Isolation of lineage depleted cord blood mononuclear cells (HSPCs)
Umbilical cord blood was obtained after informed consent from the Royal London Hospital, London, UK in accordance with the local Research Ethics Committees guidelines. The mononuclear cells were separated by density gradient centrifugation and enriched for progenitor cells using human progenitor enrichment cocktail and Stem Sep column (Stem Cell Technologies, Vancouver, Canada). The resulting lineage depleted mononuclear cells (CBLin-) termed as Hematopoietic stem/Progenitor cells (HSPCs) were used for further experiments.
Quantitative real-time PCR was performed on sub-fractions of the hematopoietic cells to quantify the transcript level of Rho GTPases RHOA. Total cellular RNA was extracted using RNeasy kit (Qiagen, Crawley, UK) and reverse transcribed into cDNA with Superscript III reverse transcriptase (Invitrogen). Real-time PCR was performed with SYBR-Green (ABI Biosystems, Carlsbad, USA) in an ABI 7900HT (ABI Biosystems) real-time PCR machine. The specificity of the product was verified in a 2% agarose gel. The primers used were: RHOA forward 5′-CTGGTGATTGTTGGTGATGG-3′ and RHOA reverse 5′-GCGATCATAATCTTCCTGCC-3′ and GAPDH forward 5′- GGGAAGGTGAAGGTCGGAGT-3′ and GAPDH reverse 5′- GGGTCATTGATGGCAACAATA-3′.
The lentiviral vectors used for the study were based on pHRcPPT SIEW Sin vector with IRES regulating eGFP reporter gene. The vector contains SFFV (Spleen Focus Forming Virus)-LTR promoter and WPRE (Woodchuck Hepatitis Virus) element for post-transcriptional processing. RHOA constitutively active (RHOAV14) and dominant negative (RHOAN19) sequences were cloned from pBluescript vectors (kindly provided by Dr. Michael Way, Cancer Research UK, London, UK) by PCR using the primers F: 5′-GCGCGGATCCATGGCTGCCATCCGGAA-3′; R: 5′-GCGCGGATCCTCACAAGACAAGGCAAC-3′. The sequences were cloned into Topo Cloning vector (Invitrogen, Paisley, UK) and subcloned subsequently into SIEW by BamHI digestion. The orientation and the presence of mutation were confirmed by DNA sequencing. Lentiviral vector with only IRES GFP was used as experimental control.
Lentiviral production and concentration
Lentiviral particles were generated by transfecting the transfer plasmid into 293 T cells with the packaging plasmids pCMVR8.94 and envelope pMD.G as described previously . Viral supernatants were collected 48 and 72 hr after transfection and concentrated by ultracentrifugation.
Lentiviral transduction of lineage depleted cord blood mononuclear cells
Freshly isolated or frozen lineage depleted mononuclear cells were stimulated for 8 hr with cytokines hFlt3L (50 ng/ml), hSCF (50 ng/ml), hIL-6 (10 ng/ml) and hTPO (20 ng/ml). After stimulation, transduction of HSPC cells were performed by the addition of the lentivirus particles containing control, RHOAV14 and RHOAN19 at a multiplicity of infection (M.O.I) of 80 in the presence of polybrene (4 μg/ml). Sixteen hours after transduction, the cells were washed and used for further experiments.
Liquid culture, LTC-IC and CFU-C assay
CFU-C assay was performed for cells transduced with RHOA constructs in methylcellulose medium (Methocult H4434, Stem Cell Tech, Vancouver, Canada). Briefly, 1 × 103 cells were seeded in 35 mm culture dishes and incubated at 37°C, 5% CO2. GFP positive cell aggregates of more than 50 cells were counted as colonies at 14 days in an inverted fluorescent microscope (Leica, Switzerland) according to the colony morphology. Long-term culture-initiating cell assay (LTC-IC) was performed by plating 1 × 104 transduced cells on a monolayer of irradiated M2-10B4 cells and half-media replaced every 7 days. At the end of 5 weeks, the cells were collected, plated in methylcellulose medium for CFU-C assay and scored after 14 days. Liquid culture was performed to maintain the cells in progenitor stage by seeding the cells in serum free medium (Stem Cell Tech) containing hSCF (300 ng/ml), hFlt3L (300 ng/ml) and hTPO (20 ng/ml)and fresh media was added every 2–3 days.
In vitro transwell migration assay
Cord blood lineage depleted cells (HSPCs) were transduced with control, constitutively active RHOA (RHOAV14) or dominant negative RHOA (RHOAN19) and cultured in serum free medium supplemented with hSCF (300 ng/ml), hFLT3L (300 ng/ml) and hTPO (20 ng/ml) for 7 days. Cytokines were added every 2–3 days. 100,000 cells were seeded in the transwell chambers (5 μm pore size) coated with fibronectin. SDF1α was added to the lower well (125 ng/ml) and the cells were allowed to migrate for 4 hours. The migrated cells in the lower well was collected and enumerated by flow cytometry (LSR II, Becton Dickinson) with the counting beads (Molecular Probes).
Phenotype and cell cycle analysis
The transduced cells were identified by their expression of the reporter gene eGFP. Cell surface markers expression was determined by staining the cells with fluorescent conjugated antibodies and analyzed by flow cytometry. Cell cycle analysis was performed by fixing the cells with 2% paraformaldehyde and permeabilised with 0.1% Triton X-100. The cells were stained with anti-Ki67 conjugated with Alexa647 (Molecular Probes) and resuspended in 2% FCS containing DAPI (4′, 6-diamidino-2-phenylindole) and analyzed by flow cytometry.
Short-term homing experiment
RHOA transduced HSPC cells were expanded in serum free media containing stem cell factor (SCF, 300 ng/ml), FMS likle tyrose kinase ligand (FLT3L, 300 ng/ml) and thrombopoietin (TPO, 20 ng/ml) for one week with addition of growth factors every second day. The cells were washed and 0.5 × 105 transduced cells were injected intravenously in NOD/SCID/β2 mice. Bone marrow homing was analysed 24 hours post-injection by staining the bone marrow cells with antibodies against human CD45.
In vivo migration and xeno-transplantation assay
All in vivo experiments were performed in accordance with the UK Home Office regulations and Cancer Research UK guidelines. NOD/SCID/β2 microglobulin null mice were purchased from Charles River Laboratories, UK. Mice aged 8–12 weeks were irradiated sub-lethally with an irradiation dose of 375 cGy from a 137Cs source. For migration experiments, 0.1 × 105 transduced HSPCs cells were injected intra-bone into one of the hind limbs of the mice. Bones from each limb was collected separately after 7–8 weeks and analyzed for human cell engraftment by staining with antibodies against human CD45. For engraftment experiments, mice were intravenously injected with 0.5 – 1 × 105 transduced HSPCs. The animals were sacrificed 12 weeks after transplantation, femurs and tibiae were collected and the cells were flushed and analyzed for human engraftment by flow cytometry.
Statistical analysis was performed using One-way ANOVA test (SPSS software) and statistical significance in in vivo experiments were assessed using generalized linear models based on the negative binomial distribution (R software).