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• W2034585706 abstract "This study aims at generating immune chicken phage display libraries and single-chain antibodies (scFvs) specifically directed against cell surface markers of cultured peripheral blood mononuclear cells (PBMCs) that contain endothelial progenitor cells (EPCs). In contrast to previous approaches that use well-defined recombinant antigens attached to plastic surfaces that may alter the structure of the proteins, the authors describe a method that maintains the cell surface markers on live cells while providing the opportunity to rapidly screen entire libraries for antibodies that bind to unknown cell surface markers of progenitor/stem cells. Chickens immunized with live EPCs, consisting of a heterogeneous population of lymphocytes and monocytes, demonstrated a robust immune response. After three rounds of biopanning, the authors purified and characterized three unique scFvs called UG1-3. Codon-optimized recombinant UG1 (gUG-1) shows binding by flow cytometry to circulating CD14-positive cells in peripheral blood consistent with predominant expression of a target protein on monocyte subsets. The authors describe the successful use of immunization of chickens for the generation of scFvs against a heterogenous population of EPCs displaying unknown cell surface markers and demonstrate the strong potential of phage display technology in the development of reagents for the isolation and characterization of stem/progenitor cells. This study aims at generating immune chicken phage display libraries and single-chain antibodies (scFvs) specifically directed against cell surface markers of cultured peripheral blood mononuclear cells (PBMCs) that contain endothelial progenitor cells (EPCs). In contrast to previous approaches that use well-defined recombinant antigens attached to plastic surfaces that may alter the structure of the proteins, the authors describe a method that maintains the cell surface markers on live cells while providing the opportunity to rapidly screen entire libraries for antibodies that bind to unknown cell surface markers of progenitor/stem cells. Chickens immunized with live EPCs, consisting of a heterogeneous population of lymphocytes and monocytes, demonstrated a robust immune response. After three rounds of biopanning, the authors purified and characterized three unique scFvs called UG1-3. Codon-optimized recombinant UG1 (gUG-1) shows binding by flow cytometry to circulating CD14-positive cells in peripheral blood consistent with predominant expression of a target protein on monocyte subsets. The authors describe the successful use of immunization of chickens for the generation of scFvs against a heterogenous population of EPCs displaying unknown cell surface markers and demonstrate the strong potential of phage display technology in the development of reagents for the isolation and characterization of stem/progenitor cells. There is considerable interest in the use of hematopoietic stem cells or other cells with angiogenic potential such as endothelial progenitor cells (EPCs) in the treatment of ischemia, restenosis, diabetic vasculopathy, and occlusive vascular disease, which includes coronary artery disease, cerebrovascular disease, and peripheral artery disease.1Dimmeler S. Zeiher A.M. Vascular Repair by Circulating Endothelial Progenitor Cells: The Missing Link in Atherosclerosis.J. Mol. Med. 2004; 82: 671-677Crossref PubMed Scopus (262) Google Scholar, 2Dong C. Goldschmidt-Clermont P.J. Endothelial Progenitor Cells: A Promising Therapeutic Alternative for Cardiovascular Disease.J. Interv. Cardiol. 2007; 20: 93-99Crossref PubMed Scopus (36) Google Scholar, 3Kawamoto A. Asahara T. Role of Progenitor Endothelial Cells in Cardiovascular Disease and Upcoming Therapies.Catheter Cardiovasc. Interv. 