DNA recombination and production of recombinant proteins
The cDNA fragment encoding the DSL domain (amino acids 127–225) of the human Dll1 (NM_005618) was amplified by PCR using a human cDNA library as a template. The product was fused at the C-terminus with a RGD-encoding fragment (hD1R) by PCR. The resultant gene fragments were cloned into pET32a (+) between the Nco I and Xho I sites, to construct pET32a-hD1R. For the production of recombinant proteins, E. coli BL21 (DE3) were transformed with the plasmids. Positive clones were expanded in Luria–Bertani (LB) medium, and cells at the exponential stage were induced with 0.5 mM isopropyl β-D-thiogalactoside (IPTG). The Trx-tagged proteins were purified by using Ni2+-NTA columns (Invitrogen, Carlsbad, CA) according to the manufacturer’s manual. To obtain the S-tagged proteins, Trx-hD1R were cleaved by using thrombin (Novagen, Darmstadt, Germany), and further purified using Ni2+-NTA columns. The hD1R protein was prepared in the Department of Medical Genetics and Developmental Biology of Fourth Military Medical University and has been detailed previously [25, 26].
Human umbilical vein endothelial cells (HUVECs) were cultured in M199 medium (GIBCO, Gaithersburg, MD) supplemented with 20 % fetal bovine serum (FBS), 30 μg/mL endothelial cell growth supplement (ECGS) (Sigma, St Louis, MO), 20 units/mL heparin, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells between passage three and five were used for experiments. For co-culture, HUVECs (2 × 104) were seeded in wells of 24-well plates and cultured to confluence. Cells were treated with mitomycin C (10 μg/mL) for 2.5 h, and were washed with PBS thoroughly for three times. Human UCB CD34+ progenitor cells were purified from human UCB samples by FACS-sorting after being stained with anti-human CD34-FITC (#581, Biolegend). The cells (2 × 103) were then plated on HUVECs and cultured in serum-free medium (StemSpan SFEM, STEMCELL Technologies, Vancouver, Canada) supplemented with a cocktail containing five types of human cytokines (h5GF) including thrombopoietin (TPO, 20 ng/mL), stem cell factor (SCF, 120 ng/mL), Flt-3 ligand (Flt-3L, 50 ng/mL), interleukin 6 (IL-6, 5 ng/mL), and interleukin 3 (IL-3, 5 ng/mL) (PeproTech, Rocky Hill, NJ). hD1R was added at the concentration of 2.5 μg/mL as previously described . In some experiments, γ-secretase inhibitor (GSI) (DAPT, Alexis Biochemicals, San Diego, CA) was included at the concentration of 10 μM. Half amount of the medium was changed every other day. Seven days after the starting of the co-culture, cells in suspension were collected by gentle pipetting and analyzed further. In some experiments, confluent HUVECs were cultured for 48 h in serum-free medium and supernatant containing soluble factor were collected and filtered through a 0.22 μm sterile filter as culture conditioned media. Live HUVECs were fixed 4 % paraformaldehyde (PFA) for 15 min and then used for co-culture experiments. Experiments associated with human samples were approved by the Ethical Committee on Medical Research-Related Affairs of the Fourth Military Medical University.
Colony-forming units (CFU) assay
CFU assay was performed by mixing freshly isolated or cultured hematopoietic cells with Methocult GF H4434 medium (STEMCELL Technologies). Cells were cultured for 14 days, and colonies (with >50 cells) containing different lineages of cells were counted under a microscope.
FACS analysis was performed routinely by using a CaliburTM flow cytometer (BD Immunocytometry Systems). Anti-mouse CD45-FITC (#104, eBioscience), anti-human CD45-APC (HI30, eBioscience), anti-human CD34-FITC (#581, Biolegend). Cell-cycle analysis was performed using DNA binding dye propidiumiodide (PI). Hematopoietic cells were fixed in 50 % ethanol and resuspended to 0.2 mL of 10 mg/mL RNAaseA and 50 µg/mL PI. Cell-cycle kinetics was performed with routine protocols using the FACS Calibur flow cytometer (Becton–Dickinson, CA). Apoptosis was analyzed by using an Annexin V-FITC Apoptosis Detection Kit (4A Biotech, Beijing, China).
