Erythropoietin-dependent Acquisition of CD71 ^hi CD105 ^hi Phenotype within CD235a ^- Early Erythroid Progenitors

The development of committed erythroid progenitors and their continued maturation into mature erythrocytes requires the cytokine erythropoietin (Epo). Here, we describe the immunophenotypic identification of a unique Epo-dependent colony-forming unit-erythroid (CFU-E) cell subtype that forms during early erythropoiesis (EE). This previously undescribed CFU-E subtype, termed late-CFU-E (lateC), lacks surface expression of the characteristic erythroid marker CD235a (glycophorin A) but has high levels of CD71 and CD105. LateCs could be prospectively detected in human bone marrow (BM) cells and, upon isolation and reculture, exhibited the potential to form CFU-E colonies in medium containing only Epo (no other cytokines) and continued differentiation along the erythroid trajectory. Analysis of ex vivo cultures of BM CD34 ⁺ cells showed that acquisition of the CD7 ^hi CD105 ^hi phenotype in lateCs is gradual and occurs through the formation of four EE cell subtypes. Of these, two are CD34 ⁺ burst-forming unit-erythroid (BFU-E) cells, distinguishable as CD7 ^lo CD105 ^lo early BFU-E and CD7 ^hi CD105 ^lo late BFU-E, and two are CD34 ^- CFU-Es, also distinguishable as CD71 ^lo CD105 ^lo early CFU-E and CD7 ^hi CD105 ^lo mid-CFU-E. The transition of these EE populations is accompanied by a rise in CD36 expression, such that all lateCs are CD36 ⁺ . Single cell RNA-sequencing analysis confirmed Epo-dependent formation of a CFU-E cluster that exhibits high coexpression of CD71, CD105, and CD36 transcripts. Gene set enrichment analysis revealed the involvement of genes specific to fatty acid and cholesterol metabolism in lateC formation. Overall, in addition to identifying a key Epo-dependent EE cell stage, this study provides a framework for investigation into mechanisms underlying other erythropoiesis-stimulating agents.