2007; 70: 477-484Crossref PubMed Scopus (108) Google Scholar, 4Werner N. Wassmann S. Ahlers P. Schiegl T. Kosiol S. Link A. Walenta K. Nickenig G. Endothelial Progenitor Cells Correlate with Endothelial Function in Patients with Coronary Artery Disease.Basic Res. Cardiol. 2007; 102: 565-571Crossref PubMed Scopus (147) Google Scholar, 5Matsuo Y. Imanishi T. Hayashi Y. Tomobuchi Y. Kubo T. Hano T. Akasaka T. The Effect of Endothelial Progenitor Cells on the Development of Collateral Formation in Patients with Coronary Artery Disease.Intern. Med. 2008; 47: 127-134Crossref PubMed Scopus (25) Google Scholar, 6Murasawa S. Asahara T. Cardiogenic Potential of Endothelial Progenitor Cells.Ther. Adv. Cardiovasc. Dis. 2008; 2: 341-348Crossref PubMed Scopus (16) Google Scholar The discovery of EPCs introduced a novel concept stating that vasculogenesis can occur postnatally, whereby, in response to various stimuli (especially ischemia), EPCs home, differentiate, proliferate, and incorporate into resident endothelial cells.7Asahara T. Murohara T. Sullivan A. Silver M. van der Zee R. Li T. Witzenbichler B. Schatteman G. Isner J.M. Isolation of Putative Progenitor Endothelial Cells for Angiogenesis.Science. 1997; 275: 964-967Crossref PubMed Scopus (7693) Google Scholar EPCs are commonly defined as a heterogenous, fibronectin-adherent mononuclear cell population derived from bone marrow, umbilical cord, or peripheral blood8Liew A. Barry F. O’Brien T. Endothelial Progenitor Cells: Diagnostic and Therapeutic Considerations.Bioessays. 2006; 28: 261-270Crossref PubMed Scopus (84) Google Scholar and have ever since been cultured using methods that are associated with cells of distinct phenotypes: colony-forming unit Hill cells (CFU-Hills), circulating angiogenic cells (CACs), and endothelial colony-forming cells (ECFCs).9Hirschi K.K. Ingram D.A. Yoder M.C. Assessing Identity, Phenotype, and Fate of Endothelial Progenitor Cells.Arterioscler. Thromb. Vasc. Biol. 2008; 28: 1584-1595Crossref PubMed Scopus (643) Google Scholar The CFU-Hills or CACs were also termed early outgrowth cells (early EPCs) because they appear in culture after 4 to 9 days, whereas ECFCs are often referred to as late outgrowth cells (late EPCs) due to their appearance in culture after 1 to 3 weeks.9Hirschi K.K. Ingram D.A. Yoder M.C. Assessing Identity, Phenotype, and Fate of Endothelial Progenitor Cells.Arterioscler. Thromb. Vasc. Biol. 2008; 28: 1584-1595Crossref PubMed Scopus (643) Google Scholar Early EPCs do not form tubules in vitro8Liew A. Barry F. O’Brien T. Endothelial Progenitor Cells: Diagnostic and Therapeutic Considerations.Bioessays. 2006; 28: 261-270Crossref PubMed Scopus (84) Google Scholar but adhere to mature endothelial cells and exert their function via paracrine effects, whereas late EPCs are capable of in vitro tubule formation without the presence of mature endothelial cells.8Liew A. Barry F. O’Brien T. Endothelial Progenitor Cells: Diagnostic and Therapeutic Considerations.Bioessays. 2006; 28: 261-270Crossref PubMed Scopus (84) Google Scholar, 9Hirschi K.K. Ingram D.A. Yoder M.C. Assessing Identity, Phenotype, and Fate of Endothelial Progenitor Cells.Arterioscler. Thromb. Vasc. Biol. 2008; 28: 1584-1595Crossref PubMed Scopus (643) Google Scholar, 10Rehman J. Li J. Orschell C.M. March K.L. Peripheral Blood “Endothelial Progenitor Cells” Are Derived from Monocyte/Macrophages and Secrete Angiogenic Growth Factors.Circulation. 