low (aqua). The names of the antiangiogenic genes are highlighted in red to emphasize that three of the six differentially expressed angiogenesis-associated genes in the Sca1– subset (bottom of figure) are antiangiogenic.

We also tested the expression of the candidate genes described above using tandem mass spectrometry proteomics with the Multidimensional Protein Identification Tool (MudPIT) [35]. Thus, we matched the protein candidates identified in LEPs (1,817 proteins) and feeder cells (1,602 proteins) to the nine growth factor/receptor pathways identified using gene expression profiling (Table S2). In total, 75 candidate proteins out of the 124 (60%) in the original set of gene expression–defined pathways were confirmed by MudPIT proteomics.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

 
Stem cells have the potential of being tools for tissue engineering and regenerative medicine. A major limitation at the present time is that endothelial progenitor frequency in bone marrow and mobilized peripheral blood cells is extremely low and the efficiency of angiogenesis is poor. Thus, a major challenge is developing strategies to engineer the differentiation and proliferative expansion of EPCs in culture. In the present paper, we describe a model of EPCs that is particularly suitable for experiments designed to investigate molecular mechanisms of EPC survival, proliferation, and differentiation.

Specifically, we demonstrate that a population of stem cells we have called LEPs are found in the murine fetal liver and can be readily isolated. We prove that CD31⁺Sca1⁺ LEPs are endothelial progenitors that form new vessels at high efficiency (approximately 26% of the total endothelial cells in a transplanted Matrigel template) and are connected to the blood stream of the host after transplantation. These data represent the first definitive demonstration that true EPCs are indeed present in fetal liver. Another cell population derived from the fetal liver, called LEP feeders, provides a set of growth factors that drive LEP proliferation and survival in culture, while maintaining their angiogenic potential.

Though EPCs have been identified in bone marrow and cord and peripheral blood, they represent only a small fraction of the cells in these compartments, typically less than 2% [12, 13, 36]. In contrast, we demonstrate that collagenase digestion of the murine fetal liver is a relatively easy method that yields a large number of readily collected LEPs (typically 5.5 ± 0.5 x 10⁶ cells per pregnant female), and the Sca1⁺ subset comprising the EPC activity represents 26% of these cells. We transplanted Sca1⁺ cells obtained from both fetal livers and adult bone marrow to assess their relative angiogenic efficiency. This comparison demonstrated that 9.3% of the mature endothelial cells were derived from the Sca1⁺ LEPs as compared with only 1.2% from the Sca1⁺ adult bone marrow cells, supporting our conclusion that the LEPs are highly efficient in angiogenesis.

Based on our demonstrations that the Sca1⁺ LEPs are functional EPCs in vitro and in vivo, we next evaluated the differences between Sca1⁺ and Sca1^– LEPs in a number of known proangiogenic molecules by gene expression profiling using RT-PCR and high density DNA microarrays. The key point is that defining the endothelial progenitor potential of a new cell population must start with an array of well-established angiogenesis assays rather than infer such activity a priori by measuring a panel of putative cell surface markers for EPCs, many of which are contradictory, mark overlapping cell populations of various lineages, and may also stain mature endothelial cells, and none of which is presently proven to be endothelial progenitor–specific as single markers.

We tested the endothelial progenitor lineage of the LEP Sca1⁺ by RT-PCR, demonstrating that only the Sca1⁺ express the VEGF receptor genes, Kdr, Flt1, and Flt4, as well as Cd133, VE-cadherin, Cd34, and Vcam-1. That VEGF receptors are expressed by the Sca1⁺ cells is important because VEGF is a key regulator of angiogenesis [37] and Kdr, Flt1, and Flt4 are involved in vascular development [36, 38, 39]. VE-cadherin and CD133 are cell surface markers generally agreed upon to be expressed by endothelial progenitors.

