Induction of megakaryocytes to synthesize and store a releasable pool of human factor VIII
2003; Elsevier BV; Volume: 1; Issue: 12 Linguagem: Inglês
10.1111/j.1538-7836.2003.00534.x
ISSN1538-7933
AutoresDavid A. Wilcox, Qizhen Shi, Paquita Nurden, Sandra L. Haberichter, Jonathan B. Rosenberg, Bryon D. Johnson, Alan T. Nurden, G C White, Robert R. Montgomery,
Tópico(s)Blood Coagulation and Thrombosis Mechanisms
ResumoSummaryvon Willebrand factor (VWF) is a complex plasma glycoprotein that modulates platelet adhesion at the site of a vascular injury, and it also serves as a carrier protein for factor (F)VIII. As megakaryocytes are the only hematopoietic lineage to naturally synthesize and store VWF within α-granules, this study was performed to determine if expression of a FVIII transgene in megakaryocytes could lead to trafficking and storage of FVIII with VWF in platelet α-granules. Isolex® selected CD34+ cells from human G-CSF mobilized peripheral blood cells (PBC) and murine bone marrow were transduced with a retrovirus encoding the B-domain deleted form of human FVIII (BDD-FVIII). Cells were then induced with cytokines to form a population of multiple lineages including megakaryocytes. Chromogenic analysis of culture supernatant from FVIII-transduced human cells demonstrated synthesis of functional FVIII. Treatment of cells with agonists of platelet activation (ADP, epinephrine, and thrombin receptor-activating peptide) resulted in the release of VWF antigen and active FVIII into the supernatant from transduced cells. Immunofluorescence analysis of cultured human and murine megakaryocytes revealed a punctate pattern of staining for FVIII that was consistent with staining for VWF. Electron microscopy of transduced megakaryocytes using immunogold-conjugated antibodies colocalized FVIII and VWF within the α-granules. FVIII retained its association with VWF in human platelets isolated from the peripheral blood of NOD/SCID mice at 2–6 weeks post-transplant of transduced human PBC. These results suggest feasibility for the development of a locally inducible secretory pool of FVIII in platelets of patients with hemophilia A. von Willebrand factor (VWF) is a complex plasma glycoprotein that modulates platelet adhesion at the site of a vascular injury, and it also serves as a carrier protein for factor (F)VIII. As megakaryocytes are the only hematopoietic lineage to naturally synthesize and store VWF within α-granules, this study was performed to determine if expression of a FVIII transgene in megakaryocytes could lead to trafficking and storage of FVIII with VWF in platelet α-granules. Isolex® selected CD34+ cells from human G-CSF mobilized peripheral blood cells (PBC) and murine bone marrow were transduced with a retrovirus encoding the B-domain deleted form of human FVIII (BDD-FVIII). Cells were then induced with cytokines to form a population of multiple lineages including megakaryocytes. Chromogenic analysis of culture supernatant from FVIII-transduced human cells demonstrated synthesis of functional FVIII. Treatment of cells with agonists of platelet activation (ADP, epinephrine, and thrombin receptor-activating peptide) resulted in the release of VWF antigen and active FVIII into the supernatant from transduced cells. Immunofluorescence analysis of cultured human and murine megakaryocytes revealed a punctate pattern of staining for FVIII that was consistent with staining for VWF. Electron microscopy of transduced megakaryocytes using immunogold-conjugated antibodies colocalized FVIII and VWF within the α-granules. FVIII retained its association with VWF in human platelets isolated from the peripheral blood of NOD/SCID mice at 2–6 weeks post-transplant of transduced human PBC. These results suggest feasibility for the development of a locally inducible secretory pool of FVIII in platelets of patients with hemophilia A. Human factor (F)VIII is a 2332 amino acid plasma protein consisting of multiple polypeptides (80 000–210 000 MW) that form a heterotrimeric complex, which plays a pivotal role in the propagation of coagulant activity at the site of a vascular injury [1Eaton D. Rodriguez H. Vehar G.A. Proteolytic processing of human factor VIII: correlation of specific cleavages by thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity.Biochemistry. 