Stat3 inhibition differentially affects primitive erythroblast maturation in vitro. Erythroid progenitor assays were performed on single-cell suspensions of individual, dissociated E8.5 embryos ( A ) and bone marrow ( B , C ) cultured in methylcellulose with appropriate media and cytokine supplementation. EryP-CFC ( A ) colonies were scored after 5 days, d3 BFU-E ( B ) were scored after 3 days and CFU-E ( C ) were scored after 2 – 3 days of culture. Primitive erythroblast maturation cultures were performed on cells pooled from dissociated E8.5 embryos ( D ) while definitive cultures were initiated with ESRE ( E ). All cultures were treated with DMSO as a vehicle control or 100 μM Stat3 inhibitor, S3I-201. Liquid cultures ( D , E ) were pretreated with DMSO or S3I-201 for 2 hours prior to EPO stimulation. Definitive erythroblasts are cultured for 2 days, versus 4 days for primitive erythroblasts, because of their more rapid maturation in vitro and in vivo. Images are representative of primitive erythroblasts at days 1 and 4 of culture ( D ) and definitive erythroblasts at days 0 and 2 of culture ( E ).
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