Fetal CSF1R⁺ ProB cells possess B- and myeloid-lineage potential. (A) Response to CSF1L in single-sorted E13.5 ProB CSF1R⁺ (black) and CSF1R^– (dark gray) cells. Mean percentage (± SEM) of clones (3 experiments, 30-60 cells per population per experiment). (B-E) Mean (± SEM) mRNA expression levels of (B) myeloid genes; (C) B-cell genes; (D) early T-cell genes; (E) early lymphoid genes, as measured by RT-qPCR in CSF1R⁺ (black) and CSF1R^– (dark gray) E13.5 FL (n = 8 litters); and 8- to 10-week-old adult BM (light gray, n = 9 mice) ProB cells (25 cells/replicate, 3 experiments, 2-3 replicates per biological sample). Results are presented relative to Hprt1 expression. #Expressed but at too low a level to be visualized on the applied scale; n.d., Not detectable. Statistical significance was tested between CSF1R⁺ (black) and CSF1R^– (dark gray) ProB population. ***P < .001; **P < .01; *P < .05; ns, not significant. (F-G) Cells generated on OP9 stroma after 7 days from sorted E13.5 FL CSF1R⁺ and CSF1R^– (n = 8 litters), and 8- to 10-week-old adult BM (n = 6 mice) ProB cells (20 cells/replicate, 2-3 experiments, 4-10 replicates per biological sample). Mean percentage (± SEM) of wells with (F) B cell (Topro^–NK1.1^–B220⁺CD19⁺) and (G) myeloid (Topro^–NK1.1^–B220^–CD19^–GR1⁺MAC1⁺) cells. (H) T-cell generation on OP9–DL1 stroma after 14 to 16 days from sorted E13.5 FL CSF1R⁺ and CSF1R^– (n = 9 litters), and 8- to 10-week-old adult BM (n = 9 mice) ProB cells or E13.5 FL-lineage negative cells (n = 2 litters) (20 cells/replicate, 2 experiments, 3-9 replicates per biological sample). Mean percentage (± SEM) of wells with T (Topro^–NK1.1^–B220^–MAC1^–GR1^–CD25⁺THY1.2⁺ or Topro^–NK1.1^–B220^–MAC1^–GR1^–CD4⁺CD8a⁺) cells.