Differentiation of hESCs into monocytes/macrophages. (A) Description of the 3-step protocol designed to induce hESC differentiation into monocyte/macrophages: step 1: EB formation with mesoderm specification in the presence of BMP-4 and VEGFA; step 2: hematopoiesis induction in the presence of SCF, TPO, FLT3-L, and IL-3; and step 3: culture of CD14⁺ cells sorted between days 14 and 21 in the presence of M-CSF, GM-CSF, FLT3-L, and IL-3. (B) hESC-derived cells were characterized by cell surface marker analysis using flow cytometry at the indicated times of the culture. (C) Methylcellulose-based colony-forming assay: (→) typical primitive erythroid (Ery-P) and macrophage (Macro-P) colonies and definitive (BFU-E, CFU-G, CFU-GM, CFU-M, and CFU-GEMM) colonies. Original magnification was 20× (Ery-P and Macro-P) and 10× (definitive colonies). Cells were assessed for their ability to form erythroid and myeloid colonies in a standard methylcellulose assay at the indicated times of the culture. The middle panel shows the percentages of the indicated colonies. The right panel shows the absolute numbers of CFU-GM + CFU-M per 10⁵ total EB cells. Results are mean ± SD of 3 independent time-course experiments with methylcellulose assays performed in triplicate and colonies enumerated at days 9-10. (D) Proliferative potential of monocytic cells; the percentage of CD14⁺ cells was measured at different times during the culture and related to the total number of cells, including adherent, floating, and those from EB cells. (E) CD14⁺ cells from step 2 were sorted, cultured, harvested at different times, and stained with May-Grünwald-Giemsa solution. Typical morphologic features of monoblasts (top panel), monocytes (middle panel), and macrophages (bottom panel) are shown. Photographs were taken with a 63× objective.