Definitive erythroid cell differentiation

When the mES cells have been cocultured with OP9 cells for 8 d in total in the presence of the appropriate growth factors and cytokine (firstly VEGF and BMP-4, and subsequently SCF, Epo, and IL-3), OP9 cells and adherent mES-cell derivatives are harvested and reseeded onto fresh monolayers of OP9 cells. The mES-cell derivatives now recommence proliferation and differentiation on the OP9 cells. Around day 12 of coculture, adherent cells start forming ostensible haematopoietic colonies as denoted by their cobblestone appearance (Figure 2C, E). Simultaneously, large numbers of cells detach from the stromal cell layer into the supernatant medium. We have ascertained that the majority of these non-adherent cells are definitive haematopoietic cells, which include mature (enucleated) erythrocytes (eryD) and cells of the myeloid lineage (16). Cell-specific features are revealed by May-Griinwald/Giemsa staining (Figure 4A); and expression of lineage-specific cell-surface antigens may be analysed by using a fluorescence-activated cell sorter (FACS1) (Figure 4B-O and Figure 5).

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Figure 4 (see Plate 3) Diversity of definitive haematopoietic cells obtained using the mES/ OP9-cell coculture system on day 12 of differentiation. For (A) to (O), non-adherent cells in the supernatant medium, representing the mature fraction of definitive haematopoietic derivatives, were harvested on day 12 of differentiation, as described in Protocol 4. (A) May-Grunwald/Giemsa staining of definitive haematopoietic cells. Both erythroid and myeloid lineages are detectable according to the morphological criteria provided in Protocol4. Eb, indicates an erythroblast; Po, late polychromatic normoblast; Re, reticulocyte; Ne, neutrophil granulocytes; Eo, eosinophil granulocytes; and Ma, macrophages. Scale bar: 25 mm. (B)-(O) Flow cytometric analysis of lineage-specific cell-surface antigens expressed by definitive haematopoietic cells. Cells were analysed for forward light scatter (FSC) versus side light scatter (SSC), revealing that three distinct populations (R1, R2, and R3) were present in the culture. (C) For the following FACS® analyses, haematopoietic cells were incubated with isotype-matched fluorochrome-conjugated antibodies, or lineage-specific fluorochrome-conjugated antibodies. Percentages of positive cells in three quadrants are indicated. Haematopoietic cells incubated with test antibodies were then gated with three distinct areas (for R1, R2, and R3 populations), and expression of c-Kit and lineage-specific markerswasexamined: for population R1, see panels D, G, J, and M; for R2, panels E, H, K, and N; and for R3, panels F, I, L, and O. Cells positive for the erythroid-specific marker, TER119, were detectable in populations R1 and R2 (at 36% and 33%, respectively). In contrast, cells positive for the macrophage marker, Mac-1, and the neutrophil marker, Gr-1, were mainly detectable in population R3, and at high frequency (over 80%). Cells positive for the B-cell marker, B220, also were observed in populations R1 and R2, but at low frequency (below 2%). Cells positive for c-Kit represent either uncommitted progenitors or lineage-committed progenitors.

Primitive haematopoiesis

Definitive haematopoiesis

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