Quantitative analysis of murine terminal erythroid differentiation in vivo: novel method to study normal and disordered erythropoiesis

Abstract

Key Points• The study establishes a reliable method to quantify differentiating mouse erythroblasts and to monitor terminal mouse erythropoiesis in vivo.• Quantitative analysis of erythropoiesis of thalassemia mice revealed stage-specific changes in terminal erythroid differentiation.Terminal erythroid differentiation is the process during which proerythroblasts differentiate to produce enucleated reticulocytes. Although it is well established that during murine erythropoiesis in vivo, 1 proerythroblast undergoes 3 mitosis to generate sequentially 2 basophilic, 4 polychromatic, and 8 orthochromatic erythroblasts, currently there is no method to quantitatively monitor this highly regulated process. Here we outline a method that distinguishes each distinct stage of erythroid differentiation in cells from mouse bone marrow and spleen based on expression levels of TER119, CD44, and cell size. Quantitative analysis revealed that the ratio of proerythroblasts:basophilic: polychromatic:orthromatic erythroblasts follows the expected 1:2:4:8 ratio, reflecting the physiologic progression of terminal erythroid differentiation in normal mice. Moreover, in 2 stress erythropoiesis mouse models, phlebotomy-induced acute anemia and chronic hemolytic anemia because of 4.1R deficiency, the ratio of these erythroblast populations remains the same as that of wild-type bone marrow. In contrast, in anemic ␤-thalassemia intermedia mice, there is altered progression which is restored to normal by transferrin treatment which was previously shown to ameliorate the anemic phenotype. The means to quantitate in vivo murine erythropoiesis using our approach will probably have broad application in the study of altered erythropoiesis in various red cell disorders. (Blood. 2013;121(8):e43-e49)

Erythroid‐extrinsic regulation of normal erythropoiesis by retinoic acid receptors

“…No significant changes were observed with CD71/Ter119 staining in conditional mutant mice (Fig 2F). Apart from subtle increases in fraction II of Epor-Cre Rara fl/fl g fl/fl mice and reductions in fraction V of Epor-Cre Rara fl/fl mice (Fig 2G), analysis using CD44 and Ter119 expression supported the conclusion that there was no major changes in erythroid populations (Liu et al, 2013). The CFU-E, PreCFU-E and megakaryocyte-erythroid phenotypic fractions did not differ significantly from the control mice (Fig 2H).…”

“…There were profound reductions in Ter119 + CD71 hi and Ter119 + CD71 med fractions in the BM of RARc À/À mice (Fig 1A-B) indicating reduced immature erythroblasts at the pro-erythroblast stage, similarly evident using CD44 and Ter119 staining (Fig 1C-D) (Liu et al, 2013). Analysis of the erythroid progenitor fractions (Pronk & Bryder, 2010) showed a reduction in the megakaryocyteerythroid progenitor fraction ( Fig 1E) with a concomitant increase in megakaryocyte-committed progenitors (Fig 1F) and a significant reduction in the erythroid colony-forming unit (CFU-E) fraction (Fig 1G).…”

Mobilization with granulocyte colony-stimulating factor blocks medullar erythropoiesis by depleting F4/80+VCAM1+CD169+ER-HR3+Ly6G+ erythroid island macrophages in the mouse

“…A 4 day G-CSF treatment, which mobilizes HSC into the blood, reduced the number of according to decreasing CD44 expression and forward scatter [25,26]. This staining and gating strategy confirmed the marked reduction in all differentiation stages of erythroblasts, In order to determine whether the effect of G-CSF on macrophages was directly mediated via the G-CSF receptor, we sorted from the BM of untreated mice F4/80 + VCAM- showed that G-CSF receptor mRNA (Csf3r) was abundantly expressed by these macrophages, whereas it was absent in IgM + and IgM -B cells (Fig.…”

Ineffective erythropoiesis caused by binucleated late-stage erythroblasts in mDia2 hematopoietic specific knockout mice

“…(C-F) Flow cytometric analysis of CD44 and forward scatter levels of CD45 negative erythroblasts from the bone marrow (C) and spleen (E) of indicated mice. Populations I-VI were defined as proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts, orthochromatic erythroblasts, reticulocytes, and red blood cells, respectively 9 . Statistical analysis of (C) and (E) were shown in (D) and (F), respectively.…”