Figure 3.

ESAM-Null HSCs Exhibited Functional Disruption of Differentiation in Culture

(A) The sorted LSK CD48^– cells of E14.5 WT or ESAM Homo KO littermates were cultured in methylcellulose medium. The number of granulocyte colony-forming units (CFU-G), macrophage colony-forming units (CFU-M), granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E), or mixed erythroid-myeloid colony-forming units (CFU-Mix) are shown (n = 3, each group).

(B) The sorted LSK CD48^– cells of E14.5 WT or ESAM Homo KO littermates were cocultured in BM stromal cell lines (MS-5), under appropriate conditions to produce erythroid cells. After 8, 11, and 14 days of culture, cells were collected and analyzed by fluorescence-activated cell sorting (FACS). The numbers of Ter119⁺ erythroid cells are shown over time (n = 4, each group).

(C) The mRNA expression levels of Hba, Hbb-b1, and Alas2 in the BFU-E colonies analyzed by qRT-PCR (n = 15, each group).

(D) Sorted LSK cells of E14.5 WT or ESAM Homo KO littermates (100 cells/well) were cocultured with MS-5 under conditions to produce B lymphoid and myeloid cells. After 10 days of culture, cells were collected and analyzed by FACS. The numbers of CD19⁺ B lymphoid cells and Mac1⁺ myeloid cells are shown (n = 4, each group).

(E) FL LSK CD48^– HSCs collected at E14.5 from WT or ESAM Homo KO fetuses were subjected to limiting dilution analyses in the MS-5 coculture system. Input cell numbers corresponding to 37% negative value are shown in rectangles.

Data are shown as means ± SEM. Statistically significant differences are represented by asterisks: ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.