1986; 25: 505-12Crossref PubMed Google Scholar]. As a result of molecular genetic defects in FVIII, individuals with hemophilia A have prolonged bleeding due to the failure of FVIII to serve as a cofactor in the 'FX-ase' activation complex of the coagulation cascade [2Ahmad S.S. Scandura J.M. Walsh P.N. Structural and functional characterization of platelet receptor-mediated factor VIII binding.J Biol Chem. 2000; 275: 13071-81Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar]. Patients' with severe hemorrhagic episodes have been historically treated with concentrates of FVIII from pooled plasma and recombinant FVIII preparations [3Kasper C.K. Hereditary plasma clotting factor disorders and their management.Haemophilia. 2000; 6: 13-27Crossref PubMed Scopus (23) Google Scholar]. Unfortunately, these treatments are inadequate due to an increased risk of viral infections, the development of inhibitors, and the creation of an enormous financial expense as a result of the necessity for multiple infusions of this short-lived molecule during the patient's lifetime [4Lusher J.M. First and second generation recombinant factor VIII concentrates in previously untreated patients: recovery, safety, efficacy, and inhibitor development.Semin Thromb Hemost. 2002; 28: 273-6Crossref PubMed Scopus (0) Google Scholar]. Hemophilia A has been successfully 'cured' using experimental therapies involving the transplantation of reticuloendothelial tissue into humans (liver and spleen) and dogs (lymph nodes, liver, and spleen) [5Webster W.P. Zukoski C.F. Hutchin P. Reddick R.L. Mandel S.R. Penick G.D. Plasma factor VIII synthesis and control as revealed by canine organ transplantation.Am J Physiol. 1971; 220: 1147-54Crossref PubMed Scopus (80) Google Scholar, 6Groth C.G. Hathaway WE, Gustafsson A. Geiss W.P. Putnam C.W. Bjorken C. Porter K.A. Starzl T.E. Correction of coagulation in the hemophilic dog by transplantation of lymphatic tissue.Surgery. 1974; 75: 725-33PubMed Google Scholar, 7Lewis J.H. Bontempo F.A. Spero J.A. Ragni M.V. Starzl T.E. Liver transplantation in a hemophiliac.N Engl J Med. 1985; 312: 1189-90Crossref PubMed Scopus (116) Google Scholar, 8Bontempo F.A. Lewis J.H. Gorenc T.J. Spero J.A. Ragni M.V. Scott J.P. Starzl T.E. Liver transplantation in hemophilia A.Blood. 1987; 69: 1721-4Crossref PubMed Google Scholar, 9Liu L. Xia S. Seifert J. Transplantation of spleen cells in patients with hemophilia A: a report of 20 cases.Transpl Int. 1994; 7: 201-6PubMed Google Scholar]. However, organ transplantation is not a practical option to correct hemophilia A due to the risk of rejection of an allogeneic graft, difficulty in finding HLA-matched donors, and the potential for other transplant related complications. Strategies for genetic replacement of FVIII are being explored since patients with severe hemophilia A have shown effective response to treatment with recombinant FVIII [10White G.C.. . . . . . . . .2.n.d. Gene therapy in hemophilia: clinical trials update.Thromb Haemost. 