Isolation and Angiogenesis by Endothelial Progenitors in the Fetal Liver - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:14 |只看该作者 |倒序浏览 |打印

作者：Stephanie Cherquia, Sunil M. Kuriana, Olivier Schusslera, Johannes A. Hewelb, John R. Yates, IIIb, Daniel R. Salomona作者单位：a Department of Molecular and Experimental Medicine;b Department of Cell Biology, The Scripps Research Institute, La Jolla, California, USA
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      【摘要】$ D5 c- ]0 G, x# j5 }( ]
   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¨Cderived 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. 7 |  T6 L; W0 n) f
      【关键词】 Endothelial progenitor cells Angiogenesis Murine fetal liver CD Sca  cells
   INTRODUCTION/ E% R" B- p1 d3 F, g9 Q2 Z' y

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 .9 y; \8 d. v( g
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Many have suggested the therapeutic potential of progenitor-driven angiogenesis . 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.: ]- ^9 [9 p2 Q2 Y7 v9 h1 w- _. A

Fetal liver is a source of stem cells that can give rise to hepatocytes and biliary epithelial cells , 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.
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MATERIALS AND METHODS+ `6 K; U6 f" t+ X% T$ P

Animals: C4 |2 S, {) F0 c( a+ M# h

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.
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Liver Endothelial Progenitor Isolation" n. x; V+ l: k' V& Y4 {- \

Livers obtained from five to seven fetal embryos (15¨C21 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¡ãC 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¡ãC 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¨C10 weeks of age.

Sorting of Sca1  Stem Cells
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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.
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Proliferation and Survival Assay. V; a% ]: a+ b- D9 g
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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 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$ f& V% j! `4 T* g

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¡ãC 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¨C1 (IGF1), human Epidermal Growth Factor (hEGF), ascorbic acid, and heparin Endothelial Cell Medium (EGM-2 Bulletkit; Cambrex). The cells were incubated at 37¡ãC, 7% CO2 for 7 days and then photographed.8 s8 ~' _& K/ Q9 R( h

Endothelial Progenitor Colony-Forming Unit Assay
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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¡ãC, 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 Templates1 q2 \$ Q5 ]0 U0 W8 ?

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.$ M  q3 [! x, c8 m% u

Estimation of Blood Vessels
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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¡ãC and resuspended by pipetting every 30 minutes. After filtering through a 70 µm filter (BD Biosciences), cells were collected and stained with blood vessel¨Cspecific 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, W¨¹rzbug, 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
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Anti-mouse FcR 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)¨Canti-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¨Canti-CD31 (Caltag Laboratories) with PE-Rat IgG2a as isotype control and FITC¨Canti-F4/80 (Caltag Laboratories) with FITC-Rat IgG2b as isotype control.

Histology7 z- M" J8 D/ d$ O3 S) E

Explanted Matrigel templates were fixed in 4% paraformaldehyde 4 hours at room temperature and incubated overnight in 10% sucrose at 4¡ãC. Specimens were frozen in Tissue-Tek OCT (optimal cutting temperature) embedding medium (Electron Microscopy Sciences, Hatfield, PA, http://www.emsdiasum.com) at ¨C80¡ãC, and 8 µm¨Cthick 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).. ^- Z. ?# g) t0 I0 j; @

Reverse Transcription¨CPolymerase 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¡ãC, 30 seconds; 55¨C60¡ãC, 1 minute; and 72¡ãC, 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 . 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).
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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  .
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Protein Extraction  N. s0 }8 H5 ~  _3 b0 ?

