Cloning of extracellular domain of sheep CD34
To obtain a cDNA clone for the extracellular domain of the sheep CD34 molecule, RNA purified from freshly isolated sheep dermal fibroblasts was provided by Dr. Paul Simmons and used to synthesize cDNA using the SuperScript® II First-Strand Synthesis System (Invitrogen, Carlsbad, CA). These resultant cDNAs were employed as templates to amplify an 858bp fragment of the extracellular domain of sheep CD34 by PCR (30 cycles) using the following primers: sense primer: 5′-atgctgggccgcaggggcgcg-3′; antisense primer: 5′-ggtcttccgggaatagctctggtg -3′. Since the sheep CD34 gene had not been sequenced, these primers were designed based on the NCBI sequence for bovine CD34. The resultant PCR product was analyzed on a 0.8% agarose gel to confirm that the correct size product had been obtained. The 858bp band corresponding to the extracellular domain of the sheep CD34 gene was excised from the gel, purified using the Qiaex II kit (Qiagen, Inc., Valencia, CA) and cloned into the pGEM-T Easy vector (Promega, San Luis Obispo, CA), according to manufacturer's instructions. The recombinant plasmid was propagated in MAX Efficiency® DH5α™ Competent Cells (Invitrogen, Carslbad, CA) and purified using the QiaPrep Midiprep system (Qiagen). Sequencing was performed using SP6 and T7 sequencing primers at the Nevada Genomics Center. Once the DNA sequence had been analyzed and aligned with bovine and goat sequences for CD34 to confirm its identity, the recombinant pGEM-T Easy plasmid was provided to Genovac AG (Freiburg, Germany).
Genetic immunization and production of monoclonal antibody to sheep CD34
The recombinant sheep CD34 plasmid (pGEM-T Easy) was employed by Genovac AG (Freiburg, Germany) for the commercial production of custom monoclonal antibodies using a proprietary procedure. Briefly, the sheep CD34 cDNA was subcloned into a proprietary expression vector and used to genetically immunize mice by repeated intra-dermal injections, thus stimulating an immune response. Lymphocytes harvested from the mice were fused to mouse myeloma cells to establish CD34-specific hybridomas. The hybridomas were screened at Genovac using proprietary recombinant cells transfected in vitro with a sheep CD34-expression vector. We then tested positive hybridoma supernatants on primary sheep hematopoietic cells and tissue sections. All sheep were a Rambouillet/Merino cross. One clone (8D11) was chosen for subsequent subcloning, yielding a pure IgG1-producing hybridoma, the supernatant of which was used for all subsequent studies.
Collection and isolation of sheep BMMNC
BM was aspirated into heparinized syringes from the posterior iliac crest of healthy control adult sheep following the procedure detailed in an IACUC-approved protocol, and BMMNC isolated by Ficoll-Hypaque (Sigma Chemicals) density gradient centrifugation, washed twice and resuspended in IMDM, 10% FCS.
Collection and isolation of sheep CBMNC
Cord blood was aspirated into heparinized syringes from the umbilical vein of healthy control sheep during delivery following the procedure detailed in an IACUC-approved protocol. CBMNC were isolated as detailed above for BMMNC.
Methylcellulose colony assays
BMMNC were cultured at a concentration of 5×105
/ml in methylcellulose (CFU-Mix, CFU-GM, BFU-E) using erythropoietin–containing MethoCult 4330 supplemented with recombinant ovine IL-3 (100U/ml), GM-CSF (100U/ml), SCF (1000U/mL) and sheep leukocyte-derived PHA-stimulated leukocyte conditioned medium (PHA-LCM) (5% vol/vol), as previously described [13
]. The plates were incubated at 37°C in a humidified atmosphere of 5% CO2
in air for 9-12 days. Hematopoietic colonies were then enumerated in situ [13
]. In the case of magnetically sorted CD34+ BM cells, cells obtained after MiniMacs sorting were cultured at 1×105
CAFC assays were performed essentially as previously described for mouse [44
] with minor modifications. In short, a sheep stromal layer was grown in 96-well microtiter plates (Costar, Cambridge, MA) in IMDM supplemented with 10% fetal calf serum, 5% horse serum, 10-5
M hydrocortisone, 3.3mM L-glutamine, 80U/ml penicillin, 80μg/ml streptomycin, and 10-4
M β–mercaptoethanol. These layers were then grow, to confluence, treated with Mitomycin C, and seeded with either whole unfractionated sheep BM or 8D11-selected BM cells at 20000, 10000, 5000, 2500, 1250, 625, 312, 156, 78, and 39 cells per well. The cells were maintained at 37°C and 5% CO2
. Half of the medium was replaced weekly, and wells were evaluated at days 28 and 35 for cobblestone areas growing underneath the stromal layer, since it is well established that day 28 and 35 CAFC represent the most primitive HSC with long-term repopulating ability.