2001; 86: 172-7Crossref PubMed Scopus (27) Google Scholar, 11Mannucci PM. Ham–Wasserman lecture: hemophilia and related bleeding disorders. a story of dismay and success. Hematology (Am Soc Hematol Educ Program) 2002; 100: 1–9.Google Scholar]. To date, three phase I clinical trials were performed in the USA for gene therapy of hemophilia A that resulted in the detection of low-levels of FVIII expression. Each trial employed a unique strategy to express the FVIII transgene. Transkaryotic Therapy (Cambridge, MA, USA) used stable transfection of patient fibroblasts [12Roth D.A. Tawa Jr, N.E. O'Brien J.M. Treco D.A. Selden R.F. Nonviral transfer of the gene encoding coagulation factor VIII in patients with severe hemophilia A.N Engl J Med. 2001; 344: 1735-42Crossref PubMed Scopus (312) Google Scholar]. Genestar (San Diego, CA, USA) utilized a gutless adenovirus vector targeted with a liver specific gene-promoter [10White G.C.. . . . . . . . .2.n.d. Gene therapy in hemophilia: clinical trials update.Thromb Haemost. 2001; 86: 172-7Crossref PubMed Scopus (27) Google Scholar, 13Zhang W.W. Josephs S.F. Zhou J. Fang X. Alemany R. Balague C. Dai Y. Ayares D. Prokopenko E. Lou Y.C. Sethi E. Hubert-Leslie D. Kennedy M. Ruiz L. Rocow-Magnone S. Development and application of a minimal-adenoviral vector system for gene therapy of hemophilia A.Thromb Haemost. 1999; 82: 562-71Crossref PubMed Scopus (23) Google Scholar]. Individuals with hemophilia A were also intravenously injected with a Moloney murine leukemia virus (MoMLV) vector encoding BDD-FVIII in a clinical trial performed by Chiron (Emeryville, CA, USA) [10White G.C.. . . . . . . . .2.n.d. Gene therapy in hemophilia: clinical trials update.Thromb Haemost. 2001; 86: 172-7Crossref PubMed Scopus (27) Google Scholar]. This resulted in the detection of vector gene sequences by PCR in the patient's peripheral blood mononuclear cells for up to 1 year post injection. In the current study, we attempt to characterize the synthesis and storage of human FVIII within hematopoietic cells following in vitro transduction of G-CSF mobilized human CD34+ PBC using Chiron's BDD-FVIII vector. Although, the viral promoter permits FVIII expression in multiple hematopoietic lineages, this study exclusively examines FVIII synthesis in megakaryocytes and platelets: because (i) megakaryocytes are the only hematopoietic cell that synthesize and store VWF, the carrier protein for FVIII and (ii) platelets mediate the primary response to vascular injury, thereby potentially providing FVIII at sites of complication in hemophilia A patients. Our results reveal that synthesis of functional FVIII occurred in megakaryocytes leading to its storage in association with VWF in the α-granules of platelets. This outcome suggests that FVIII could undergo regulated release from platelets following a physiological hemostatic response to vessel injury. A phycoerythrin (PE)-conjugated anti-human-glycoprotein (GP)Ibα antibody (mouse anti-human CD42b), PE-anti-GPIIIa antibody (mouse anti-human CD61), PE-anti-P-selectin (mouse anti-human CD62) and isotype standards (PE-IgG) were obtained from Becton Dickinson (San Jose, CA, USA). The monoclonal antibody, MBC 103.3, which recognizes an epitope on human FVIII light chains [14Rosenberg J.B. Foster P.A. Kaufman R.J. Vokac E.A. Moussalli M. Kroner P.A. Montgomery R.R. Intracellular trafficking of factor VIII to von Willebrand factor storage granules.J Clin Invest. 1998; 101: 613-24Crossref PubMed Google Scholar]; AP2, which recognizes the human GPIIb–IIIa complex [15Pidard D. Montgomery R.R. Bennett J.S. Kunicki T.J. Interaction of AP-2, a monoclonal antibody specific for the human platelet glycoprotein IIb–IIIa complex, with intact platelets.J Biol Chem. 1983; 258: 12582-6Abstract Full Text PDF PubMed Google Scholar]; and a polyclonal antibody to human VWF that cross-reacts with murine VWF were produced by our Hybridoma Core Laboratory [14Rosenberg J.B. Foster P.A. Kaufman R.J. Vokac E.A. Moussalli M. Kroner P.A. Montgomery R.R. Intracellular trafficking of factor VIII to von Willebrand factor storage granules.J Clin Invest. 