2003; 107: 1164-1169Crossref PubMed Scopus (1500) Google Scholar All EPCs take up acetylated low-density lipoprotein (acLDL) and Ulex europeus agglutinin I (UEA-1) and share the expression of cell surface markers such as vascular endothelial growth factor receptor-2 (VEGFR2), CD31, von Willebrand factor (vWF), and endothelial nitric oxide synthase (eNOS), whereas they differ in the expression of other cell surface markers (late outgrowth are CD133–, CD115–, CD45–, CD14–, CD34+ and early outgrowth are CD133+ and CD115+) with conflicting reports on the expression of some early EPC markers (CD45+/–, CD14+/–, CD34+/–).7Asahara T. Murohara T. Sullivan A. Silver M. van der Zee R. Li T. Witzenbichler B. Schatteman G. Isner J.M. Isolation of Putative Progenitor Endothelial Cells for Angiogenesis.Science. 1997; 275: 964-967Crossref PubMed Scopus (7693) Google Scholar, 8Liew A. Barry F. O’Brien T. Endothelial Progenitor Cells: Diagnostic and Therapeutic Considerations.Bioessays. 2006; 28: 261-270Crossref PubMed Scopus (84) Google Scholar, 9Hirschi K.K. Ingram D.A. Yoder M.C. Assessing Identity, Phenotype, and Fate of Endothelial Progenitor Cells.Arterioscler. Thromb. Vasc. Biol. 2008; 28: 1584-1595Crossref PubMed Scopus (643) Google Scholar, 10Rehman J. Li J. Orschell C.M. March K.L. Peripheral Blood “Endothelial Progenitor Cells” Are Derived from Monocyte/Macrophages and Secrete Angiogenic Growth Factors.Circulation. 2003; 107: 1164-1169Crossref PubMed Scopus (1500) Google Scholar, 11Hill J.M. Zalos G. Halcox J.P. Schenke W.H. Waclawiw M.A. Quyyumi A.A. Finkel T. Circulating Endothelial Progenitor Cells, Vascular Function, and Cardiovascular Risk.N. Engl. J. Med. 2003; 348: 593-600Crossref PubMed Scopus (3114) Google Scholar, 12Ribatti D. The Discovery of Endothelial Progenitor Cells: A Historical Review.Leuk. Res. 2007; 31: 439-444Crossref PubMed Scopus (110) Google Scholar, 13Liew A. McDermott J.H. Barry F. O’Brien T. Endothelial Progenitor Cells for the Treatment of Diabetic Vasculopathy: Panacea or Pandora’s Box.Diabetes Obes. Metab. 2008; 10: 353-366Crossref Scopus (11) Google Scholar, 14Rohde E. Malischnik C. Thaler D. Maierhofer T. Linkesch W. Lanzer G. Linkesch W. Guelly C. Strunk D. Blood Monocytes Mimic Endothelial Progenitor Cells.Stem Cells. 2006; 24: 357-367Crossref PubMed Scopus (232) Google Scholar, 15Rohde E. Bartmann C. Schallmoser K. Reinisch A. Lanzer G. Linkesch W. Guelly C. Strunk D. Immune Cells Mimic the Morphology of Endothelial Progenitor Colonies In Vitro.Stem Cells. 2007; 25: 1746-1752Crossref PubMed Scopus (142) Google Scholar Research in this area is hindered, however, by the absence of an EPC marker, and there is a clear need to develop novel assays and highly specific reagents. Murine monoclonal antibody (MAb) technology has found wide acceptance but suffers from limitations such as randomly identified antibodies against immobilized antigens or the absence of a robust rodent immune response against proteins that are highly conserved in closely related species. We sought an alternative system for generating EPC-specific MAbs. Phage display has become a powerful technology for the generation of high-affinity reagents to study angiogenesis.16Smith J. Kontermann R.E. Embleton J. Kumar S. Antibody Phage Display Technologies with Special Reference to Angiogenesis.FASEB J. 2005; 19: 331-341Crossref PubMed Scopus (27) Google Scholar Chickens have become effective hosts for generating high-specificity recombinant antibodies to mammalian proteins using phage display, as they can circumvent many of the common problems encountered with murine immunizations.17Andris-Widhopf J. Rader C. Steinberger P. Fuller R. Barbas III., C.F. Methods for the Generation of Chicken Monoclonal Antibody Fragments by Phage Display.J. Immunol. Methods. 2000; 242: 159-181Crossref PubMed Scopus (176) Google Scholar,18Van Wyngaardt W. Malatji T. Mashau C. Fehrsen J. Jordaan F. Miltiadou D. du Plessis D.H. A Large Semi-Synthetic Single-Chain Fv Phage Display Library Based on Chicken Immunoglobulin Genes.BMC. Biotechnol. 