CD34+ cells isolated from the bone marrow (BM) of patient P3 were transduced with either two bicistronic lentivirus vectors (encoding AK2A and AK2B, respectively, together with a GFP reporter gene: AK2A+B+GFP) or a lentivirus encoding the GFP reporter only (GFP). As a positive control, cord-blood-derived CD34+ cells were transduced with GFP. After lentiviral transduction, 104 CD34+ cells from the BM of P3 were seeded in semi-solid medium for each condition, corresponding to 500 complemented cells (based on a transduction efficiency of 5%). As a control, 500 CD34+ cord blood cells were seeded in semi-solid medium (transduction efficiency of 35%).
(a) After 13 days, CFU-G/GM colonies were collected and cytospin preparations of each condition (P3 BM GFP; P3 BM AK2A+B+GFP; CB GFP) were analyzed by May-Grunwald/Giemsa staining. Representative pictures of various stages of neutrophil maturation are shown at magnifications of 50× (left panel) or 100× (right panel).
(b) Enumeration of myeloid subpopulations among CFU-G/GM colonies. Myeloid precursors (blasts, myeloblasts and promyelocytes) and differentiated myeloid cells (myelocytes, metamyelocytes and granulocytes) are represented in percentage and in absolute cell numbers.
(c) Representative flow cytometry analysis of CD15 and CD11b expression in CFU-G/GM colonies (left-hand dot plots) and on transduced GFP+ CFU-G/GM colonies (right-hand dot plots)
(d) Absolute number of double-positive CD15+CD11b+ labelled cells are indicated for each condition (left panel). The absolute number of GFP+ cells isolated from the CFU-G/GM colonies and from the CFU-M colonies are indicated (right panel).