1998; 101: 613-24Crossref PubMed Google Scholar]. A rabbit anti-human polyclonal antibody that recognizes human and murine VWF was purchased from Dako (Carpinteria, CA, USA). Alexa Fluor® 488 F(ab′)2 fragment of goat anti-mouse IgG (H + L) and Alexa Fluor® 594 F(ab′)2 fragment of goat anti-rabbit IgG (H + L) were purchased from Molecular Probes (Eugene, OR, USA). Texas red (TXR)-donkey anti-rabbit F(ab′)2 conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). The human promegakaryocytic cell line, Dami, was previously described [16Greenberg S.M. Rosenthal D.S. Greeley T.A. Tantravahi R. Handin R.I. Characterization of a new megakaryocytic cell line: the Dami cell.Blood. 1988; 72: 1968-77Crossref PubMed Google Scholar]. CD34 antigen positive cells were immuno-selected (89% CD34 + purity) from the apheresis product of normal healthy subjects on an Isolex® 300i Magnetic Cell Separator (Nexell Therapeutics Inc., Irvine, CA, USA; distribution through Baxter Healthcare Corp.) and stored as previously described [17Wilcox D.A. Olsen J.C. Ishizawa L. Griffith M. White II, G.C. Integrin alphaIIb promoter-targeted expression of gene products in megakaryocytes derived from retrovirus-transduced human hematopoietic cells.Proc Natl Acad Sci USA. 1999; 96: 9654-9Crossref PubMed Scopus (0) Google Scholar]. A MoMLV provirus, HALB-FVIII (HALB-F8/CC/9801H1 isolate), was generously provided at a titer of 6.6 × 108 cfu mL−1 by Chiron Corporation (Emeryville, CA, USA) [18DePolo N.J. Harkleroad C.E. Bodner M. Watt A.T. Anderson C.G. Greengard J.S. Murthy K.K. Dubensky Jr, T.W. Jolly D.J. The resistance of retroviral vectors produced from human cells to serum inactivation in vivo and in vitro is primate species dependent.J Virol. 1999; 73: 6708-14Crossref PubMed Google Scholar]. The provirus was stored frozen at −80 °C until utilized at a total of 100 cfu cell−1 over a 24-h period to transfer a DNA cassette encoding the BDD-FVIII into hematopoietic cells under control of the promoter for the retroviral long-terminal repeat (LTR) [19Greengard J.S. Jolly D.J. Animal testing of retroviral-mediated gene therapy for factor VIII deficiency.Thromb Haemost. 1999; 82: 555-61Crossref PubMed Google Scholar]. Mice (deficient in Fc receptors, complement function, B- and T-cell function) were used as recipients of the human hematopoietic cells. Mice of 5–7 weeks of age were purchased from Jackson Laboratory (Bar Harbor, ME, USA). All animals were handled under sterile conditions and maintained in microisolators with autoclaved food and acidified water at the Medical College of Wisconsin's American Association for the Accreditation of Laboratory Animal Care approved Animal Resource Center (Milwaukee, WI, USA). Platelets were isolated using Fico/Lite Platelets (Atlanta Biologicals, Norcross, GA, USA) from 200 µL of whole blood collected by retro-orbital bleed of an anesthetized NOD/SCID mouse using a heparinized microhematocrit capillary tube (Fisher, Pittsburgh, PA, USA). Platelets were resuspended in Tyrodes buffer containing 2% bovine serum albumin (BSA) and used directly for flow cytometry or centrifuged onto a microscope slide using a cytospin at 50 × g for 6 min, air-dried overnight, and then used for immunofluorescence analysis as described below. Bone marrow was collected from normal C57 BL/6 mice maintained in microisolators with autoclaved food and acidified water at the Medical College of Wisconsin Animal Resource Center as previously described [20Johnson B.D. Becker E.E. LaBelle J.L. Truitt R.L. Role of immunoregulatory donor T cells in suppression of graft-versus-host disease following donor leukocyte infusion therapy.J Immunol. 