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¡ãC) and sedimented at 12,000g (10 minutes, 4¡ãC). The protein pellet was washed three times in two volumes of 0.3 M guanidine hydrochloride in 95% ethanol for 20 minutes at 25¡ãC and then centrifuged at 7,500g (5 minutes, 4¡ãC). The pellet was then vortexed in 2 ml of 80% ethanol and centrifuged at 7,500g (5 minutes, 4¡ãC). The protein pellet was dried at room temperature for 10 minutes and stored at ¨C20¡ãC.6 z4 x/ G. X, M; t3 {- X1 v( _. r7 |$ V+ d
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Multidimensional Protein Identification Technology, l7 ]' w: y2 }' B% B
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We used multidimensional protein identification technology (MudPIT) for this analysis as described previously . 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.0 N; N& q( G+ p5 Z
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Analysis of Tandem Mass Spectra
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MS/MS spectra were analyzed using the SEQUEST software analysis protocol as described . 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.7 n( Z$ M( ~; K- ?8 O

Statistical Analysis
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Data are expressed as mean ¡À 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

RESULTS4 c2 d0 N# v# [9 {8 V0 a4 V. I
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Phenotypic Characterization of the Isolated LEPs0 N0 R" T; p: k$ u4 E6 L
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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 ¡À 0.5 x 106 cells per five to seven murine fetal embryos harvested 15¨C18 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% ¡À 1.6%) and Sca1 (Ly6a, 26.0% ¡À 7.0%). On the other hand, LEPs were essentially hematopoietic lineage¨Cnegative. Flow cytometry analysis demonstrated that the cells are not macrophages (F4/80, 1.8% ¡À 0.6% vs. isotype control, 1.2% ¡À 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% ¡À 0.3% versus the isotype control of 1.2% ¡À 0.2%. LEPs are found only in fetal, not in adult, liver preparations.

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¨Cderived 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.
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Our working hypothesis was that the monolayer of cells that underlies the LEP clusters represents a feeder cell population. To test this hypothesis, supernatant from the feeder cells was added at various concentrations: 0%, 10%, 20%, and 40% by volume to harvested LEPs. After 72 hours, we collected the cells for counting and stained with annexin V and propidium iodide to determine the quantity of dead cells by flow cytometry. The number of LEPs increased significantly between 0% and 10% conditioned media (p = .04) and it was maximal at 20% (Fig. 1B, upper panel). Similarly, we observed a significant decrease in the quantity of dead cells between 0% and 10% of conditioned media (p = .004) after which this measure reached a plateau (Fig. 1B, lower panel). The same data were obtained with a proliferation assay using 3H-Thymidine (4,606 ¡À 285 cpm for 0% vs. 16,557 ¡À 525 cpm for 10%; p = .04). These data prove that factors produced by the underlying feeder cell monolayer are essential for the survival and proliferation of LEPs. One possibility is that the conditioned media might support LEP survival and proliferation but not the maintenance of their endothelial progenitor function. To test this hypothesis, we cultured LEPs in 20% conditioned media for 2 weeks after which we demonstrated they could still form blood vessels in vivo after transplantation into immunodeficient mice (data not shown) in the Matrigel template assay described below.

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 . 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.$ S% p; z) a9 @! {

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.
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LEPs Can Form Blood Vessels In Vivo After Transplantation
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We used an angiogenesis assay based on collagenase digestion and flow cytometry to measure the potential of LEPs to form new vessels in vivo after transplant. The underlying premise is that endothelial cells released by collagenase digestion of a tissue and stained with endothelial cell¨Cspecific antibodies can be counted by flow cytometry and expressed as the percentage of positive cells in the unlabeled background. Thus, the number of endothelial cells in a given tissue is a function of the number of vessels. To validate this assay, we harvested kidney, lung, heart, and brain from four transgenic Tie2-GFP mice, in which only endothelial cells express GFP . Cells harvested from the collagenase-digested tissues were then analyzed for expression of CD31, as an independent marker for mature endothelial cells, and GFP to detect the Tie2  cells. These results confirmed that all GFP  cells were also CD31  and represent mature endothelial cells (data not shown).1 ^% f1 n1 Y0 t  }0 N

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% ¡À 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% ¡À 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¨CCD31 ) cells in both the LEP and control templates is consistent with the fact that Matrigel is a proangiogenic material.
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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¨CG): 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.

