Supplemental Methods
Generation and expression of retrovirus
The PINCO expression vector co-expressing AML1-ETO and green
fluorescent protein (GFP) from an internal CMV promoter was created as previously
described.(14;15)
Replication-defective
retrovirus
was
generated
by
transient
transfection of Phoenix packaging cells (gift of Garry Nolan, Stanford University
School of Medicine, CA). Expression of the AML1-ETO transduced gene in myeloid
cells was performed by RT-PCR analysis as previously described.(14)
Generation of human CD34+ cells expressing AML1-ETO
Neonatal cord blood was obtained from normal full-term pregnancies at the
Maternity Unit of the University Hospital of Wales, Cardiff, U.K. Human CD34+ cells
(>95% pure) were derived from cord blood mononuclear cells using MiniMACS
(Miltenyi Biotec, Surrey, U.K.) according to the manufacturer's instructions. These
cells were subsequently cultured overnight at 1105 cells/ml in IMDM containing 20%
FCS and the following growth factors (from R&D Systems, Abingdon, U.K.): IL-3 (5
ng/ml), IL-6 (10 ng/ml), SCF (20 ng/ml), GM-CSF (5 ng/ml), G-CSF (5 ng/ml), Flt3
ligand (5 ng/ml). On the following day cells were transduced with retrovirus on
RetroNectin (Takara Shuzo, Shiga, Japan)-coated wells as previously described.(14)
In this way two cultures were derived from each CD34 + preparation: control
(expressing GFP alone) and AML1-ETO transduced. Following infection, cultures
were maintained in growth medium containing 5 ng/ml of IL-3, SCF and G/GM-CSF.
Colony assays were initiated on days 3, 6, 9 and 16 by limiting dilution in 96U plates
(0.3 cells/well) in the same liquid medium containing IL-3, SCF and G/GM-CSF and
incubated at 37oC with 5% CO2. Individual colonies (>50 cells) were harvested after 7
or 14 days following plating and were scored and analysed for differentiation (see
“Cell surface phenotype and differentiation analysis”).
Cell surface phenotype and differentiation analysis
At the time-points indicated, liquid cultures were analysed by four-colour
immunophenotypic
analysis.
Cells
were
stained
with
CD13-APC
(Leinco
Technologies, St Louis, MO) in combination with M-CSF-R-biotin (Santa Cruz
Biotechnology Inc, Santa Cruz, CA) and one of the following PE-labelled antibodies
(Dako, Ely, U.K.): CD11b, CD14, CD19, CD34 or CD56; biotinylated M-CSF-R was
subsequently labelled with Streptavidin-PerCP-Cy5.5 (BD Biosciences, Oxford, U.K.).
Individual colonies were aspirated and stained with CD15-biotin (Sigma, Poole,
Dorset, U.K.) and CD14-APC (Leinco); biotinylated CD15 was subsequently labelled
with Neutravidin-PE (Molecular Probes, Eugene, OR). Colonies were identified as
monocytes (CD15lo, CD14hi) or granulocytes (CD15hi, CD14lo). For the analysis of
intracellular myeloperoxidase (MPO), control and AML1-ETO transduced cultures
were purified by cell sorting based on GFP positivity. Sorted cells were stained for
MPO using Fix & Perm® (Caltag, Towcester, U.K.) according to the manufacturer’s
instructions in combination with a PE conjugated antibody to MPO (Dako). All
reactions were controlled with the appropriate isotype-matched irrelevant antibody.
Incubations were carried out at 4oC for 30 minutes in the presence of 0.5% human
gamma globulin (Sigma). Reagent concentrations were as recommended by the
manufacturer. Data acquisition and analysis is described below.
Data analysis
Flow cytometric data (acquired using a FACSCalibur® cytometer, BD) were
analysed using WinMDI (Joe Trotter, Pharmingen, San Diego, CA). The threshold for
GFP positivity was determined from the autofluorescence of GFP negative cells in
mock transduced cultures; for antibody labelled cells, this was determined from
control stained cells (in each case, set to 95% of controls). Significance of difference
was tested using the Students t-test. Minitab software version 12.0 (Minitab Inc, State
College, PA) was used for all analyses.