Cells and cell culture
Human cord blood (CB) cells from volunteer donors were obtained from the New York Blood Center (New York, NY) and Tokai Cord Blood Bank (Nagoya, Japan). CD34+ cells were purified as described previously [39
], by density gradient centrifugation using Ficoll-Paque Plus and then positive selection using the Miltenyi MACS CD34 Isolation Kit (Miltenyi Biotec). Cytokine-driven liquid (Delta) cultures were performed in QBSF-60 serum-free media (Quality Biological) containing 10 ng/mL recombinant human TPO (rhTPO), recombinant human SCF (rhSCF) and recombinant human Flt3 ligand (rhFlt3L). 293T cells were grown in modified Eagle medium (MEM) supplemented with 10% FBS and 2 mM L-glutamine. MS-5 murine stroma cells were grown in alpha MEM (α-MEM) supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 U/mL streptomycin. MS-5 stromal co-culture and long-term culture-initiating cell (LTC-IC) experiments were performed in α-MEM containing 12.5 % FBS, 12.5 % horse serum and 2 mM glutamine, 40μg/ml gentamicin, 250 μg/ml amphotericin B and 1 μM hydrocortisone.
Lentiviral vectors and reagents
The lentiviral vector FG12 (which expresses GFP under the Ubiquitin C promoter and an shRNA under the human U6-RNA Pol III promoter) was kindly provided by David Baltimore [40
]. To generate shRNA-expressing lentiviral vectors capable of targeting human cyclin C, cyclin D1, cyclin D2 or cyclin D3, oligonucleotides directed against cyclin C mRNA at nucleotides 468–486 and 582–600 (GenBank accession number gi: 61676090), cyclin D1 mRNA at nucleotides 5383–5401 (Gen Bank accession number gi: 166795258), cyclin D2 mRNA at nucleotides 1454–1472 (GenBank accession number gi: 16950656) and cyclin D3 mRNA at nucleotides 1585–1603 (GenBank accession number gi: 16950657) were subcloned into the FG12 vector (FG12 shCCNC, FG12 shCCNC2, FG12 shCCND1, FG12 shCCND2 and FG12 shCCND3, respectively). For the control shRNA, a shRNA against LacZ was used (FG12 shLacZ), with the target sequence being the 1915–1933 region of the bacterial galactosidase gene (LacZ; gtgaccagcgaatacctgt). Recombinant human SCF, interleukin (IL) -3, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF) and TPO were kindly provided by Kirin (Japan). Recombinant human granulocyte CSF (rhG-CSF) and erythropoietin (rhEpo) were obtained from Amgen, and Ortho Biotech, respectively. Recombinant human Flt3 ligand was purchased from Peprotech. An antibody against human cyclin C (sc-1061) was purchased from Santa Cruz. Fluorescence-conjugated antibodies to human CD34, CD44 and CXCR4 were purchased from BD Biosciences.
Lentiviral production and transduction into CD34+ cells
293T cells were used to produce lentiviruses by co-transfection of packaging plasmids and the FG12 lentiviral vector. Human CD34+ cells were cultured for 5 to 8 hours in the presence of rhTPO (10 ng/ml), rhSCF (10 ng/ml) and rhFlt3L (10 ng/ml) in QBSF-60 serum-free media, and then infected by spin-inoculation in the presence of 4 μg/ml of polybrene (Sigma), at 1300 rpm for 45 min two or three times with 12-hour intervals. Forty-eight hours following the last infection, GFP positive cells were purified by FACS and used for further experiments. For the transplantation experiments, only one round of transduction was performed to minimize the loss of stemness during the transduction period.
siRNA electroporation into CD34+ cells
A control non-silencing scrambled siRNA and an siRNA directed against human CCNC mRNA (siCCNC) were synthesized by the High-Throughput Drug Screening Facility at MSKCC (sequences available upon request). Nucleofection was performed using the Nucleofector™II kit (Amaxa), according to the manufacturer’s directions. Briefly, 1 × 106 CD34+ cells were mixed with 1.5 μg of siRNA and electroporated, using the U-08 program.
RNA isolation and Quantitative PCR
RNA extraction was performed using the RNeasy Plus kit (Qiagen). cDNA was synthesized using the SuperScript™ III First-Strand Synthesis System for RT-PCR kit (Invitrogen), according to the manufacturer’s instructions. Quantitative PCR was performed using an ABI 7500 instrument and analyzed using Sequence Detector Software (Applied Bioscience). The expression level for each gene was normalized to the level of hypoxanthine phosphoribosyltransferase (HPRT) expression. The PCR primer sequences used are available upon request.
Western blotting analysis
Cells were washed with phosphate-buffered saline (PBS), and lysed in lysis buffer (1% TritonX-100, 150 mM NaCl, 20 mM Tris (pH 8.0), 1 mM EDTA, and protease inhibitors). The protein lysates were briefly sonicated and centrifuged for 15 minutes at the maximum speed. The protein lysates were mixed with 4x SDS loading buffer, boiled for 5 minutes, and run on a 10 % NuPAGE Bis-Tris gel (Invitrogen, Carlsbad, CA). The protein was transferred to a polyvinylidene fluoride membrane, and the membrane blocked in 5% non-fat dry milk in PBS. The blot was incubated with a primary antibody, washed with PBS containing 0.05% Tween-20, and incubated with a secondary antibody conjugated to horseradish peroxidase (Amersham). The blot was developed using a chemiluminescent substrate and exposed on autoradiography film.
