Immature erythroblasts with extensive ex vivo self-renewal capacity emerge from the early mammalian fetus

Abstract

In the hematopoietic hierarchy, only stem cells are thought to be capable of long-term self-renewal. Erythroid progenitors derived from fetal or adult mammalian hematopoietic tissues are capable of short-term, or restricted (10(2)- to 10(5)-fold), ex vivo expansion in the presence of erythropoietin, stem cell factor, and dexamethasone. Here, we report that primary erythroid precursors derived from early mouse embryos are capable of extensive (10(6)- to 10(60)-fold) ex vivo proliferation. These cells morphologically, immunophenotypically, and functionally resemble proerythroblasts, maintaining both cytokine dependence and the potential, despite prolonged culture, to generate enucleated erythrocytes after 3-4 maturational cell divisions. This capacity for extensive erythroblast self-renewal is temporally associated with the emergence of definitive erythropoiesis in the yolk sac and its transition to the fetal liver. In contrast, hematopoietic stem cell-derived definitive erythropoiesis in the adult is associated almost exclusively with restricted ex vivo self-renewal. Primary primitive erythroid precursors, which lack significant expression of Kit and glucocorticoid receptors, lack ex vivo self-renewal capacity. Extensively self-renewing erythroblasts, despite their near complete maturity within the hematopoietic hierarchy, may ultimately serve as a renewable source of red cells for transfusion therapy.

The embryonic origins of erythropoiesis in mammals

“…[6][7][8]62 The second wave of hematopoiesis also includes multipotential, highly proliferative progenitors (HPP-CFC) identified in vitro. 62 HPP-CFC and more extensively self-renewing erythroid progenitors that expand in culture 63 are both found in the yolk sac at about the same time of development. These progenitors are maintained in culture under different growth factor and cytokine conditions, 62,63 however, and their lineage relationship in vivo (if any) is unclear.…”

“…62 HPP-CFC and more extensively self-renewing erythroid progenitors that expand in culture 63 are both found in the yolk sac at about the same time of development. These progenitors are maintained in culture under different growth factor and cytokine conditions, 62,63 however, and their lineage relationship in vivo (if any) is unclear. In addition, HPP-CFC lack HSC activity and are found in the yolk sac 12 to 24 hours earlier than the first newborn repopulating HSCs 64 Recently, it has been demonstrated that erythropoiesis in the fetal liver begins before its colonization by HSCs.…”

Neomorphic effects of the neonatal anemia (Nan-Eklf) mutation contribute to deficits throughout development

“…Erythroblasts were derived from wild-type and Nan/+ E12.5 fetal livers and depleted of adherent cells as previously described (England et al, 2011;Gnanapragasam et al, 2016). Briefly, cells were harvested, and plated on gelatin dishes for 2 days.…”

Kruppel-like transcription factors KLF1 and KLF2 have unique and coordinate roles in regulating embryonic erythroid precursor maturation

“…To study primitive erythroid progenitors, E8.25-E8.5 (6-12 somite pairs) implants including embryo and yolk sac were dissected in PB2, 32 and dissociated into single cells using 0.01% trypsin (Worthington Biochemical Corporation, NJ, USA) and mechanical trituration. Half of the cells from each implant were plated in 1% methylcellulose supplemented with 10% plasma derived serum (Animal Technologies, TX, USA), 5% protein-free hybridoma medium (Gibco/BRL), IL-3 (20 ng/mL), IL-6 (20 ng/mL), stem cell factor (60 ng/mL) (IL-3, IL-6 and SCF-Peprotech, NJ, USA), erythropoietin (2 U/mL), MTG and L-glutamine.…”