2004; 4: 6Crossref Scopus (43) Google Scholar Most importantly, however, the amino acid homology between the mammalian and avian orthologues of a given protein is typically low, and some mammalian proteins may not even exist in avians.19Wallis J.W. Aerts J. Groenen M.A. Crooijmans R.P. Layman D. Graves T.A. et al.A Physical Map of the Chicken Genome.Nature. 2004; 432: 761-764Crossref PubMed Scopus (157) Google Scholar The immunoglobulin response of chickens to highly conserved mammalian proteins is therefore reliably robust, often exhibits high affinity, and potentially targets a broad spectrum of epitopes on protein immunogens.17Andris-Widhopf J. Rader C. Steinberger P. Fuller R. Barbas III., C.F. Methods for the Generation of Chicken Monoclonal Antibody Fragments by Phage Display.J. Immunol. Methods. 2000; 242: 159-181Crossref PubMed Scopus (176) Google Scholar,20Yamanaka H.I. Inoue T. Ikeda-Tanaka O. Chicken Monoclonal Antibody Isolated by a Phage Display System.J. Immunol. 1996; 157: 1156-1162PubMed Google Scholar,21Finlay W.J. deVore N.C. Dobrovolskaia E.N. Gam A. Goodyear C.S. Slater J.E. Exploiting the Avian Immunoglobulin System to Simplify the Generation of Recombinant Antibodies to Allergenic Proteins.Clin. Exp. Allergy. 2005; 35: 1040-1048Crossref PubMed Scopus (40) Google Scholar In this study, we aimed at applying phage selections to in vitro cultured, intact EPCs displaying the full repertoire of potential cell surface markers and set out to generate antibodies to heterogeneous proangiogenic endothelial progenitor cell preparations that contain mainly lymphocytes and monocytes using chicken immune phage display technology. The ultimate goal was to develop single-chain antibodies that bind to the surface of live EPCs and internalize. These scFvs could be used not only as reagents to detect and enrich cells prior to applications in regenerative medicine such as diabetic wound healing or cardiac repair after acute myocardial infarction but also preferably as tools that aid in the specific targeting of gene delivery systems to circulating angiogenic cells to enhance their therapeutic potential. We identified and characterized three novel scFvs, termed UG 1-3. Codon-optimized UG1, called gUG1, was obtained in higher yields and binds predominantly to monocyte subsets in peripheral blood. The strategy outlined here can be applied to generate specific antibodies against other mammalian cells that are of therapeutic interest such as embryonic stem cells or human mesenchymal stem cells and will greatly aid in the clinical translation of well-characterized, cell-based therapeutics in regenerative medicine applications. This study was approved by the Clinical Research Ethics Committee, University College Hospital, Galway, Ireland. Peripheral blood was obtained from donors with informed consent. EPCs were cultured as previously described.22Tepper O.M. Galiano R.D. Capla J.M. Kalka C. Gagne P.J. Jacobowitz G.R. Levine J.P. Gurtner G.C. Human Endothelial Progenitor Cells from Type II Diabetics Exhibit Impaired Proliferation, Adhesion, and Incorporation into Vascular Structures.Circulation. 2002; 106: 2781-2786Crossref PubMed Scopus (1292) Google Scholar Briefly, peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation (Ficollpaque, GE Healthcare, Uppsala, Sweden). After purification with three washing steps, 10 × 106 PBMCs were plated on fibronectin-coated, 6-well plates and cultured in endothelial cell basal medium-2 (Clonetics, Lonza, Walkersville, MD) supplemented with EGM-2 single aliquots (Clonetics, Lonza) consisting of fetal bovine serum, vascular endothelial growth factor, fibroblast growth factor-2, epidermal growth factor, insulin-like growth factor-1, heparin, gentamicin/amphotericin-B, and ascorbic acid. After 4 days in culture at 37 °C and 