One possible concern for use of anti-CD31 staining as a parameter for estimating tissue angiogenesis is that this molecule is also expressed on platelets. To exclude the possibility that marginated platelets and/or postmortem thrombi are contributing to the staining, we stained with an antibody reacting with murine integrin IIbß3 (Leo.D2), specifically expressed by platelets . The results were that staining with this antibody represents 2%¨C3% of the total CD31 staining observed, excluding platelets as a source of error.( U2 ?. z3 `  m1 k$ ^  |
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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¨Cspecific anti-vWF antibody (Figs. 3B¨C3D). 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).( n0 g% V# B0 m+ d

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.: ~; W- e3 R' P9 `
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Finally, for the sake of a functional comparison, the same experimental design was performed with bone marrow¨C derived Sca1  cells from adult Tie2-GFP mice. Flow cytometric analysis of the explanted Matrigel templates demonstrated that only 1.2% ¡À 0.03% of the total cells were GFP /CD31  endothelial cells. These results demonstrate the high angiogenic efficiency of the fetal liver¨C derived LEPs as compared with the adult bone marrow¨C derived Sca1  cells to form blood vessels in vivo.
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LEP Sca1  Cells Contain the Endothelial Progenitor Cells
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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¨C 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 ¡À 0.5, LEP Sca1  56.5 ¡À 5.5, and LEP Sca1¨C 3.5 x 0.5. The quantity of CFU is significantly enriched in the Sca1¨C subset (p = .01) and lower in Sca1¨C (p = .001).

Figure 4. Endothelial progenitor cell (EPC) colony-forming assay comparing liver endothelial progenitor (LEP), LEP Sca1 , and LEP Sca1¨C. 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 ¡À 5.5) (B), fewer were observed in the unseparated LEPs (23.5 ¡À 0.5) (A), and almost none in LEP Sca1¨C (3.5 ¡À 0.5) (C). Magnification x200.$ X) U; I8 E+ `8 g% ^# O

Sca1  and Sca1¨C LEPs from Tie2-GFP mice were then transplanted (106 cells per Matrigel template). Two weeks after transplant, we injected biotinylated isolectin B4 by tail vein and then explanted the templates, collagenase digested, and examined the cells by flow cytometry. The results were that GFP /Tie2  endothelial cells represent 0.9% ¡À 0.8% of the total cells analyzed in the empty template control, 1.6% ¡À 0.9% for Sca1¨C and 8% ¡À 2.4% for the Sca1 . Two-color analysis for PE-labeled isolectin staining revealed 0.6 ¡À 0.5% for the empty template control, 1.5% ¡À 0.4% for Sca1¨C, and 6.1% ¡À 2.1% for the Sca1 . Therefore, all these data prove that the LEP Sca1  cell subset contains the endothelial progenitors.

Gene Expression Profiling of LEP Sca1  and Sca1¨C Cells
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To investigate potential factors that drive EPC differentiation, we compared the genes expressed by these two populations. We first performed reverse transcription (RT)¨CPCR on LEP Sca1  and Sca1¨C 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¨C 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  (data not shown).2 U; S, N# Y, M; z; B

Figure 5. Comparisons of liver endothelial progenitor (LEP) Sca1  and LEP Sca1¨C. Reverse transcription¨Cpolymerase chain reaction analysis of hematopoietic and vascular-specific gene expression.6 x' S4 L) {3 P, [" S$ G: a+ j

