Figure 2.

Men1^−/− ES cell lines are equally efficient in early differentiation. A) Men1^−/− ES cell lines are similar to wildtype cells in forming embryoid bodies (EBs). Parental TC1 and the three null ES cell lines, 3.2N, 173N and 179N, were plated for the initiation of the differentiation process, and the total numbers of EBs formed 10 days after plating were counted and represented as a percentage relative to TC1; TC1 is indicated as 100% (total EBs). The numbers of EBs that showed blood islands, hematopoietic EBs, were also counted (Hemat. EBs), and presented as a percentage of the total EBs. The results from four independent experiments are summarized here. A knock-in mutant ES cell line, KIKL55, which has a Cbfb allele replaced with the leukemia fusion gene Cbfb-MYH11, is used as a control in all the in vitro differentiation experiments, as it is described to be competent to form EBs but not differentiated hematopoietic colonies (Castilla et al., 1996). B) Loss of menin does not affect the expression of early hematopoietic markers Flk-1 and c-Kit. Single cell suspensions of 6-day EBs were made after harvesting them from methylcellulose culture and dissociating with trypsin/EDTA. 1 × 10⁶ cells were labeled with fluorescently tagged antibodies for Flk-1 or c-Kit (BD Pharmingen) and analyzed on a FACS Calibur (Becton Dickenson). The numbers in the left corner indicate the percentage of positive cells for each marker. C) The percentage of cells that express both CD41 and c-Kit is unaffected by the loss of menin. Representative data for a null (3.2N) and the wild-type (TC1) cell lines are shown. The numbers in the upper-right (boxed) corner indicate the percentage of cells positive for both CD41 and c-Kit. Flow cytometric analysis of day 6 EBs from the wildtype TC1 and the three Men^−/− cells show a similar fraction of cells expressing the cell surface markers of hematopoietic differentiation.