An optimized system for studies of EPO-dependent murine pro-erythroblast development

Objective. Objectives were to develop new means to isolate useful numbers o f primary progenitor cells and to quantitatively assay the stepwise maturat ion of erythroblasts. Methods. Approaches involved dosing mice with thiamphenicol (TAP) to yield staged cohorts of pro-erythroid cells; optimizing conditions for their EPO- dependent in vitro growth and survival; developing assays for CFU-E maturat ion; analyzing stage-specific transcript expression; and expressing a heter ologous, erythroid-specific tag (EE372) in transgenic mice. Results. Per TAP-treated mouse, 3 x 10(7) highly EPO-responsive erythroid p rogenitor cells were generated that represented up to 30% of total splenocy tes and showed strict dependence on EPO for survival, growth, and immediate response gene expression. In this developing cohort, a tightly programmed sequence of gene expression was observed, and maximal expression of c-kit, EPO receptor, and beta -globin transcripts occurred at 72, 96, and 120 hour s post-TAP withdrawal, respectively. Also, the newly discovered erythroid-s pecific dual-specificity kinase, DYRK3, was revealed to be expressed at a l ate CFU-E stage. In vitro, these progenitor cells matured stepwise from hig h FALS Ter119(-) cells (24-hour culture) to high FALS Ter119(+) cells (24-3 6 hours) to low FALS Ter119(+) maturing erythroblasts (40-48 hours) and sha rp differences in their morphologies were observed. Finally, a MACS-based p rocedure for the purification of erythroid progenitor cells from TAP-treate d EE372 transgenic mice also was developed. Conclusions. A comprehensive new system for isolating large numbers of prim ary murine erythroid progenitor cells and quantitatively monitoring their d evelopment is established that should serve well in investigations of endog enous and pharmacological regulators of red blood cell development. (C) 200 1 International Society for Experimental Hematology. Published by Elsevier Science Inc.