1999; 163: 6479-87PubMed Google Scholar]. Anesthetized mice were sacrificed by cervical dislocation, bone marrow was flushed from each femur with DMEM (Life Technologies, Grand Island, NY, USA) and approximately 1.5 × 107 mononuclear cells were purified with ficoll (Atlanta Biological). Cells were resuspended in Dulbecco's modified Eagle's medium (DMEM) containing 25% fetal bovine serum and 10% dimethyl sulfoxide (DMSO) and stored in liquid nitrogen until needed. Cells (2.5 × 105) were transduced with 500 µL of (HALB-FVIII) retrovirus supernatant. The multiplicity of infection was 150 virions (HALB-FVIII) per cell in the presence of 4 µg mL−1 polybrene (Sigma Co., St Louis, MO, USA) in a single well of a six-well plate at 37 °C in 5% CO2. After 2.5 h, retroviral supernatant was removed and cells were resuspended in IMDM (Life Technologies) containing 10% horse serum and cultured at 37 °C in 5% CO2 until used for analysis of FVIII:C as described below. Human CD34+ PBC were transduced as previously described [17Wilcox D.A. Olsen J.C. Ishizawa L. Griffith M. White II, G.C. Integrin alphaIIb promoter-targeted expression of gene products in megakaryocytes derived from retrovirus-transduced human hematopoietic cells.Proc Natl Acad Sci USA. 1999; 96: 9654-9Crossref PubMed Scopus (0) Google Scholar]. Cells were prestimulated in IMDM (Life Technologies) containing 20% fetal bovine serum (Life Technologies), 10 ng mL−1 recombinant human (rh) interleukin (IL)-3, 100 ng mL−1 rhIL-6, 100 ng mL−1 stem cell factor (rhSCF) and 50 ng mL−1 rhflk2/flt3 ligand (PeproTech, Rocky Hill, NJ) and PEGylated-rh megakaryocyte growth and development factor (PEG-rhMGDF) (generous gift of Kirin Brewery, Maebashi-shi, Gunma, Japan) for 48 h at 37 °C in 5% CO2. Cells were transduced at 2.5 × 106 per well of a sterile, six-well non-tissue culture-treated plate (Falcon-Becton Dickinson, Franklin Lakes, NJ, USA) coated with 20 µg cm−1[2Ahmad S.S. Scandura J.M. Walsh P.N. Structural and functional characterization of platelet receptor-mediated factor VIII binding.J Biol Chem. 2000; 275: 13071-81Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar] RetroNectin™[21Kimizuka F. Taguchi Y. Ohdate Y. Kawase Y. Shimojo T. Hashino K. Kato I. Sekiguchi K. Titani K. Production and characterization of functional domains of human fibronectin expressed in Escherichia coli.J Biochem. 1991; 110: 284-91Crossref PubMed Scopus (0) Google Scholar, 22Hanenberg H. Xiao X.L. Dilloo D. Hashino K. Kato I. Williams D.A. Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells.Nature Med. 1996; 2: 876-82Crossref PubMed Scopus (0) Google Scholar] (generous gift of Takara Bio, Otsu, Shiga, Japan) with a calculated multiplicity of infection of 100 virions (HALB-FVIII) per cell in IMDM plus 20% FCS and rhIL-3, rhIL-6, rhSCF, rhflk2/flt3 ligand, PEG-rhMGDF. Fresh viral supernatant was added after 2 h. This procedure was repeated 24 h later. Two hours after the final transduction, cells were collected, washed and injected via the tail vein into NOD/SCID mice (see below). A fraction of the FVIII-transduced PBC (5 × 105 cells mL−1) was induced with cytokines (rhIL-3, -6, -11, rhSCF, rhflk2/flt3 ligand, PEG-rhMGDF) to form megakaryocytes for up to 10 days in vitro. Bone marrow from C57BL/6 mice were transduced using the protocol as described for human CD34+ PBC with the exception that recombinant murine (rm) growth factors rmIL-3, rmIL-6, rmSCF replaced rh growth factors in the media. Levels of biologically active FVIII (FVIII:C) were measured in tissue culture supernatant or Tyrode's buffer using a fluid assay kit, Coatest VIII:C/4 kit (DiaPharm, Franklin, OH, USA) as previously described [14Rosenberg J.B. Foster P.A. Kaufman R.J. Vokac E.A. Moussalli M. Kroner P.A. Montgomery R.R. Intracellular trafficking of factor VIII to von Willebrand factor storage granules.J Clin Invest. 