Real time reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted by using the Trizol reagent (Invitrogen). cDNA was prepared by using a kit from TOYOBO (Osaka, Japan) with random primers. Real time PCR was performed by using a kit (SYBR Premix EX Taq, Takara) and the ABI Prism 7500 real time PCR system, with β-actin as a reference control. Primers used in RT-PCR were as follows: β-actin-F: 5′-TGGCACCCAGCACAATGAA; β-actin-R: 5′-CTAAGTCATAGTCCGCCTAGAAGCA; CXCR4-F: 5′-CCTATGCAAGGCAGTCCATGT; CXCR4-R: 5′-CTAAGTCATAGTCCGCCTAGAAGCA; Hes1-F: 5′-TGGAAATGACAGTGAAGCACCTC; Hes1-R: 5′-TCGTTCATGCACTCGCTGAAG; α4integrin-F: 5′-GGAATATCCAGTTTTTACACAAAGG; α4integrin-R: 5′-AGAGAGCCAGTCCAGTAAGATGA; α6integrin-F: 5′-ATGCACGCGGATCGAGTTT; α6integrin-R: 5′-TTCCTGCTTCGTATTAACATGCT.
NOD/SCID mice of 6–8 weeks old were purchased from Beijing HFK Bioscience Co. Ltd and were maintained in axenic conditions and sublethally (300 cGy) irradiated by total-body irradiation with γ-ray from a 60Co irradiation apparatus. Freshly isolated BM cells or in vitro expanded cells were infused via the tail vein. Mice were then maintained with gentamycin sulfate-containing water until further experiments. The homing efficiency of the transplanted cells was estimated by flow cytometry. For limit dilution assays, serial numbers of uncultured CD34+ UCB-derived cells or a fraction of the final culture equivalent to starting cells (103, 6 × 103, 10 × 103 cells) were transplanted. Positively engrafted mice were regarded as to have ≥0.5 % of human CD45+ (hCD45+) cell in peripheral blood 12 weeks after the transplantation. The percentage of the negatively engrafted mice was plotted against the initial numbers of cells infused. SCID-repopulating cell (SRC) frequency was estimated by using the L-Calc software (STEMCELL Technologies) as the cell number by which 37 % of the mice were non-engrafted. For secondary engraftment, 50 % of the bone marrow (from both femurs and tibiae) from each recipient mouse (10 × 103 cells group) was transplanted into one secondary sublethally irradiated mouse. 12 weeks after transplantation, bone marrow was harvested from the secondary mice and analyzed by flow cytometry. All animal experiments were approved by the Animal Experiment Administration Committee of the Fourth Military Medical University.
Chemotaxis experiments were performed in polycarbonate transwell inserts (5-mm pore, Corning Costar Corp.). Migration medium with 100 ng/mL SDF-1 (Peprotech) was added in the lower chamber. Freshly purified CD34+ cells or cells expanded ex vivo (2 × 105) were seeded in the upper compartment. After 4 h of incubation at 37 °C, migrated cells in the lower chamber were enumerated by flow cytometry. The percentage of migration was calculated by dividing total cells migrated to the lower well by the cell input multiplied by 100.
Peripheral blood analysis
Retro-orbital peripheral blood was collected using microhematocrit capillary tubes (Sanger Biotech, China). Differential blood counts were obtained using PE-6000 Hematology Analyzer (Prokan Electronics Inc., China).
Tissues were fixed in 4 % paraformaldehyde overnight. Femurs were decalcified for 21 days using an EDTA decalcifying solution for paraffin section. H&E staining of bone sections were performed using standard procedures. Images were taken under a fluorescence microscope (Olympus BX51, Japan).
Data were analyzed with the SPSS 17.0 software. Comparisons between groups were undertaken by using the unpaired Students’ t test. Results were expressed as the mean ± standard deviation (SD). P < 0.05 was considered statistically significant.