In vitro hematopoietic differentiation. (A) Clonogenic potential of murine KL and human AFKL cells. The number of total CFUs obtained 14 days after seeding 105 candidate cells in 7 independent experiments is shown, together with the relative percentages of the different types of colonies generated. Representative images of day 14 mKL cells and hAFKL cell–derived hematopoietic BFU-E, CFU-M, and CFU-GEMM cells (cultured in semisolid methylcellulose-based medium and supplied with hematopoietic cytokines) are shown. (B) Representative results from 1 of 6 myeloid differentiation experiments performed with murine and human KL cells (left and right panels, respectively). Cells were stained with specific mAbs for murine Gr1 myeloid antigen and human CD13 and CD33. (C) Surface phenotype analysis of T- and B-cell differentiation of murine KL cells assessed with OP9-mDelta1 coculture of 5000 candidate cells (left panel). Single-cell suspensions were obtained after 10 days (CD4/CD8), 15 days (CD3/TCRγδ), and 20 days (CD3/TCRαβ) of T-cell differentiation or after 10 days (CD19/IgM) of B-cell differentiation. A forward-scatter/side-scatter gate was set to eliminate stromal cells from the analysis. Only GFP+ 7-AAD− viable cells were taken into account. The results shown are representative of 6 independent experiments performed with donors at various ages of gestation (E11.5, n = 1; E12.5, n = 2; E13.5, n = 1; E14.5, n = 1; E15.5, n = 1; E19.5, n = 1). On the right panels, surface phenotype analysis of T- and NK-cell differentiation of hAFKL (from donors at 16, 18, 19, 21, and 23 weeks of amenorrhea) assessed with OP9-hDelta1 coculture of 5000 candidate cells. Human cells were recovered after 7 days (CD5/CD7), 15 days (CD4/CD8), and 25 days (CD8/CD16-CD56) of coculture. Representative results from 1 of 5 experiments are shown.
