Figure 5. MiR-150 expression promotes myeloid differentiation of primary BC CML patient cells.

Primary BC CML patient samples were transduced with pre-miR-150 or empty control lentiviral (ECV) supernatants, sorted for GFP expression, and then assayed by flow cytometry for CD34 and CD11b expression or colony formation by colony-forming unit (CFU) assays. CD34 expression decreases with myeloid differentiation, whereas CD11b expression increases. (A) CD34+ cells from two BC CML patients were assessed for CD34 and CD11b expression 7 days after transduction in the presence of rhSCF, rhIL3, rhIL6, rhGCSF, and rhGMCSF (50 ng/ml each). Data are shown separately for each patient. Duplicates measurements are reported for patient 1 and triplicate measurements for patient 2. (B) BC CML progenitor cells from patient 1 were sorted and plated in triplicate 5 and 7 days after transduction in Methocult™ containing 20% lot-tested FBS, 10% BSA, and cytokines stated above. Colonies were counted 12-16 days after plating. CFU assays demonstrated decreased myeloid CFUs (CFU-M, CFU-G, and CFU-GM combined) in miR-150 versus control primary BC CML patient cells. Individual colonies were plucked, prepared by cytospin, and stained with Wright-Giemsa to validate morphology. A representative example of cells from a CFU-GM colony (from miR-150 expressing BC CML patient 1 cells) shows monocytes, bands, and granulocytes (top photo) and bands and metamyelocytes (bottom photo). BC CML progenitor cells from patient 2 were sorted and plated in triplicate 4 and 6 days after transduction on Methocult™ as above with the addition of EPO (2 U/ml). A similar decrease in myeloid CFUs was observed. Additionally, BFU-E and CFU-E were also decreased in numbers. The mean colony numbers from triplicate plates are shown; error bars indicate standard deviations (*P≤0.005 for comparisons, Student’s t-test).