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

Abstract

• Increasing receptor stability of an Mpl-based cell growth switch improves ex vivo expansion from cord blood CD34 1 cells.• Expansion includes Epoindependent, macrophageassociated erythropoiesis from a novel erythroidmegakaryocytic precursor population.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 ( /CD41a1 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 1 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 erythropoietinindependent, macrophage-associated pathway supporting terminal erythropoiesis in this expansion system. (Blood. 2015;125(6):1025-1033

Cytokine-free rapid red cell regeneration

“…1 T he properties of the cells they describe resemble those of cells previously observed during stress erythropoiesis in mice, also initially reported in Blood, 2,3 suggesting the existence of a novel cellular response to erythropoietic stress in both mice and humans.…”

“…1 Whereas smaller VWF multimers, especially dimers, tetramers, and hexamers, enhance C3b inactivation by CFI, large and unusually large VWF multimers are devoid of this cofactor activity and, therefore, they enhance complement activation by the alternative pathway C3 convertase, C3bBb.…”

“…Further understanding of the mechanisms controlling HbF and F cell numbers could impact the treatment of sickle cell disease and b-thalassemia, where increased HbF and F cell numbers are beneficial. One of the potentially promising applications of the current findings is to exploit the expansion of erythroid cells from bipotent PEM progenitors free of exogenous cytokines in culture using the CGS-CID system as described by Belay et al 1 Currently, scaling up to produce 1 unit of packed cultured red blood cells (RBCs) from human peripheral blood CD34 1 cells is feasible but is limited by excessive production costs, which are largely due to the requirement for exogenous cytokines. 8 Therefore, exogenous cytokine-free ex vivo-cultured human RBC production would enable a significant reduction in costs.…”

GATA1 insufficiencies in primary myelofibrosis and other hematopoietic disorders: consequences for therapy

“…We then demonstrated that the bone marrow and spleen from Gata1 low mice do not contain stress-specific progenitor cells but a “unique erythroid” precursor with the morphology of erythroblasts but still capable to mature within 24 hours into megakaryocytes in response to thrombopoietin (TPO) [78,79]. The phenotype of this precursor is similar to that of a bipotent erythroid-megakaryocyte precursors specifically generated in vitro by human CD34 + cells in response to a hyperstimulator of MPL, the receptor for TPO [80], suggesting that chronic stress may activate the TPO/MPL axis. In agreement with this hypothesis, numerous attempts to generate Gata1 low mice lacking Mpl were consistently unsuccessful (ARM, unpublished observations).…”

Novel targets to cure primary myelofibrosis from studies on Gata1low mice

“…23,24 These precursor cells are similar to those observed in vitro when human CD34+ cells are cultured with a recombinant hyperactive TPO mimetic. 25 Proof that almost all the red blood cells circulating in the blood of Gata1 low mice are generated by the spleen is provided by the observation that splenectomy induces death of the mice by profound anemia within 2 weeks. 26 In addition, in Gata1 low mice almost all the hematopoietic stem cells are present in their spleen.…”