5% CO2, adherent cells were stained for the uptake of diI-labeled acLDL (Molecular Probes, Carlsbad, CA) and the binding of fluorescein isothiocyanate (FITC)–labeled Ulex europaeus agglutinin I (Sigma, St. Louis, MO). For further characterization, we examined the EPCs with a 1:50 sheep anti-human vWF-FITC antibody and with a 1:200 rabbit anti-human VEGFR2 in combination with a 1:100 dilution of a goat anti-rabbit antibody (all from Abcam, Cambridge, MA). Images were acquired at room temperature using an Olympus DP70 color camera (Tokyo, Japan) with Olympus Biosystems AnalySISD software. EPCs were cultured for 7 days, nonadherent cells removed by phosphate-buffered saline (PBS) wash, the adherent cells detached with 5 mM EDTA and frozen down at –80 °C in a 10% DMSO/90% fetal bovine serum solution. Two ISA brown chickens were supplied by and immunizations conducted at Agro-Bio (La Ferté, St. Aubin, France). Cryopreserved fibronectin adherent cells were thawed and centrifuged at 400g for 5 min room temperature (RT). The freezing medium was removed and cells resuspended in a total of 10 mL of supplemented medium and incubated at 37 °C for 45 min. Cells were washed twice with sterile PBS solution. Both chickens were injected intradermally (under the wing) with 5 × 105 cells in a total volume of 200 μL PBS at day 0, 21, and 42. Chickens were prebled at day 0 and bled again at day 49. At day 49, the chickens were sacrificed and the spleen and bone marrow were harvested, homogenized, and stored in TRIzol reagent (Invitrogen, Carlsbad, CA). ScFv library generation was performed as described previously.18Van Wyngaardt W. Malatji T. Mashau C. Fehrsen J. Jordaan F. Miltiadou D. du Plessis D.H. A Large Semi-Synthetic Single-Chain Fv Phage Display Library Based on Chicken Immunoglobulin Genes.BMC. Biotechnol. 2004; 4: 6Crossref Scopus (43) Google Scholar,23Hillier L.W. Miller W. Birney E. Warren W. Hardison R.C. Ponting C.P. et al.Sequence and Comparative Analysis of the Chicken Genome Provide Unique Perspectives on Vertebrate Evolution.Nature. 2004; 432: 695-716Crossref PubMed Scopus (2155) Google Scholar Briefly, RNA was extracted from spleen and bone marrow samples using TRIzol reagent, and first-strand cDNA was synthesis performed using the Superscript First Strand synthesis system for RT-PCR (Invitrogen). Antibody VH and VL genes were amplified using the primers CSCVHo-F (5′-GGTCAGTCCTCTAGATCTTCCGCCGTGACGTTG GACG AG-3′) and CSCG-B (5′-CTGGCCGGCCTGGCCACT AGTGGAGGAGAC GATGACTTCGGTCC 3′) to amplify the chicken VH domains. Primers CSCVK (5′-GTGGCCCAGGCG GCCCTG ACCTAGGACGGTCAGG 3′) and CKJo-B (5′-GGA AGATCTAG AGGACTGACC TAGGACGGT CAG-3′) were used to amplify the VL domains. The single-chain antibody library was assembled in a VL-VH scFv format, with a flexible linker (peptide sequence GGSSRSS) by SOE-PCR using the pooled VH and VL PCR products and subcloned into the pComb3X vector (Scripps Institute, La Jolla, CA). Electrocompetent XL1 Blue bacteria (Stratagene, La Jolla, CA) were mixed with the library prior to electroporation with a Micropulser (Bio-Rad, Hercules, CA). The bacteria were incubated at 37 °C at 220 rpm for 1 h. Ampicillin (25 μg/mL) and tetracycline (10 μg/mL) were added for 1 h. After transfer to 183 mL terrific broth containing ampicillin and tetracycline, 2 mL of VCSM13 helper phage was added and the samples were incubated for 2 h at 37 °C, 220 rpm. Kanamycin was added at 25 μg/mL, and all cultures were incubated for 6 h at 37 °C and 220 rpm. The bacteria were spun down at 3000g for 15 min and phages precipitated from the supernatant by addition of 8 g PEG, 6 g NaCl, followed by an incubation period of 30 min on ice and centrifugation at 15 000g for 15 min at 4 °C. The phage pellet was washed with 1% (w/v) bovine serum albumin (BSA) in PBS and passed through a 0.2-μm filter. We devised the following protocol for selecting and enriching (panning) antibody displaying phage on the surface followed by their uptake into live cells, a process we refer to here as biopanning of libraries. The phage library was dialyzed in PBS for 1 h and blocked in 1% BSA, PBS solution for 1 h at 4 °C. The library was added to the supernatant of the cultured EPCs and incubated with gentle shaking at 4 °C for 45 min. The cells were washed once with PBS and once with 100 mM glycine (pH 2.7) to reduce the high numbers of antibodies that bound weakly to the cell surface. The EPCs were incubated at 37 °C for 1 h to allow for internalization of antibody-displaying phage. Cells were washed once with PBS, trypsinized for 10 min (37 °C, 5% CO2), washed with serum containing medium, and spun down at 1500 rpm for 5 min. All cells were lysed with 100 mM TEA, neutralized with 2M Tris (pH 7.2) and added to 5 mL of XL1 Blue for 1 h to allow phage reinfection. The bacteria were plated on superbroth/ampicillin plates and incubated overnight at 37 °C. Two further rounds of phage preparations and panning of phage libraries were performed using cultured EPCs as screening targets. The library was transformed into XL1 blue after three rounds of biopanning as described above. Clones were randomly picked from a plate and the antibody amplified by PCR using primers ompseq (5′-AAGACAGCTATCGCGATTGCAG-3′) and gback (5′-GCCCCCTTA TTAGCGTTT GCCATC-3′). PCR products were digested with Alu I for 4 h at 37 °C and analyzed on a 4% agarose gel for unique restriction digest patterns. All unique antibody clones from the third round of biopanning were submitted to MWG (Germany) for sequencing analysis using primers ompseq and gback. Clones were grown in 2 mL terrific broth (TB), 50 μg/mL carbenicillin, 1 mM MgCl2 at 37 °C, 220 rpm overnight, transferred to 250 mL of fresh TB/carbenicillin, 1 mM MgCl2, 1% glucose, and incubated at 37 °C, 250 rpm for 8 h. The cultures were centrifuged at 2500g at 4 °C for 15 min, the pellets were resuspended in 250 mL TB, 50 μg/mL carbenicillin, 1 mM MgCl2, and induction of antibody expression was initiated by the addition of 0.5 mM IPTG. After incubation at 30 °C, 250 rpm for 24 h, the bacteria were spun down at 6000g for 15 min. The soluble, secreted antibody containing a tag composed of six histidines was isolated from the filter-sterilized bacterial supernatant by Ni-NTA agarose column purification (Qiagen, Venlo, the Netherlands) via FPLC (GE Healthcare). An Ni-NTA column (Qiagen) was equilibrated with 10 mM imidazole, 0.5 M NaCl in PBS (pH 7.4); the antibody sample was applied and eluted in a stepwise gradient at 80% and then 100% of a 1 M imidazole elution buffer (1 M imidazole, 0.5 M NaCl in PBS, pH 7.4). Eluted fractions of the antibodies were either dialyzed against PBS or desalted using FPLC gel permeation chromatography (Superdex 200 10/300 gel filtration column, GE Healthcare). Flow cytometry. The chicken immune response after injection of EPCs was determined by FACS (flow cytometry). EPCs were harvested by cell scraping, incubated with 1:200 dilutions of serum from chicken bleeds with FACS buffer (1% BSA, 0.02% sodium azide in PBS) and incubated on ice for 30 min. Cells were washed twice with FACS buffer and incubated with a 1:100 dilution of rabbit anti-IgY FITC (Pierce, Rockford, IL) preincubated in FACS buffer containing 20% human serum to reduce the background binding of the anti-IgY antibody. Cells were washed and resuspended in 200 μL of FACS buffer plus 20 μL of 30 μM 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma) for dead cell exclusion. Surface marker characterization of EPC cultures before and after cryopreservation and binding of scFvs to PBMCs was examined