1998; 101: 613-24Crossref PubMed Google Scholar, 23Hillery C.A. Mancuso D.J. Evan Sadler J. Ponder J.W. Jozwiak M.A. Christopherson P.A. Cox Gill J. Paul Scott J. Montgomery R.R. Type 2M von Willebrand disease: F606I and I662F mutations in the glycoprotein Ib binding domain selectively impair ristocetin- but not botrocetin-mediated binding of von Willebrand factor to platelets.Blood. 1998; 91: 1572-81Crossref PubMed Google Scholar]. Cells were seeded at 5 × 105 mL−1 per well of 24-well tissue culture plates. Conditioned media was collected from separate wells at days 7–10 in culture and polybrene (Sigma) was added at 64.5 µg mL−1 of media to inactivate heparin. Triplicate samples of supernatant were placed in uncoated wells of a 96-well microtiter plate (25 µL per well) and assay components, including phospholipid, FIXa, FX, and calcium chloride were added to each well, and the plates were incubated for 10 min at 37 °C. The chromogenic FXa substrate S-2222 was added, and the plate was transferred immediately to a ThermoMax microplate reader (Molecular Devices, Menlo Park, CA, USA) preset at 37 °C. The FXa-dependent conversion of S-2222 is directly related of the amount of FVIII:C in each well. A standard curve was constructed by plotting known amounts of recombinant human FVIII (rh-BDD-FVIII) (Refracto™, Pharmacia, Columbus, OH, USA) diluted in tissue culture media using Vmax at 405 nm. The Vmax of each reaction was converted to units of FVIII:C activity using the kinetic software program, SOFTmax, v.2.34 (Molecular Devices). Cultured human PBC were harvested for physiologic studies at 8–10 days post-transduction, washed, and megakaryocytes were activated according to a previously described protocol [24Wilcox D.A. Olsen J.C. Ishizawa L. Bray P.F. French D.L. Steeber D.A. Bell W.R. Griffith M. White II, G.C. Megakaryocyte-targeted synthesis of the integrin beta(3)-subunit results in the phenotypic correction of Glanzmann thrombasthenia.Blood. 2000; 95: 3645-51Crossref PubMed Google Scholar]. Cells were resuspended in Tyrode's buffer (2.5 × 106 mL−1) containing 1 mmol L−1 CaCl2, 1 mmol L−1 MgCl2, 25 µmol L−1 each of, ADP, epinephrine and TRAP (H-Ser-Phe-Leu-Leu-Arg-Asn-OH peptide) for 30 min at 25 °C. Separate aliquots were incubated in Tyrode's buffer without agonist as a negative control. The percentage of activated megakaryocytes was determined by flow cytometry and two-color analysis with a PE-conjugated antibody to P-selectin (FL2 channel) gated on cells that stained positive with a FITC-conjugated antibody to human megakaryocyte marker, GPIIIa (FL1 channel). At least 5000 FL1 positive events were collected for each sample and analyzed as described in the method for flow cytometric analysis. Supernatant was collected from agonist treated and negative control samples and tested for FVIII:C activity using the coatest assay (as described above) and VWF antigen levels determined as previously described [23Hillery C.A. Mancuso D.J. Evan Sadler J. Ponder J.W. Jozwiak M.A. Christopherson P.A. Cox Gill J. Paul Scott J. Montgomery R.R. Type 2M von Willebrand disease: F606I and I662F mutations in the glycoprotein Ib binding domain selectively impair ristocetin- but not botrocetin-mediated binding of von Willebrand factor to platelets.Blood. 1998; 91: 1572-81Crossref PubMed Google Scholar]. The intracellular location of VWF and FVIII was detected using a MRC 600 confocal laser imagining system equipped with a krypton-argon laser (Bio-Rad, Hercules, CA, USA) using our previously described protocol [14Rosenberg J.B. Foster P.A. Kaufman R.J. Vokac E.A. Moussalli M. Kroner P.A. Montgomery R.R. Intracellular trafficking of factor VIII to von Willebrand factor storage granules.J Clin Invest. 