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. 2013 Sep 24;8(9):e75815. doi: 10.1371/journal.pone.0075815

Figure 3. MiR-150 expression promotes myeloid differentiation of CD34+ PBMCs from healthy donors.

Figure 3

Primary CD34+ PBMCs were transduced with pre-miR-150 or empty control lentiviral (ECV) supernatants, sorted for GFP expression, and then assayed by flow cytometry for CD11b, CD34, and CD14 expression or colony formation by colony-forming unit (CFU) assays. (A) Cells were assessed by flow cytometry at the indicated days after cell transduction cultured in the presence of rhSCF, rhIL3, rhIL6, rhGCSF, rhGMCSF (50 ng/ml each), and EPO (2 U/ml). CD11b and CD14 expression increased in both patient samples in the miR-150 vs. control transduced cells, while CD34 did not significantly change. The means from combined patients each run in duplicate experiments are shown; the error bars represent standard deviations (*P≤0.05, for comparisons, Student’s t-test). (B) At day 14 after transduction, miR-150 or ECV transduced CD34+ PBSC were stained with Wright-Giemsa after cytospin preparations and showed morphological evidence of myeloid differentiation, including immature cells (large red arrows), intermediate differentiated cells (large black arrows), and mature myeloid cells (small black arrows). (C) Cells were sorted and plated in triplicate 3 days post transduction in Methocult™ containing 20% lot-tested FBS, 10% BSA, and cytokine concentrations as above. Colonies were counted 12-16 days after plating. The mean colony numbers from triplicate plates are shown for each patient sample (#1 or #2); error bars indicate standard deviations. MiR-150 transduced cells had significantly lower colony numbers compared to control transduced cells, with decreased erythroid colonies (BFU-E and CFU-E), decreased CFU-G and CFU-GM, but increased monocyte/macrophage colonies (CFU-M) (P≤0.05 for comparisons, Student’s t-test). (D) Expression of genes associated with myeloid differentiation was assessed in both CD34+ PBSC patient samples transduced with either miR-150 or ECV 9 days after transduction by QPCR. Relative fold-difference in expression for miR-150 vs. ECV cells is displayed for each patient sample; error bars represent standard deviations of technical duplicates.