by flow cytometry. EPC cultures, cryopreserved EPCs, and PBMCs were prepared as described above, washed, and resuspended in FACS buffer (PBS, 2% FCS, 0.05% NaN2, pH 7.4) in the presence of human FcR blocking reagent (Miltenyi Biotech, Germany) at 4 °C for 10 min. Cells were again washed and resuspended in FACS buffer at 5 × 106 cells/mL and transferred into FACS tubes (Fisher Scientific, Dublin, Ireland) at 100 μL/tube. EPCs were incubated at 4 °C for 20 min with three to four color combinations of the following monoclonal antibodies: mouse anti-human CD45-PE (clone 5B1; Miltenyi Biotec), mouse anti-human CD3-AF488 (clone UCHT1), mouse anti-human CD14-PE-Cy7 (clone M5E2), mouse anti-human CD19-PE (clone HIB19), mouse anti-human CD56-APC (clone B159), and mouse anti-human CD34-APC (clone 581/CD34; all from BD Biosciences, San Jose, CA); mouse anti-human Tie-2/TEK/CD202b-APC (clone 83715; R&D Systems, Minneapolis, MN), and mouse anti-human CD309 (VEGFR-2/KDR; clone ES8-20E6, Miltenyi Biotec). PBMCs were first incubated with and without a previously optimized amount (2.5 μg) of purified HA-tagged gUG1 at 4 °C for 30 min and then were washed in FACS buffer followed by addition of mouse anti-HA-PE (Miltenyi Biotec) and two to three color combinations of antibodies directed against CD3, CD14, CD19, CD56, CD 34, Tie-2, and CD309 as described above for 20 min at 4 °C. Following all staining steps, cells were washed and resuspended in FACS buffer; the dead cell stain, SYTOX Blue (Invitrogen, Molecular Probes), was added, and the cells were immediately analyzed on a BD Biosciences FACSAria II flow cytometer. Between 105 and 5 × 105 events were acquired for each individual sample. Flow cytometry data were analyzed using FlowJo software (TreeStar Inc., Olten, Switzerland). For all analyses, nonviable (SYTOX-positive) cells and cell aggregates were excluded, and appropriate negative staining controls (fluorescent minus one and/or matched isotype controls) were used to distinguish positive and negative staining populations. Cells grown on slides were washed in PBS, fixed in 2% PFA for 1 h at RT, washed again with PBS, and blocked in 2% BSA/PBS for 30 min at RT. Immunolabeling of heterogenous EPCs mainly consisting of CD14+ monocytes and CD3+ T lymphocytes was performed by incubating the cells with a 1:300 dilution of mouse anti-CD3 (eBioscience) or mouse anti-CD14 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) in 2% BSA/PBS for 1 h at RT. Cells were washed with PBS twice for 5 min and then incubated with a 1:200 dilution of biotinylated, horse anti-mouse IgG antibody in 2% BSA/5% horse serum/PBS for 30 min at RT. Slides were washed twice with PBS for 5 min and incubated with a 1:100 streptavidin Qdot 605 solution (Invitrogen) in 2% BSA/PBS for 1 h at RT. Slides were washed with PBS, stained with a 1:1000 dilution of Hoechst dye for 10 min at RT, washed twice with PBS, mounted with VECTASHIELD (Vector Laboratories, Burlingame, CA), and dried overnight at 4 °C. Images were acquired with a Zeiss LSM510 META confocal microscope as previously described.24Ferrara D.E. Weiss D. Carnell P.H. Vito R.P. Vega D. Gao X. Nie S. Taylor W.R. Quantitative 3D Fluorescence Technique for the Analysis of En Face Preparations of Arterial Walls Using Quantum Dot Nanocrystals and Two-Photon Excitation Laser Scanning Microscopy.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2006; 290: R114-R123Crossref PubMed Scopus (51) Google Scholar Matrigel tubule assay was performed as previously described with modifications.22Tepper O.M. Galiano R.D. Capla J.M. Kalka C. Gagne P.J. Jacobowitz G.R. Levine J.P. Gurtner G.C. Human Endothelial Progenitor Cells from Type II Diabetics Exhibit Impaired Proliferation, Adhesion, and Incorporation into Vascular Structures.Circulation. 