Flow cytometric analysis of sheep peripheral blood and bone marrow
1×106 sheep PB, CB, or BM MNC were transferred to separate tubes, pelleted, and resuspended in PBS, 0.1% sodium azide. Cells were fixed in 2% paraformaldehyde, blocked for 15 minutes with 10% normal goat serum in TBS, and stained for 30 minutes at room temperature with a 1:10 dilution of supernatant containing monoclonal anti-sheep CD34. CD34 staining was then visualized with a PE-labeled anti-mouse IgG secondary antibody (Southern Biotech, Birmingham, AL) diluted 1:20, washed, and the cells stained with a 1:10 dilution of FITC-labeled antibody to sheep CD45 (AbD Serotec, Raleigh, NC). Following a final wash, cells were analyzed on a FACScan (Becton Dickinson Immuno-Systems, San Jose, CA). Negative controls consisted of PB, CB, and BM MNC stained with IgG1 control antibody and the identical PE-labeled secondary antibody used for CD34 visualization.
Mobilization of chimeric and control sheep with granulocyte–colony-stimulating factor
Human recombinant granulocyte–colony-stimulating factor (G-CSF) Neupogen (Amgen, Inc., Thousand Oaks, CA) was administered for 4 days once a day subcutaneously in the morning, weighing animals before injection, and using a dose of 4.8-5.7μg/kg. PB was drawn daily prior to injection and analyzed by flow cytometry to evaluate the total white blood cell (WBC) count and CD34+ progenitor cell content within the circulation.
Magnetic sorting of sheep CD34+ cells
BMMNC were washed twice with MiniMacs (Miltenyi Biotec, Inc., Auburn, CA) wash buffer, counted, and brought to a concentration of 108 cells per 300μl of buffer. 300μl of cell suspension was stained for 30min at 4°C with 50μl of monoclonal anti-ovine CD34. Samples were washed with MiniMacs buffer and the cell pellet resuspended in 300ul of MiniMacs buffer and incubated with 50μl of Goat anti-mouse IgG microbeads for 15min at room temperature. Samples were again washed and resuspended in 500μl of MiniMacs buffer. CD34+ cells were then separated on a MiniMacs magnetic column as per the manufacturer's instructions (Miltenyi Biotec, Inc.).
Immunohistochemical labeling of endothelial cells with anti-ovine CD34
Paraffin-embedded sections were de-waxed in xylene and rehydrated through a graded ethanol series to diH2O. Sections were blocked for 15-minutes with serum-free Protein Block (Dako, Carpentaria, CA) and incubated at room temperature for 1 hour with a 1:10 dilution of 8D11 supernatant. Residual unbound primary antibody was removed by three washes in TBS, containing 0.05% Tween 20. Sections were then incubated for 30 minutes at room temperature in the dark with PE-conjugated anti-mouse IgG secondary antibody (Southern Biotech) diluted 1:20. After three 1 minute washes, sections were counterstained with DAPI/Antifade solution (Vector Laboratories, Burlingame, CA) and coverslipped. Slides were viewed on an Olympus BX60 microscope. Sections processed in the absence of primary antibody served as controls.
To characterize the hematopoietic protein recognized by clone 8D11, sheep BMMNC were magnetically sorted with clone 8D11. The resultant cell pellet was resuspended in 200μl of PBS and 200μl of 2X protein sample buffer (0.5M Tris-HCl pH 6.8, 5% glycerol, 2% SDS and 100 mM DTT). Samples were boiled for 5 minutes and centrifuged at 16,000g for 15 min to remove insoluble material. Samples were loaded onto a pre-cast 7.5% Ready gel with a 4.5% stacker (BioRad Laboratories, Hercules, CA) and run for 1 hour at 200 volts. After electrophoresis, the proteins were transferred to nitrocellulose membranes in a Bio-Rad Electroblotter Apparatus. Membranes were blocked in TBST (TBS, 0.05% Tween 20) containing 5% milk for 1 h at room temperature and then reacted overnight at 4°C with 8D11 diluted 1:50 in TBST-milk. After three washes in TBST, the membranes were treated with affinity-purified horse radish peroxidase (HRP)-conjugated goat anti-mouse IgG (Promega, Madison, WI) diluted 1:1,000. Membranes were washed three times in TBST, and then developed with a DAB-based HRP detection kit (Vector Laboratories, Burlingame, CA).
Digital Image Acquisition
All images were captured with an Olympus DP70 CCD camera attached to an Olympus BX60 microscope, using Olympus DP Controller Software version 188.8.131.52 (Olympus America, Melville, NY, USA). Images were then subjected to minimal global processing, such as brightness and contrast adjustment and color balance in Adobe Photoshop CS.