Next, we performed gene expression profiling comparing LEP Sca1  and LEP Sca1¨C using Affymetrix MOE430A GeneChips (n = 2 Sca1  and n = 3 Sca1¨C). The signal intensities were normalized using RMA Express, and a class comparison for differentially expressed genes was performed with BRB ArrayTools with the p value filter set at .005. This analysis demonstrated 445 differentially expressed genes: 228 genes were upregulated in the Sca1  subset, and 217 genes were upregulated in the Sca1¨C subset. We then filtered the entire set of differentially expressed genes for functions related to angiogenesis and differentiation on the basis of the published literature as identified in Online Mendelian Inheritance in Man (OMIM), PubMed, and Mouse Genome Informatics (MGI) as well as functional classifications of known genes listed at Ne-tAffyx and Gene Ontology (GO) database. There are 54 proangiogenic genes expressed by Sca1  cells and only three by Sca1¨C (all different than those expressed by Sca1 ). Figure 6 depicts a heat map and cluster analysis of the proangiogenic and antiangiogenic genes differentially expressed by both LEP subsets. A full set of all differentially expressed genes is available as Supplemental Data (Table S1).$ |) J- C1 S* M* J( q' G8 o& ]% U  A
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Figure 6. Heat map for differential gene expression of 62 angiogenesis-associated genes comparing liver endothelial progenitor (LEP) Sca1  and LEP Sca1¨C. We identified 445 differentially expressed genes: 228 genes were upregulated in the Sca1  subset, and 217 genes were upregulated in the Sca1¨C 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¨C 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¨C subset (bottom of figure) are antiangiogenic.9 ]0 v* `; I  D" a5 n
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Candidate Growth Factor Pathways
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LEPs depend for survival and proliferation on the underlying feeder cells or their supernatants. We propose that this dependence creates a model system to identify growth factor and differentiation signal pathways for EPCs that may also be relevant to angiogenesis. Therefore, we performed gene expression profiling using Affymetrix MOE430A GeneChips to compare the LEPs to the feeder cell monolayer. To focus on potential proangiogenic growth factors secreted by the feeder cell monolayer, we selected candidates whose genes were expressed only by the feeder cells and where the genes for their receptors were expressed by the LEPs. This analysis identified nine growth factor/receptor pathways potentially involved in the survival and proliferation of LEPs: bone morphogenic protein¨C4 (BMP-4), epidermal growth factor (EGF), leukemia inhibitory factor (LIF), fibroblast growth factor¨C1 and ¨C2 (FGF1, FGF2), insulin-like growth factor¨C2 (IGF2), platelet-derived growth factor alpha (PDGF), transforming growth factor beta (TGFß), and VEGF. We then used the literature and Web-based tools (as already described) as well as Biocarta (www.biocarta.com) to create gene lists for each growth factor¨Cstimulated receptor signaling pathway potentially activated in the LEPs. The results of this analysis are provided as Supplementary Data (Table S2) and demonstrate that 92 out of 124 total genes in this candidate pathway table (74%) are found "Present" by gene expression profiling in the LEPs.

We also tested the expression of the candidate genes described above using tandem mass spectrometry proteomics with the Multidimensional Protein Identification Tool (MudPIT) . 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¨Cdefined pathways were confirmed by MudPIT proteomics.
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DISCUSSION& b! K, z$ ~! J: y0 L) e

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.( T& P. N) l2 w, Z$ R6 P
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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.
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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% . 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 ¡À 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.7 s# ]% [- t5 k, L+ n3 I
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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¨C 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¨Cspecific as single markers.* e: _3 c# ^6 Y0 }! n4 @

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 . VE-cadherin and CD133 are cell surface markers generally agreed upon to be expressed by endothelial progenitors.- ?' `1 I1 V- Z* s5 |5 l. V
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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¨Cassociated 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¨Cderived mouse endothelial progenitor . On the other hand, only six genes are upregulated and angiogenesis-associated in the Sca1¨C LEPs, including three that are antiangiogenic (Fig. 6).( D1 O! A( ~; J# f' a
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In conclusion, the genomic profiling of LEP Sca1  versus Sca1¨C cells distinguished these two populations by differential gene expression. Specifically, LEP Sca1  cells are characterized by upregulation of primarily angiogenesis-associated and endothelial lineage¨Cspecific genes, while the LEP Sca1¨C cells reveal a majority of hematopoietic lineage¨Cspecific 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.
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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.
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CONCLUSION" U, Y, D2 d: G, l

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." r1 |% W8 X, H$ W6 Q7 P" O: r

ACKNOWLEDGMENTS9 ?1 P( y# ^8 `0 p9 N  [5 \
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We acknowledge the kind gift of the anti-phosphotyrosine¨Cspecific 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
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The authors indicate no potential conflicts of interest.
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