2002; 106: 2781-2786Crossref PubMed Scopus (1292) Google Scholar Briefly, matrigel (BD Biosciences) was thawed and placed in 4-well glass slides or in 12-well plates at RT for 30 min to allow solidification. DiI- and FITC-labeled EPCs (2 × 104 and 4 × 104, respectively) were co-plated with 4 × 104 human umbilical vein endothelial cells (HUVECs) and incubated at 37 °C. Tubule formation was defined as a structure exhibiting a length four times its width, and the number of tubules was counted per field under the microscope. Tubule count was performed for three fields per well in triplicate. The data shown are representative of several similarly performed experiments that consistently gave comparable results. Clonal sequence of UG1-3 obtained from MWG (Germany) were submitted to GENEART (Germany) for codon optimization in Escherichia coli. The codon-optimized clone (gUG1) was ligated into pComb3X for expression. EMBL:FN555207 Both chickens were immunized successfully with fibronectin-adherent EPC (Fig. 1a) preparations that, based on single-color immunofluorescence microscopy, consisted of mainly T lymphocytes (Fig. 1b) and monocytes (Fig. 1c). Early EPCs also took up acLDL and UEA-1 (Fig. 1d). These heterogenous cultures of EPCs stained positive for endothelial markers such as vWF (Fig. 1e) and VEGFR2 (Fig. 1f). The angiogenic potential of cultured EPCs was assessed in standard matrigel tubule assays. Cultured EPCs did not form tubules (data not shown). However, the numbers of angiogenic tubules formed by HUVECs in matrigel (Fig. 1g) increased twofold when co-cultured with EPCs (Fig. 1h, j), indicating the retention of functionality of cultured progenitor cells after isolation from peripheral blood of healthy adult donors prior to immunizations of avians. Multicolor flow cytometry was used to more comprehensively analyze the cellular constituents of EPC cultures following cryopreservation and thawing (Fig. 2). This demonstrated a high level of viability, predominant expression of the pan-leukocyte marker CD45, and co-existence of a large, CD14+ monocytic population (5%–10%) with a smaller, CD14–lymphocytic population (Fig. 2a). Subanalyses of these two populations (Fig. 2b) indicated that the lymphocytic populations consisted primarily of CD3+ T cells with lesser proportions of CD19+ B cells and CD56+ NK cells. The monocytic population contained a subset of CD14+/CD3+ cells but minimal numbers of cells staining positive for CD19 and CD56. Neither population contained significant numbers of CD34+ cells. Similar results were obtained for EPCs from a separate donor and for EPCs analyzed prior to cryopreservation (data not shown). The immunoglobulin response of both chickens was assessed via flow cytometry, by comparing binding of the preimmune and terminal bleed IgY to the cells used for immunization (Fig. 3). Both chickens showed a robust immune response as demonstrated by a shift in fluorescent intensity between the terminal bleed and preimmune serum.Fig. 2Multicolor flow cytometric analysis of cryopreserved and thawed endothelial progenitor cell (EPC) culture. (A) Sequential dot plots are shown for viability gating based on exclusion of SYTOX Blue vital dye, surface staining of viable cells for the pan-leukocyte marker CD45, and surface staining of viable, CD45+ cells for the monocyte marker CD14. Relevant proportions are indicated for each def" @default.
• W2034585706 created "2016-06-24" @default.
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• W2034585706 date "2011-08-01" @default.
• W2034585706 modified "2023-10-05" @default.
• W2034585706 title "Developing Cell-Specific Antibodies to Endothelial Progenitor Cells Using Avian Immune Phage Display Technology" @default.
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