We extended our analysis of differential gene expression by using high-density DNA microarrays. A large number of genes (228) are differentially upregulated in the Sca1⁺ cells, and 56 are linked functionally to angiogenesis, including only two that are antiangiogenic (Fig. 6). Among the 54 proangiogenic genes upregulated, we note the well-known endothelial progenitor–associated genes, Kdr and VE-cadherin (cdh5), consistent with our RT-PCR data. Indeed, Sca1⁺Kdr⁺VE-cadherin⁺ cells have been shown to define a bone marrow–derived mouse endothelial progenitor [9]. A number of the upregulated, angiogenesis-associated genes in the Sca1⁺ LEPs represent candidates for further studies. Pleiotrophin and Midkine represent a family of mitogenic and angiogenic growth and differentiation factors expressed in cells in early differentiation [40, 41]. The secreted frizzled related sequence protein–1 (sFRP-1) is strongly expressed during early phases of the vascularization process in embryonic vasculature and modulates vascular cell proliferation [42]. It is interesting that the Frizzled homologue 2 (Fzd2) is also upregulated in Sca1⁺ LEPs, but its precise physiologic function has not been determined yet, and so this was not counted in our list of angiogenesis-associated gene candidates [43]. Tweak receptor (Tnfrsf12a) induces angiogenesis in vivo [44]. Laminin alpha4 (lama4) and Nidogen (Nid1) are both upregulated in Sca1⁺ LEPs and form a stable complex that plays an important modulatory role in angiogenesis [45]. On the other hand, only six genes are upregulated and angiogenesis-associated in the Sca1^– LEPs, including three that are antiangiogenic (Fig. 6).

In conclusion, the genomic profiling of LEP Sca1⁺ versus Sca1^– cells distinguished these two populations by differential gene expression. Specifically, LEP Sca1⁺ cells are characterized by upregulation of primarily angiogenesis-associated and endothelial lineage–specific genes, while the LEP Sca1^– cells reveal a majority of hematopoietic lineage–specific genes. This separation by gene expression profiling supports the biological results we obtained showing that the Sca1⁺ comprise the endothelial progenitor activity of the LEPs.

Taking advantage of the evidence that LEPs need the feeder cells to survive and proliferate, we investigated the key question: what are the growth factors required to maintain EPCs in culture? Commonly, monoclonal antibodies or supplementation of cultures with recombinant growth factors is used to identify expression of specific growth factor receptors. In the present study, we used a combined strategy of gene transcript, protein expression profiling, and bioinformatics to identify candidate pathways. Therefore, we organized the gene expression profiling by selecting pathways in which the feeder monolayer cells expressed the growth factor and the LEPs expressed the receptor. This integrated analysis with bioinformatics allowed us to identify nine different growth factor pathways. All these growth factors are well known and can be involved in cell proliferation and survival. The LEP feeder cells may also have a role in EPC differentiation. Indeed, among the nine growth factors, five have at least one of their receptors expressed only on the Sca1⁺: Acvr1, Egfr, Fgf1, Tgfbr2, and Vegfr-1, -2, and -3. Beside VEGF receptors that are well known for enhancing angiogenesis, the function of the others in endothelial differentiation is less well known and they represent promising candidates for further studies of endothelial differentiation.

	CONCLUSION

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

 
We have proven that Sca1⁺ endothelial progenitors are found in the fetal liver and that these form blood vessels integrated with the host’s vasculature when transplanted in Matrigel templates. We have developed an isolation strategy that is easy and yields a significant number of progenitors for study that proliferate in culture and also have a high efficiency of angiogenesis in vivo. Thus, we suggest that this murine system has promise as a means for advancing studies of endothelial progenitor differentiation, angiogenesis, and transplantation. Finally, we have used gene expression profiling and tandem mass spectrometry proteomics as a first approach in identifying candidate genes potentially involved in EPC growth and survival and endothelial differentiation and angiogenesis.

	ACKNOWLEDGMENTS

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

 
We acknowledge the kind gift of the anti-phosphotyrosine–specific antibodies from Dr. Martin Schwartz (University of Virginia). This work was funded by the National Institutes of Health, R21 DK62598 (D.R.S.), the Juvenile Diabetes Research Foundation, 3-2003-738 (S.C.), and the Molly Baber Research Fund.

DISCLOSURES
The authors indicate no potential conflicts of interest.

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Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

 

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