Hoechst 33342/Pyronin Y staining
Purified CD34+ cells were stained with 2.5 μM Hoechst 33342 (Molecular Probes) for 45 minutes at 37 °C. Pyronin Y was added at the final concentration of 1 μM and cells were further incubated for 45 minutes at 37 °C. Thereafter, cells were kept on ice for 30 minutes. Cells were washed 2 times and stained with APC-conjugated anti human CD34 antibody.
Ki-67 expression analysis
Forty-eight hours after nucleoporation, human CD34+ cells were collected, washed and resuspended in PBS containing 1% formaldehyde, and incubated for 30 minutes at 4 °C. An equal volume of PBS containing 0.2 % Triton X-100 was then added, and the cells were stored overnight at 4 °C. The cells were washed twice and incubated with FITC-conjugated anti-human Ki-67 monoclonal antibody (BD) for 30 minutes. The cells were then washed twice with PBS, resuspended and incubated at 4 °C in PBS containing 5 μg/ml 7-aminoactinomycin-D (7-AAD) for 3 hours.
Cell division tracking and Cell-cycle analysis
CD34+ cells were labeled with 2.5μM carboxyfluorescein diacetate succinimidyl ester (CFSE, Molecular Probes) in PBS for 10 min at 37 °C, as described previously. After a variable period of time in liquid culture, the cells were analyzed for fluorescence intensities of CFSE using flow cytometry.
For propidium iodide (PI) staining, cells were centrifuged and fixed in 70 % ethanol for 2 hours. Cells were washed twice with PBS containing 2 % BSA and finally resuspended in PBS containing 2 % BSA, 140 μg/ml RNase A and 50 μg/ml PI.
Colony-forming unit (CFU) assay
CFU assays were performed in semi-solid medium as previously described [39
], with modifications. Briefly, 500 sorted cells were plated in 35-mm plates using 0.9 % methylcellulose containing 20 % BIT9500 (Stem Cell Technologies), 2 mM glutamine, 50 ng/mL rhSCF, 20 ng/mL rhIL-3, 20 ng/mL rhIL-6, 20 ng/mL rhG-CSF, 20 ng/mL rhGM-CSF and 6 U/mL rhEpo. Colonies were scored 14 days after plating. For secondary CFU assays, cells were harvested and washed with PBS on day 14 after the first plating and 5 × 104
cells were replated as described above.
The Delta assay is a short-term suspension culture based assay used to detect hematopoietic stem/primitive progenitor cells. Cells with colony-forming unit potential are first grown in liquid culture before they are replated in semi-solid medium. The numbers of colonies obtained are considered an indicator of the number of hematopoietic stem/progenitor cells present. For the Delta assay, 5 × 103 cells growing in liquid culture were plated on methylcellulose, and colonies were scored on day 14.
We performed CFU assays using bone marrow cells obtained from mice transplanted with human CD34+ cells, by plating 5 × 104 cells in methycellulose containing rhSCF and rhIL-3 (which do not cross-react with murine receptors) and rhEPO. Colonies were scored on day 14.
Cobblestone area–forming cell (CAFC) and long-term colony-initiating cell (LTC-IC) assays
CAFC assays were performed by plating CD34+ cells onto MS-5 stroma cells. Weekly demidepopulation was performed, with phenotypic analysis of the non-adherent cells. Cobblestone areas, defined as groups of at least 5 phase-contrast dark cells tightly associated beneath the MS-5 monolayer, were scored at week 5.
Limiting dilution LTC-IC assays were performed by plating sorted FG12 shLacZ and FG12 shCCNC transduced CD34+ cells in 96-well tissue-culture plates containing an MS-5 cell monolayer, at 6 progressive dilutions with 10 replicate wells per dilution. After 5-weeks of co-culture, the cells were harvested and plated in methylcellulose; CFUs were counted on day 14. The frequency of LTC-ICs was calculated using L-calc™ software (Stem Cell Technologies).
Transplantation into NOD/SCID IL-2Rγ null (NOG) mice
NOG mice were purchased from Jackson Laboratories and bred in the SKI core animal facility. Mice between 8 and 12 weeks old were irradiated with a single dose of 3.5 Gy. 1 × 105 transduced human CD34+ cells were injected into the mice via their tail vein. After 6 weeks, bone marrow cells were harvested and analyzed for human CD45 expression, to define the engraftment efficiency.
Results obtained from multiple experiments are expressed as the mean ± standard deviation (SD), except as otherwise noted. The mean ± standard error of the mean (SEM) is used for describing the chimerism of human CD45+ cells in the transplanted NOG mice. The data were analyzed using a two-tailed unpaired Student’s t-test. Probability values < .05 defined significant differences between test points