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, erythrocytes 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 high-expansion 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.