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Blood, Vol. 113, Issue 9, 2104-2107, February 26, 2009
Endothelial progenitor cell infusion induces hematopoietic stem cell reconstitution in vivo
Blood Salter et al.
113: 2104
Supplemental materials for: Salter et al
Generation and characterization of fetal blood EPCs
To establish primary murine fetal blood EPC cultures, we first collected fetal blood from n=20 C57Bl6 pups (Jackson Laboratory, Bar Harbor, ME; 20–30 µL per pup) following the method described by Chen et al.17 Fetal blood MNCs were isolated via density centrifugation using Lymphoprep (Axis-Shield, Oslo, Norway). 100mm culture plates (Corning, Corning, NY) were coated with 0.2% gelatin (Sigma, St Louis, MO) in PBS for 1 hour at room temperature, then washed twice with DPBS, and 20 mL/dish of EC growth media, containing M199 (Invitrogen, Carlsbad, CA), 10% fetal bovine serum (FBS; Hyclone, Logan, UT), 2 mM L-glutamine (Invitrogen), 100 U/mL penicillin, 100 µg/mL streptomycin (1% pcn/strp; Invitrogen), 4 U/mL heparin (Sigma), and 60 mg/L endothelial cell growth supplement (Sigma) was added. Fetal blood MNCs were seeded at 10 × 106/dish and then incubated for 24 hours at 37°C in 5% CO2. After 24 hours, the dishes were washed, all non-adherent cells were removed and new EC growth media was added; this was repeated every 24 hours for 7 days per the method described by Yoder et al. to generate primitive ECFCs.18 Between 3–4 weeks of culture, primary adherent colonies were observed. Primary EPC colonies were trypsinized and passaged several times and a bank of early passage EPCs was created. Immunohistochemical analysis was performed to determine whether the cells possessed the phenotype of primitive ECFCs.18 For immunocytochemical staining, the EPC monolayers were fixed and incubated with 1–10 µg/ml of primary antibodies as previously described.18 Nuclei were counterstained with 100 ng/ml Hoechst 33342 (Molecular Probes, Eugene, OR). We used the following primary antibodies for immunocytochemical staining: rat anti-mouse CD31 (Chemicon, Temecula, CA), rabbit anti-VEGFR2 (Abcam, Cambridge, MA), FITC rat anti-mouse CD14 (BD), FITC sheep anti-mouse VWF (Serotec), FITC rat anti-mouse 115 (eBiosciences), FITC rat anti-mouse CD45 (eBiosciences), rat anti-mouse VE-cadherin (R&D, Minneapolis, MN), and rat anti-mouse CD105 (BD), which has been used to detect mesenchymal stem cells (MSCs).23 Secondary staining for visualization was performed with Alexa 555 or Alex 488 conjugated antibodies, as previously described.18 For analysis of uptake of LDL, media was changed to ECCM containing 10g/ml 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine-labeled acetylated LDL (DiI-Ac-LDL; Biomedical Tech., Stoughton, MA) and cells were incubated at 37°C for 2 hours prior to analysis, as previously described.18 For analysis of lectin binding, cells were stained with 20g/ml FITC-labeled lectin from Ulex europaeus (UEA-1 lectin; Sigma) as previously described.18 Visualization of antibody staining was performed using a Zeiss Axiovert 200 inverted microscope (Carl Zeiss Microimaging, Inc, Thornwood, NY) with an LD-plan NEOFLUAR 40×/0.6 objective lens at 25° C in Cytoseal XYL imaging medium (Richard Allen Scientific). Image acquisition and analysis was conducted using an AxioCam MRc camera and Zeiss Axiovision 4.5 software Carl Zeiss Microimaging). EPCs were also plated in 14 day methylcellulose cultures with 100 ng/mL VEGF and 5 ng/mL GMCSF to measure colony forming unit-EC (CFU-EC) production, which distinguishes EPCs from monocyte-derived cells.24 Complete blood counts and differentials were measured in each sample using an Abbott CELLDYN 3700 Analyzer (Abbott Laboratories, Abbott Park, IL). A minimum of 10–15 mice were analyzed per time point per condition to allow statistical comparisons. Statistical comparisons