1998; 101: 613-24Crossref PubMed Google Scholar]. Control samples (untransduced cells) were processed in parallel under identical conditions. Human PBC or murine bone marrow were induced to form megakaryocytes for up to 10 days after FVIII-transduction in tissue culture media containing rhIL-3, -6, -11, rhSCF, rhflt3/flk2 ligand, and PEG-rhMGDF [24Wilcox D.A. Olsen J.C. Ishizawa L. Bray P.F. French D.L. Steeber D.A. Bell W.R. Griffith M. White II, G.C. Megakaryocyte-targeted synthesis of the integrin beta(3)-subunit results in the phenotypic correction of Glanzmann thrombasthenia.Blood. 2000; 95: 3645-51Crossref PubMed Google Scholar]. Cells were allowed to adhere to poly d-lysine-treated coverslips, fixed in 3.7% buffered formalin, permeabilized in 1% Triton X-100 solution (20 mmol L−1 Hepes/300 mmol L−1 sucrose/50 mmol L−1 NaCl/3 mmol L−1 MgCl2·6H2O, pH 7.0), and blocked in 2% normal serum/PBS. Dual immunofluorescent labeling of the cells was accomplished using a sequential antibody staining method as previously described [14Rosenberg J.B. Foster P.A. Kaufman R.J. Vokac E.A. Moussalli M. Kroner P.A. Montgomery R.R. Intracellular trafficking of factor VIII to von Willebrand factor storage granules.J Clin Invest. 1998; 101: 613-24Crossref PubMed Google Scholar]. Affinity-purified anti-VWF polyclonal antibodies and anti-FVIII monoclonal antibody (MBC 103.3) were used as primary antibodies and diluted at 2.5 and 5.0 µg mL−1 in 1% BSA/HBSS, respectively. The Alexa Fluor® 488-conjugated F(ab′)2 fragment of goat anti-mouse IgG (H + L) was used as a secondary antibody to detect FVIII and Alexa Fluor® 594-conjugated F(ab′)2 fragment of goat anti-rabbit IgG (H + L) or Texas red (TXR)-donkey anti-rabbit F(ab′)2 conjugated secondary antibodies were used to detect the presence of VWF. Nonspecific isotype control antibodies served as negative controls (not shown). Cells were imaged by a series of Z sections taken for each field and the entire Z series (12–25 images) combined into a stacked projection. The projections for each emission were merged using the Confocal Assistant software program (Bio-Rad). Computer-assigned colors were based on the intensities of bitmap overlaps, with Alexa488-fluorochrome represented by green pixels, TXR- or Alexa594-fluorochrome represented by red pixels, and colocalization of the two fluorochrome-conjugated antibodies represented by yellow pixels. Sample preparation and analysis were performed similar to a previously described protocol [25Nurden P. Poujol C. Winckler J. Combrie R. Pousseau N. Conley P.B. Levy-Toledano S. Habib A. Nurden A.T. Immunolocalization of P2Y1 and TPalpha receptors in platelets showed a major pool associated with the membranes of alpha-granules and the open canalicular system.Blood. 2003; 101: 1400-8Crossref PubMed Scopus (0) Google Scholar]. Sample preparation Transduced and untransduced cells were induced with cytokines to form megakaryocytes in vitro for 8 days (murine bone marrow) or 10 days (human CD34+ PBC). Cell samples were collected, washed, and fixed in 1.25% glutaraldehyde (Fluka AG, Buchs, Switzerland) in phosphate-buffered saline (PBS) followed by overnight transport from Milwaukee to Bordeaux at 4 °C in PBS containing 0.1% glutaraldehyde. Each sample contained approximately 2 × 106 total cells. Immunogold labeling After washing, cells were infused with 2.3 mol L−1 sucrose (Fluka) before being frozen in propane and then in liquid nitrogen with a Reichert KF 80 freezing system (Leica, Vienna, Austria). Ultra-thin sections of approximately 80 nm were cut at −120 °C with the Ultracut E ultramicrotome equipped with a FC 4E cryokit attachment and placed on