Reconstitution of the Myeloid and Lymphoid Compartments after the Transplantation of Autologous and Genetically Modified CD34 Bone Marrow Cells, Following Gamma Irradiation in Cynomolgus Macaques - Genetic Engineering: Recent Developments in Applications - page 129

titer (IT), expressed as transducing units/ml, was calculated as: IT = %GFP+cells
× 4 × 105/100 × d.
Transduction of Immunoselected CD34 + cells
Following immunoselection, CD34+ cells were cultured in a proliferation me-
dium composed of Iscove's MDM supplemented with 1% bovine serum albu-
min (BSA), bovine pancreatic insulin (10 µ g/ml), human transferrin (200 µ g/
ml), 2-mercaptoethanol (10-4M) and L-glutamine (2 mM; Stemspan, Stem Cell
Technologies, Meylan, France). The medium was supplemented with 50 ng/ml
recombinant human (rh) SCF (Stem Cell Technologies, Meylan, France), 50 ng/
ml rh Flt3-L (Stem Cell Technologies, Meylan, France), 10 ng/ml rh IL-3 (R&D
Systems, Minneapolis, USA),10 ng/ml rh IL-6 (R&D Systems, Minneapolis,
USA) and 4 µ g/ml polybrene (Sigma, Saint Louis, USA) in plates coated with
retronectin (Cambrex Bio Science, Paris, France). The CD34+cells were then
transduced by 24 hours of coculture with the vector (multiplicity of infection
(MOI) = 100).
Myeloid Differentiation of CD34 + cells
Following the coculture of CD34 + cells with lentiviral vector, part of the cell cul-
ture was fixed in CellFix solution (Becton Dickinson, Erembodegem, Belgium)
for evaluation of the rate of transduction of undifferentiated CD34 + cells. Part
of the cell culture was cultured for 14 days in 35 mm Petri dishes containing
semi-solid medium (Methocult GF H4434, Stem Cell Technologies, Meylan,
France) composed of Iscove's MDM medium supplemented with 1% methylcel-
lulose, 30% fetal bovine serum, 10-4 M 2-mercaptoethanol, 2 mM L-glutamine,
50 ng/ml rhSCF, 10 ng/ml rhGM-CSF, 10 ng/ml rhIL-3 and 3 IU/ml rhEPO.
Cells were cultured at a density of 104 cells/ml (in triplicate) at 37°C, under an
atmosphere containing 5% CO2, to allow the myeloid differentiation of colony-
forming cells (CFC).
The remaining cells were cocultured in 96-well plates for 35 days at 37°C,
under an atmosphere containing 5% CO2, on a layer of stromal cells of the mu-
rine fibroblastic cell line M2-10B4, in a medium composed of α MEM supple-
mented with 12.5% horse serum (HS), 12.5% FBS, 2 mM L-glutamine, 10-4 M
2-mercaptoethanol, 0.16 M I-inositol and 16 µM folic acid (Myelocult H5100,
Stem Cell Technologies, Meylan, France) and 10-6 M hydrocortisone. Cells were
cultured at a concentration of 103 cells per well (24 wells per condition per mon-
key), to allow long-term culture-initiating cells (LTC-IC) to undergo myeloid
differentiation to generate progenitor cells or CFC. The CFC were cultured for 14