Role of miR-29a and miR-142-3p in CD34⁺ HSPCs derived from UCB. CD34⁺ cells purified from human UCB samples were infected with Lenti-control, Lenti-29a, or Lenti-142. Cells were cultured for 17 days. Granulocytic differentiation was induced by human G-CSF (20 ng/mL) and rhIL-6 (10 ng/mL), and monocytic differentiation by rhM-SCF (50 ng/mL), rhIL-6 (1 ng/mL), and rhFlt-3L (100 ng/mL). Cells were collected for morphologic, colony formation, and myeloid marker expression analysis. (A) Real-time PCR analysis of miR-29a and miR-142-3p expression during differentiation. Samples were collected at the indicated days after induction. The relative expression of miR-142-3p and miR-29a was normalized to the expression level at day 0. (B) CD14 mRNA levels for monocytic differentiation and CD11b mRNA levels for granulocytic differentiation were analyzed by real-time PCR. (C) Colony formation assay after an 11-day colony formation period. A total of 1 × 10⁴ of infected CD34⁺ cells were plated in complete methylcellulose medium without erythropoietin. The arrows indicate typical CFU-GM (colony-forming unit-granulocyte/macrophage), CFU-G (colony-forming unit-granulocyte), and CFU-M (colony-forming unit-macrophage; left). Quantification of formation of blood colonies was shown (right). Images were captured with microscopy Nikon eclipse TS100 using 10× phase-contrast objective with numeric aperture 0.3 and acquired through CCD DS-Qi1 camera and NIS-Elements F Version 3.0 software (Nikon) at room temperature. (D) Changes in the morphology of May-Grünwald-Giemsa–stained CD34⁺ cells infected with the viruses at day 13 of the differentiation culture. Images were captured at room temperature with Olympus BX51 microscope using 20× with numeric aperture 0.5. (E) The number of cells in various differentiation stages. In total, 100 cells were counted per sample. The averages from 2 independent experiments are shown. MB indicates myeloblasts; PM, promyelocytes; MM, metamyelocytes; Band and Seg, band neutrophils and segmented neutrophils for granulocytic differentiation; Mob, monoblasts; PMo, promonocytes; Mo, monocytes; and Ma, macrophage for monocytic differentiation.