A hyperactive Mpl-based cell growth switch drives macrophage-associated erythropoiesis through an erythroid-megakaryocytic precursor

Abstract

Several approaches for controlling hematopoietic stem and progenitor cell expansion, lineage commitment, and maturation have been investigated for improving clinical interventions. We report here that amino acid substitutions in a thrombopoietin receptor (Mpl)- containing cell growth switch (CGS) extending receptor stability improve the expansion capacity of human cord blood CD34+ cells in the absence of exogenous cytokines. Activation of this CGS with a chemical inducer of dimerization (CID) expands total cells 99-fold, eryth-rocytes 70-fold, megakaryocytes 0.5-fold, and CD34+ stem/progenitor cells 4.4-fold by 21 days of culture. Analysis of cells in these expanded populations identified a CID-dependent bipotent erythrocyte-megakaryocyte precursor (PEM) population, and a CID-independent macrophage population. The CD235a+/CD41a+ PEM population constitutes up to 13% of the expansion cultures, can differentiate into erythrocytes or megakaryocytes, exhibits very little expansion capacity, and exists at very low levels in unexpanded cord blood. The CD206+ macrophage population constitutes up to 15% of the expansion cultures, exhibits highexpansion capacity, and is physically associated with differentiating erythroblasts. Taken together, these studies describe a fundamental enhancement of the CGS expansion platform, identify a novel precursor population in the erythroid/megakaryocytic differentiation pathway of humans, and implicate an erythropoietin-independent, macrophage-associated pathway supporting terminal erythropoiesis in this expansion system.