Phenotypic and functional analysis of CD43⁺ subsets. (A) CFC potential of FACS-sorted CD43⁺ subsets on day 6 and day 9 of hESC/OP9 cocultures. CFC-E/Mks and CFC-GEMM/GM/Ms were determined by serum-free MegaCult collagen assay and FBS-containing MethoCult GF+ methylcellulose assay, respectively. Results are the mean ± SD from 9 independent experiments (H1, n = 6; H9, n = 3). NA indicates not applicable (subset was not detected/sorted). Photographs show typical E and small Mk colonies detected in CD43⁺CD41a/CD235a⁺CD45^–/+ subsets (i, scale bar represents 200 μm; inset shows Mk colony stained with anti-CD41a mAb, scale bar represents 50 μm) and multilineage GEMM (ii, scale bar represents 200 μm) and large Mk colonies (iii, scale bar represents 50 μm) detected in CD43⁺CD41a/CD235a^–CD45^–/+ subsets. Images were captured with an inverted DMIRB microscope (Leica Microsystems) equipped with a 5×/0.12 (i-iii) or a 20×/0.3 (inset) objective lens, and were acquired with a MagnaFire camera and software (Optronics). (B) qRT-PCR analysis of FACS-sorted CD43⁺ subsets on day 8 of H1/OP9 coculture. The stacked bar graph shows expression levels of indicated transcripts represented by relative units (see “Materials and methods” for details). Results are the means of 2 independent experiments. Representative agarose gel electrophoresis of qPCR products is shown. (C) FACS analysis of CD43⁺ subsets. CD43⁺ cells were isolated on day 8 of H1/OP9 coculture by direct CD43 MACS microbeads. Color-matching combinations of CD43, CD41a, CD235a, CD45, and indicated mAbs were used for CD43⁺ subset gating and analysis. Plots show isotype control (open) and specific mAb (tinted) histograms. Values within plots indicate ΔMFI values. Representative analysis of 3 independent experiments is shown. (D) Lymphoid and myeloid differentiation of FACS-sorted CD43⁺ subsets in coculture with MS-5 stromal cells. CD43⁺ subsets were isolated on day 8 of hESC/OP9 cocultures and cultured with MS-5 cells in presence of cytokines supporting either lymphoid or myeloid differentiation (see “Materials and methods” for details). Lymphoid MS-5 cultures were examined for expression of NK cell (CD3E, CD3Z) and B-cell (MB1, VPREB, PAX5) specific transcripts by qRT-PCR on the fourth week of culture. The relative expression of each GAPDH-normalized target gene was calculated in comparison with isolated CD43⁺ cells before coculture (MB1, very low levels of CD3E/Z, but no detectable VPREB and PAX5 were found in CD43⁺ cells before coculture). Results are the mean ± SD from 3 independent experiments with H1 (n = 2) and H9 (n = 1) cells. A representative agarose gel of qPCR products is shown. Myeloid MS-5 cocultures were examined for total CD43⁺ cells and myeloid CFCs (GM/M) during 6 weeks of culture. Results are the means ± SD from 4 independent experiments (H1, n = 2; H9, n = 2).