Isolation of b potential (mega and erythroid) precursors from the spleen of mice treated for i day with phenyl-hydrazine

The existence of precursors bipotent for the megakaryocytic and the erythroid differentiation lineage has been suggested by several lines of evidence. However, it is not clear if this precursor represents a stochastic subpopulation of all the mega and erythroid precursors or if it is an obligatory step of the two differentiation programs. In fact, although all the erythroid bursts and mega-karyocytic colonies (out of 70 single colonies investigated) express (by RT-PCR) at day 7 of culture both erythroid (β globin, EpoR) and megakaryocytic (AchE, GPnb, Mpl) markers, none of the colonies formed at day 3 from CFU-E (out of 30 single colonies analyzed) express megakaryocytic markers. In order to identify the frequency of the common precursor in the erythroid differentiation program, we have isolated cells from the spleen of anemic mice after 1 day of phenyl-hydrazine (PHZ) treatment. PHZ-cells synchronously progress in vivo and in vitro along the erythroid differentiation program. A fraction of PHZ-cells (25-45%) survives in culture for 18 hours in the absence of growth factors (GF). These cells are 4A5~ Ter-119~ (megakaryocyte and erythroid surface markers, respectively), benzidine' cells stopped along all the phases of the cell cycle which do not express levels of βglobin detectable by RT-PCR. We have characterized the differentiation potential of these cells by exposing them to either erythropoietin (Epo) or trombopoietin (Tpo) for up to 24 hrs. Both growth factors induce the cells to progress along the cell cycle and to accumulate in Go/Gt with no further proliferation. The cells exposed to either Epo or Tpo express similarly high level of βglobin and GPflB by RT-PCR with 60% of them co-expressing 4A5 and Ter-119 (by FACS analysis). These results suggest that the day 1 PHZ-cells which survive 24 hrs of GF starvation are highly enriched for the common mega and erythroid precursor which requires either Tpo or Epo for survival.