were performed using a t test. One femur from each animal was decalcified in Cal-Ex Decalcifying Solution (Fisher, Pittsburgh, PA) for 30 minutes on ice and frozen in Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA) for hematoxylin and eosin staining and MECA-32 antibody staining. For estimation of HSC content within EPC-treated mice versus control mice, whole BM was collected from B6.SJL (CD45.1+) mice at day +20 following 700 cGy TBI and EPC infusion and from B6.SJL mice at day +20 following 700 cGy TBI and PBS injections. The BM cells were treated with RBC lysis buffer (Sigma), washed, resuspended and transplanted in limiting dilutions via tail vein injection into lethally irradiated (950 cGy) C57Bl6 (CD45.2+) recipients. Donor cells were co-transplanted with 1 × 105 recipient BM cells to allow competitive repopulation. Donor-derived hematopoietic reconstitution was measured in the peripheral blood (PB) by flow cytometry (FACS Aria, BD) every 4 weeks over time, as previously described.5 PB was collected from isofluorane-anesthetized mice via retro-orbital puncture; cells were stained with FITC- or PE-CD45.1, FITC-CD45.2, PE–anti-Thy 1.2, PE–anti-B220, PE–anti–Ter-119 or PE–anti–Mac-1. Animals were considered to be engrafted if donor CD45.1 cells were present at ≥1% in the PB.16 Competitive Repopulating Unit (CRU) calculations were performed using L-Calc software (Stem Cell Technologies).16 GFP+ EPCs were generated from fetal blood collected from C57BL/Ka-Thy1.1 GFP+ transgenic mice that were provided courtesy of Dr. Jos Domen (Duke University). GFP+ EPCs were administered IV at 1 × 106 cells/mouse into 10-week-old BALB/c mice after total body irradiation with 550cGy as described above. Mice that received intravenously administered GFP+ EPCs (n=3) and control, non-treated mice (n=3) were sacrificed at 24 hours, 4 days and 7 days post-infusion and the lungs, femurs, spleen and liver were collected. All tissues were fixed with 4% paraformaldehyde (Sigma) overnight at 4°C. The liver, spleen, and lungs were then placed in 30% sucrose (Fisher Biotech, Fair Lawn, NJ) dissolved in DPBS, overnight at 4°C. These tissues were then placed in Tissue-Tek cryomolds (Sakura Finetek), embedded in Tissue-Tek OCT compound and frozen on dry ice. Femurs were decalcified using daily changes of 14% EDTA (EMD Chemicals, Gibbstown, NJ) (pH 7.1) for 4 to 7 days at 4°C. The femurs were washed thoroughly with DPBS and soaked in 30% sucrose overnight at 4°C. They were then frozen in OCT as described above. Cryosectioning was performed on a Leica CM1850 cryotome (Meyer Instruments, Houston, TX), and 10µm serial sections were adhered to Poly-prep poly-L-lysine coated slides (Sigma). Sections were then permeabilized for 15 min in PBS with 0.05% Tween-20 (Calbiochem, San Diego, CA) and stained for 45 minutes with 100ng/ml Hoechst 33342 in PBS with 0.05% Tween-20. Slides were visualized on a Zeiss Axiovert 200 microscope. Examination of BM vasculature
Femurs were collected from EPC-treated BALB/c mice and saline-treated BALB/c controls over time following irradiation and immediately decalcified in Cal-Ex Decalcifying Solution (Fisher, Pittsburgh, PA) for 30 minutes on ice and fresh frozen as previously described.25 Sections were cut on a cryostat and placed on Poly-Prep slides. Slides were placed in cold (−20°C) acetone (VWR Scientific, West Chester, PA) for 5 to 10 minutes and then stored at −80°C until immunostaining. Slides were removed from −80°C freezer and thawed in PBS for 5 minutes. Slides were washed twice in PBS with 0.5% Tween 20. Endogenous peroxidases were blocked in 3% hydrogen peroxide solution (EMD Biosciences, San Diego, CA) for 30 minutes at room temperature, and samples were then washed 3× in PBS with 0.5% Tween 20. Non-specific binding was then blocked with 20% rabbit serum (Sigma) in PBS for one hour at room temperature. Blocking agent was removed, and samples were stained overnight at 4°C with a 1:80 dilution of either biotin-conjugated MECA-32 (BD) or rat IgG2a (BD).15 Additional processing and counterstaining with streptavidin-HRP (BD) was performed as previously described.15 Images were acquired with an AxioCam MRc digital camera mounted on an Axiovert 200 inverted microscope. Anti–VE-cadherin administration in irradiated BALB/c mice
