Fig. 4: Lineage restriction events during erythropoiesis reflect the sequence of regulatory programs.

a Schematic diagram of lineage potential assays during the first 7 days of ex vivo erythropoiesis. Cells were sampled daily and transferred to lineage-permissive media. Multilineage capacity was determined as frequency of CFU-GEMM progenitors. Erythroid potential as frequency of BFU-Es and megakaryocytic potential as frequency of CD41⁺ cells. b Frequency of multipotent CFU-GEMM in methylcellulose assay from cells sampled over the course of erythroid differentiation. c Frequency of unipotent erythroid progenitor colonies (BFU-E) in methylcellulose assay (red line) and frequency of CD41⁺ cells after transplantation in secondary megakaryocytic media (blue line). d Changes in lineage potential coincide with the transitions between the regulatory modules identified earlier. Transition from modules 1 and 2 to module 3 reflects the loss of multipotency occurring between days 3 and 4, while transition from module 3 to erythroid modules 4 and 5 coincides with the depletion of unipotent progenitors and entry to erythroid maturation (days 5 to 6). Data represent mean values. Error bars denote ±1 SE of the mean from n = 4 experiments. Asterisks denote statistically significant difference in CFU-GEMM and BFU-E counts from day 1 (two-sided Student’s t test p-value <0.05). CFU-GEMM Colony Forming Unit - Granulocytic, Erythroid, Macrophage, Megakaryocyte. BFU-E Burst Forming Unit-Erythroid.