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

Abstract

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 β-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.

Figure 1

Flow cytometric analysis and isolation of erythroblasts of wild-type mice bone marrow cells. (A) Plot of FSC versus SSC of all the cells. (B) Plot of 7AAD versus SSC of all the cells. (C) Plot of CD45CD11bGR1 versus SSC of all the cells. (D) Histogram of TER119 of CD45⁻CD11b⁻GR1⁻ cells. (E) Plot of CD44 versus TER119 of the TER119 positive cells without gating. (F) Plot of CD44 versus TER119 of the TER119 positive cells with the gating on CD44^hiTER^low population I. (G) Plot of CD44 versus FSC of the TER119 positive cells, showing naturally occurring clusters. (H) Plot of CD44 versus FSC of the TER119 positive cells with gating the population II, III, IV, V and VI. (I) Representative cytospin images of the sorted populations. (J) Histogram of FSC of distinct stage of erythroblasts. (K) Proportion of distinct stage of erythroblasts in bone marrow.

Figure 2

Representative erythropoiesis profiles of bone marrow and spleen of 4.1R^−/− and phlebotomy-induced mice. (Top panel) Representative erythropoiesis profiles of bone marrow. Note that there is an increased proportion of nucleated erythroblasts (I+II+III+IV) which is accompanied by a decreased proportion of enucleated erythrocytes (V+VI) in both 4.1R^−/− and phlebotomy-induced WT bone marrow compared with WT bone marrow. (Bottom panel) Representative erythropoiesis profiles of spleen. Note that there are very few erythroblasts in WT spleen but a large proportion of nucleated erythroblasts in the spleens of both 4.1R^−/− and phlebotomy-induced WT anemic mice.

Figure 3

Altered terminal erythroid differentiation of Hbb^th1/th1 mice and normalization after thansferrin therapy. (A) Representative erythropoiesis profiles of bone marrow and spleen of Hbb^th1/th1 mice. (Top panel) The representative erythropoiesis profiles of bone marrow. A decrease in fraction I (proerythroblast) and fraction VI (mature red cells) is clearly seen in PBS-treated thalassemia bone marrow and is restored in transferrin-treated bone marrow. (Bottom panel) Representative erythropoiesis profiles of spleen. There are very few erythroblasts in WT spleen, but the spleen of thalassemia mouse is highly erythropoietic and is restored in transferrin-treated spleen. (B) Western blot revealed an increase in soluble serum TfR1 in Hbb^th1/th1 relative to WT mice and is normalized after transferrin treatment.