Adult BALB/c mice were injected IP with 750 µg of a neutralizing anti-VE Cadherin (E4G10, ImClone Systems, NY)20 every 3 days beginning on day +3 following 550 cGy TBI and every 3 days thereafter for a total of 5 doses, as previously described.22 Mice did not receive IP EPC injections on day +3 post-irradiation to avoid potential interaction between the antibody and the infused EPCs. Control mice were identically irradiated and then received 5 doses of PBS via the same route and schedule of administration. PB was collected from all mice treated with VE-cadherin antibody beginning at day +9 post-irradiation and complete blood counts were compared with irradiated control mice and EPC-treated mice that did not receive VE-cadherin antibody injections. Fibroblast infusion studies
Primary murine dermal fibroblasts (FBs) were generated from 10 week old female C57Bl6 mice as previously described.26 Early passage (8 to 10) FBs were generated in culture over several weeks. Prior to injection, FBs were irradiated with 3000 cGy in vitro using a Cs137 irradiator. One hour after irradiation, the FBs were centrifuged and resuspended in fresh media just prior to IV injection. Adult BALB/c mice were irradiated with 550 cGy TBI and, after 4 hours, were injected IV with 1 × 106 FBs. On days +1 to +4, the mice also received 2.5 × 106 irradiated FBs daily via IP administration. PB was collected from all FB-treated mice and PBS-treated control mice for measurement of complete blood counts beginning at day +9 post-irradiation as described above. In vitro cultures of BM hematopoietic progenitor cells with EPCs
Primary C57Bl6 fetal blood EPCs (1 × 105) were plated on gelatin-treated 6 well culture dishes in complete EC media and after 72 hours, confluence was achieved. The cells were then washed with PBS and the media was replaced with IMDM supplemented with 10% FBS and thrombopoietin 20 ng/mL, SCF 120 ng/mL, Flt-3 ligand 50 ng/mL (TSF, R & D Systems, Minneapolis, MN) and 1% penicillin/streptomycin. BM c-kit+sca-1+lin− (KSL) cells (4 × 104) were isolated via sterile FACS sorting from adult (non-irradiated) C57Bl6 mice and plated in contact and non-contact (transwell) cultures with EPCs or cytokines alone (TSF) for 10 days. Total viable cells counts and KSL content were measured in each condition compared to input. A group of C57Bl6 mice were also irradiated with 700 cGy TBI and 4 hours post-irradiation, BM cells were collected, washed and BM MNCs were isolated by density centrifugation. 1 × 105 BM MNCs were then plated in contact and non-contact culture with EPCs or cytokines alone and total viable cell recovery was compared between the groups at day +10.
Files in this Data Supplement:
- Table S1. Immunophenotype of Fetal Blood EPCs (PDF, 10.4 KB)
- Table S2. EPC infusions increase BM CRU content (PDF, 40.4 KB)
- Figure S1. Fetal blood-derived EPCs have features of ECFCs (JPG, 53.5 KB)
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(A) Primary fetal blood EPCs at day 21 in culture, demonstrate the phenotype of endothelial cells (i). The cells display cobblestone appearance (50×, top left), strongly express UEA-lectin and take up Ac-LDL (400×), express VEGFR2 (200×), CD31 (200×), VE cadherin (200×) and lack expression of CD14 (200×). (ii) EPCs exclusively give rise to EC colonies in 14 day methylcellulose culture with VEGF and GM-CSF. Data represents the mean ± SD of n=3 cultures.
