Identification of two CD45R^- B lineage precursor populations

While murine stem cells and CD45R⁺ pro-B cells can be isolated for in vitro culture studies, precursors intermediate between these two stages have not been resolved on the basis of surface markers. Osmond and colleagues used intranuclear terminal deoxynucleotide transferase (TdT) in fixed and permeabilized cells to identify a CD45R^- ToT⁺ "early pro-B" population in bone marrow. We have now found two bone marrow populations highly enriched for CD45R^- B lineage precursors by correlating surface c-kit with intranuclear TdT in sorted bone marrow cells lacking lineage markers (Lin^-). Lin^-c-kit^Lo and Lin^-C-kit^- bone marrow includes almost all of the Lin^-TdT⁺ cells with TdT⁺ cells making up 35.5% and 7.4% of these populations respectively. When placed in culture, both Lin^-c-kit^Lo and Lin^-c-kit^- cells generated CD45R⁺ CD19⁺ pro-B cells and did so more rapidly than the stem cell enriched, Lin^-c-kit⁺ population. B lineage progeny from Lin^-c-kit^Lo and Lin^- c-kit^- cells exhibited a more mature phenotype (based on CD24 and BP-1) than those derived from Lin^-c-kit⁺ cells. The intermediate nature of the Lin^-c-kit^Lo and Lin^- c-kit^- populations was verified by the observation that Lin^-c-kit⁺ cells gave rise to a Lin^-c-kit^{- to Lo} population within 18 hours, and the latter differentiated to pro-B cells when sorted and recultured. Identification of these developmental intermediates now makes it possible to probe early differentiation events and the microenvironmental signals required to initiate them.