Erythropoietin critically regulates the terminal maturation of murine and human primitive erythroblasts

Abstract

Primitive erythroid cells, the first red blood cells produced in the mammalian embryo, are necessary for embryonic survival. Erythropoietin and its receptor EpoR, are absolutely required for survival of late-stage definitive erythroid progenitors in the fetal liver and adult bone marrow. Epo-and Epor-null mice die at E13.5 with a lack of definitive erythrocytes. However, the persistence of circulating primitive erythroblasts raises questions about the role of erythropoietin/EpoR in primitive erythropoiesis. Using Epor-null mice and a novel primitive erythroid 2-step culture we found that erythropoietin is not necessary for specification of primitive erythroid progenitors. However, Epornull embryos develop a progressive, profound anemia by E12.5 as primitive erythroblasts mature as a synchronous cohort. This anemia results from reduced primitive erythroblast proliferation associated with increased p27 expression, from advanced cellular maturation, and from markedly elevated rates of apoptosis associated with an imbalance in pro-and anti-apoptotic gene expression. Both mouse and human primitive erythroblasts cultured without erythropoietin also undergo accelerated maturation and apoptosis at later stages of maturation. We conclude that erythropoietin plays an evolutionarily conserved role in promoting the proliferation, survival, and appropriate timing of terminal maturation of primitive erythroid precursors. Erythropoietin critically regulates the terminal maturation of murine and human primitive erythroblasts

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

“…Therefore the method of Malik et al was used to count nascent colonies on Day 2 of culture. 33 There are similar numbers of EryP-CFC in KLF1-/-KLF2-/-and KLF1+/-KLF2+/-on Day 2 ( Figure 5C). This suggests that normal numbers of primitive erythroid progenitors are specified in KLF1-/-KLF2-/-embryos, but the simultaneous ablation of KLF1 and KLF2 leads to a stochastic defect in maturation of erythroid precursors, leading to fewer colonies on Day 6.…”

“…Similar results have been reported by Malik et al for E10.5 erythroblasts. 33 The cell cycle distribution of KLF2-/-erythroblasts is similar to wild-type, suggesting that KLF2 ablation does not affect cell cycle progression. KLF1-/-and KLF1-/-KLF2-/-embryos have an approximately 20% reduction in cells in S-phase compared to wild-type, and a concomitant 2-fold increase in cells in G0/G1 phase, suggesting a block in S-phase entry.…”

“…The protocol was adapted from Malik et al 33 Briefly, E9.5 erythroblasts were cultured in erythroid maturation medium containing BrdU for 90 min, fixed, and stained with APCconjugated anti-BrdU antibody and 7-amino-actinomycin D (7-AAD). The BD FACSCanto™ II Analyzer was used to measure total DNA content and BrdU incorporation using 7-AAD and APC fluorescence intensity, respectively.…”

Placental expression of erythropoietin in preeclampsia

“…Согласно данным современной литературы [14], максимальное число рецепторов к ЭПО обнаруживается на эритроидных клетках-предшественницах, в процессе эри-тропоэза количество рецепторов уменьшается и считается, что у ретикулоцита и у эритроцита они отсутствуют. Одна-ко существует ряд исследований, свидетельствующих о на-личии рецепторов к ЭПО на мембранах эритроцитов, в том числе содержащих фетальный гемоглобин, однако их плот-ность очень низкая [15,16].…”

Interplay between SOX7 and RUNX1 regulates hemogenic endothelial fate in the yolk sac

“…Hematopoietic differentiation of yolk sac explants and cell suspensions E9.5 yolk sac explants were cultured for 2 days in explant medium adapted from Malik et al (2013), containing: IMDM, 16% BIT serum substitute (Stem Cell Technologies), 50 µg/ml ascorbic acid (Sigma), 0.5% PFHMII (Invitrogen), 15% serum replacement (Invitrogen), 5% platelet-derived serum, 2 mM L-glutamine (Gibco), 150 µM MTG (Sigma), 2 U/ml EPO (Eprex, Janssen-Cilag) and 100 ng/ml SCF (Peprotech) (protocol kindly provided by Professor James Palis, University of Rochester Medical Center, Rochester, USA). For dissociated yolk sac cultures, E9.5 yolk sacs were dissociated with collagenase dispase (Roche) diluted to 1 mg/ml in PBS for 20 min at 37°C.…”