- Figure S2. Multilineage engraftment in mice transplanted with BM from EPC-treated mice (JPG, 38.6 KB)
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C57Bl6 (CD45.2+) mice were irradiated with 950 cGy TBI and then transplanted with BM cells collected at Day +20 from Bl6.SJL (CD45.1+) donor mice that had been irradiated with 700 cGy TBI and treated with EPC infusions alone or with PBS. Increased donor-derived myeloid (Mac-1/Gr-1), B-cell (B220), T-cell (Thy 1.2) and erythroid progenitor cell (Ter119) engraftment was observed at 12 weeks in the mice that were transplanted with BM cells from EPC-treated donors (open circles) versus BM cells from PBS-treated controls (filled circles). Plots show PB lineage engraftment in mice transplanted with 100K donor BM cells. Lines represent mean levels of CD45.1+ cell engraftment in the PB in each group.
- Figure S3. Infused GFP+ EPCs do not contribute to the BM vascular niche in irradiated mice (JPG, 53.7 KB)
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Cross sections from lungs, femurs and spleens were analyzed for evidence of GFP+ cells in mice treated with 1 × 106 GFP+ EPCs or PBS via tail vein infusion. At 24 hours, 4 days and 7 days post-infusion, GFP+ cells were detected in the lungs of treated mice, but no GFP+ cells were observed in the BM or spleen.
- Figure S4. Infusion of C57Bl6-derived, irradiated fibroblasts does not promote hematologic recovery in irradiated mice (JPG, 27.7 KB)
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PB WBCs (top left), neutrophil counts (top right) and platelet counts (bottom) are shown over time in BALB/c mice following 550 cGy TBI and subsequent treatment with either irradiated C57Bl6 fibroblasts (gray line) or PBS (black line). Data represent the mean ± SEM of n=10–12 mice PB samples per time point. * P=0.02 and P=0.01 for differences in WBC and neutrophil count between fibroblast-treated mice and controls at day 16.
- Figure S5. Soluble factors produced by EPCs support the expansion of BM progenitor cells in culture and the recovery of irradiated BM progenitors (JPG, 31.5 KB)
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BM MNCs from normal or irradiated C57Bl6 mice were placed in culture with TSF alone (see Materials and Methods) or EPC-contact and non-contact cultures supplemented with TSF. (Top) EPC non-contact cultures (transwell, TW) supported a significant increase in total cells compared to cultures with TSF alone (d10,* P=0.005). (Middle) EPC non-contact cultures also induced a 5-fold expansion of BM KSL cells at day 10 (*P=0.003 vs. TSF). (Bottom) BM MNCs were harvested from 700 cGy irradiated C57Bl6 mice and then placed in 10 day culture with TSF alone or EPC cultures supplemented with TSF in contact and non-contact conditions. Both contact and non-contact EPC cultures (TW) supported the recovery of viable BM progenitor cells whereas no viable cells were recovered in TSF cultures. Data represent the means ± SD of n=3–6 experiments per study.
REFERENCES
23. Olivier EN, Rybicki AC, Bouhassira EE. Differentiation of human embryonic stem cells into bipotent mesenchymal stem cells. Stem Cells. 2006;24:1914–1922.
24. Urbich C, Heeschen C, Aicher A, Dernbach E, Zeiher AM, Dimmeler S. Relevance of monocytic features for neovascularization capacity of circulating endothelial progenitor cells. Circulation. 2003;108:2511–2516.
25. Congdon KL, Voermans C, Ferguson EC, et al. Activation of Wnt Signaling in Hematopoietic Regeneration. Stem Cells. 2008;26:1202–1210.
26. Salmon AB, Murakami S, Bartke A, Kopchick J, Yasumura K, Miller RA. Fibroblast cell lines from young adult mice of long-lived mutant strains are resistant to multiple forms of stress. Am J Physiol Endocrinol Metab. 2005;289:E23–29.
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