collodion-coated nickel grids. Then, the grids were incubated for 10 min on drops of washing buffer consisting of PBS supplemented with 0.5% or 1% BSA before being incubated with antibodies. The rabbit polyclonal antibody to human VWF (Dako) and mouse monoclonal antibody to human FVIII used for confocal microscopy were also used for electron microscopy. The grids were first placed on drops containing purified IgG (10 µg mL−1) of the antibodies to VWF and FVIII for 1 h at room temperature. Sections were washed and incubated for 1 h with a goat anti-rabbit secondary antibody adsorbed onto 5 nm gold particles (1/50 dilution of AuroProbe EM G5; Amersham, Les Ulis, France) and a goat anti-mouse secondary antibody adsorbed onto 10 nm gold particles (1 : 100 dilution of AuroProbe EM G10). Controls included the use of an irrelevant IgG of the same species and at the same concentration. Electron microscopy The grids were floated several times on PBS and then on water. The cryosections were stained by uranyl acetate and osmium according to our standard procedures and embedded in a thin film of methylcellulose prior to observation with a Jeol JEM-1010 transmission electron microscope (Jeol, Croissy-sur-seine, France) at 80 kV. Six- to 8-week-old NOD/SCID mice were conditioned for cellular transplantation with a sublethal dose of 350 cGy total body irradiation using a small animal cesium irradiator (Sheperd Mark I, J.L. Shepherd, San Fernando, CA, USA). Twenty-four hours after irradiation, retrovirus-transduced human CD34+ PBC were resuspended in IMDM containing recombinant murine SCF, rhIL-6, -11 and rhFlk2/Flt3 Ligand and PEG-rhMGDF. A cell dose of 3.5 × 107 cells was transplanted into each mouse by tail vein injection in a volume of 600 µL per mouse. Mice were treated with these growth factors plus 1.0 µg PEG-rhMGDF by interperitoneal injection once per week for the duration of the studies to induce production of human platelets as previously described [26Perez L.E. Rinder H.M. Wang C. Tracey J.B. Maun N. Krause D.S. Xenotransplantation of immunodeficient mice with mobilized human blood CD34+ cells provides an in vivo model for human megakaryocytopoiesis and platelet production.Blood. 2001; 97: 1635-43Crossref PubMed Scopus (35) Google Scholar]. All animal experiments were performed in accordance with institutional guidelines approved by the Animal Care and Use Committee of the Medical College of Wisconsin. Whole blood was collected at 2–6 weeks post-transplant from retrovirus-transduced human CD34+ PBC recipients via retro-orbital bleed using a heparinized capillary tube and platelets were isolated using FicoLite (Atlanta Biologicals, Atlanta, GA, USA). Transduced and untransduced samples (∼1.0 × 106) were blocked for 15 min with 2% BSA in PBS, and incubated for 20 min at 25 °C with phycoerythrin-conjugated antibodies that specifically recognize human GPIIIa or GPIb (Becton Dickinson, San Jose, CA, USA). Cells were resuspended in 500 µL of PBS containing 2% BSA and collected on a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA). The data was analyzed with Win MDI software. A minimum of 2 × 104 events were collected for the analysis. Background staining was determined using isotype-specific control antibodies. Chimeric conversion of murine peripheral blood to human hematopoietic cells was determined by dividing the fraction of platelets expressing human GPIIIa or GPIb by the total platelet count. To determine if human hematopoietic cells could serve as a primary tissue source for the synthesis of a functional form of FVIII (FVIII:C), normal individuals were treated with granulocyte colony stimulating factor to mobilize bone marrow stem cells into the peripheral blood and
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