Differential regulatory and compensatory responses in hematopoiesis/erythropoiesis in alpha- and beta-globin hemizygous mice

Characterization of hematopoiesis/erythropoiesis in thalassemias from multipotent primitive cells to mature erythrocytes is of fundamental importance and clinical relevance. We investigated this process in alpha- and beta-globin hemizygous mice, lacking the two adult tandemly organized genes from either the alpha- or beta-globin locus. Although both mice backcrossed on a homogeneous background exhibited similar reduced red blood cell (RBC) survival, beta-globin hemizygous mice had less severe reticulocyte loss and globin chain imbalance, suggesting an apparently milder thalassemia than for alpha-globin hemizygous mice. In contrast, however, beta-globin hemizygous mice displayed a more marked perturbation of hematologic parameters. Quantification of erythroid precursor subpopulations in marrow and spleen of beta-globin hemizygous mice showed more severely impaired maturation from the basophilic to orthochromatophilic erythroblasts and substantial loss of these late precursors probably as a consequence of a greater susceptibility to an excess of free alpha-chain than beta-chain. Hence, only erythroid precursors exhibiting stochastically moderate chain imbalance would escape death and mature to reticulocyte/RBC stage, leading to survival and minimal loss of reticulocytes in the beta-globin hemizygous mice. Furthermore, in response to the ineffective erythropoiesis in beta-globin hemizygous mice, a dynamic compensatory hematopoiesis was observed at earlier differentiation stage as evidenced by a significant increase of erythroid progenitors (erythroid colony-forming units approximately 100-fold) as well as of multipotent primitive cells (day 12 spleen colony-forming units approximately 7-fold). This early compensatory mechanism was less pronounced in alpha-globin hemizygous mice. The expansion of multipotent primitive and potentially stem cell populations, taken together with ineffective erythropoiesis and increased reticulocyte/RBC destruction could confer major cumulative advantage for gene targeting/bone marrow transplantation. Therefore, this study not only corroborated the strong potential effectiveness of transplantation for thalassemic hematopoietic therapy but also demonstrated the existence of a differential regulatory response for alpha- and beta-thalassemia.     

Endothelial Cell-Selective Adhesion Molecule Expression in Hematopoietic Stem/Progenitor Cells Is Essential for Erythropoiesis Recovery after Bone Marrow Injury

Numerous red blood cells are generated every second from proliferative progenitor cells under a homeostatic state. Increased erythropoietic activity is required after myelo-suppression as a result of chemo-radio therapies. Our previous study revealed that the endothelial cell-selective adhesion molecule (ESAM), an authentic hematopoietic stem cell marker, plays essential roles in stress-induced hematopoiesis. To determine the physiological importance of ESAM in erythroid recovery, ESAM-knockout (KO) mice were treated with the anti-cancer drug, 5-fluorouracil (5-FU). ESAM-KO mice experienced severe and prolonged anemia after 5-FU treatment compared to wild-type (WT) mice. Eight days after the 5-FU injection, compared to WT mice, ESAM-KO mice showed reduced numbers of erythroid progenitors in bone marrow (BM) and spleen, and reticulocytes in peripheral blood. Megakaryocyte-erythrocyte progenitors (MEPs) from the BM of 5-FU-treated ESAM-KO mice showed reduced burst forming unit-erythrocyte (BFU-E) capacities than those from WT mice. BM transplantation revealed that hematopoietic stem/progenitor cells from ESAM-KO donors were more sensitive to 5-FU treatment than that from WT donors in the WT host mice. However, hematopoietic cells from WT donors transplanted into ESAM-KO host mice could normally reconstitute the erythroid lineage after a BM injury. These results suggested that ESAM expression in hematopoietic cells, but not environmental cells, is critical for hematopoietic recovery. We also found that 5-FU treatment induces the up-regulation of ESAM in primitive erythroid progenitors and macrophages that do not express ESAM under homeostatic conditions. The phenotypic change seen in macrophages might be functionally involved in the interaction between erythroid progenitors and their niche components during stress-induced acute erythropoiesis. Microarray analyses of primitive erythroid progenitors from 5-FU-treated WT and ESAM-KO mice revealed that various signaling pathways, including the GATA1 system, were impaired in ESAM-KO mice. Thus, our data demonstrate that ESAM expression in hematopoietic progenitors is essential for erythroid recovery after a BM injury.     

A novel way to induce erythroid progenitor self renewal: cooperation of c-Kit with the erythropoietin receptor

Red blood cells are of vital importance for oxygen transport in vertebrates. Thus, their formation during development and homeostasis requires tight control of both progenitor proliferation and terminal red cell differentiation. Self renewal (i.e. long-term proliferation without differentiation) of committed erythroid progenitors has recently been shown to contribute to this regulation. Avian erythroid progenitors expressing the EGF receptor/c-ErbB (SCF/TGFalpha progenitors) can be induced to long-term proliferation by the c-ErbB ligand transforming growth factor alpha and the steroids estradiol and dexamethasone. These progenitors have not yet been described in mammals and their factor requirements are untypical for adult erythroid progenitors. Here we describe a second, distinct type of erythroid progenitor (EpoR progenitors) which can be established from freshly isolated bone marrow and is induced to self renew by ligands relevant for erythropoiesis, i.e. erythropoietin, stem cell factor, the ligand for c-Kit and the glucocorticoid receptor ligand dexamethasone. Limiting dilution cloning indicates that these EpoR progenitors are derived from normal BFU-E/CFU-E. For a detailed study, mEpoR progenitors were generated by retroviral expression of the murine Epo receptor in bone marrow erythroblasts. These progenitors carry out the normal erythroid differentiation program in recombinant differentiation factors only. We show that mEpoR progenitors are more mature than SCF/TGFalpha progenitors and also do no longer respond to transforming growth factor alpha and estradiol. In contrast they are now highly sensitive to low levels of thyroid hormone, facilitating their terminal maturation into erythrocytes.     

BMP4/Smad5 dependent stress erythropoiesis is required for the expansion of erythroid progenitors during fetal development

The rapid growth of the embryo places severe demands on the ability of the cardiovascular system to deliver oxygen to cells. To meet this need, erythroid progenitors rapidly expand in the fetal liver microenvironment such that by E14.5, erythropoiesis predominates in the fetal liver. In this report we show that the BMP4/Smad5 dependent stress erythropoiesis pathway plays a key role in the expansion of erythroid progenitors in the fetal liver. These data show that the fetal liver contains two populations of erythroid progenitors. One population resembles the steady state erythroid progenitors found in the adult bone marrow. While the second population exhibits the properties of stress erythroid progenitors found in adult spleen. Here we demonstrate that defects in BMP4/Smad5 signaling preferentially affect the expansion of the stress erythroid progenitors in the fetal liver leading to fetal anemia. These data suggest that steady state erythropoiesis is unable to generate sufficient erythrocytes to maintain the rapid growth of the embryo leading to the induction of the BMP4 dependent stress erythropoiesis pathway. These observations underscore the similarities between fetal erythropoiesis and stress erythropoiesis.     

Fetal anemia and apoptosis of red cell progenitors in Stat5a-/-5b-/- mice: a direct role for Stat5 in Bcl-X(L) induction.

The erythropoietin receptor (EpoR) is essential for production of red blood cells; a principal function of EpoR is to rescue committed erythroid progenitors from apoptosis. Stat5 is rapidly activated following EpoR stimulation, but its function in erythropoiesis has been unclear since adult Stat5a-/-5b-/- mice have normal steady-state hematocrit. Here we show that Stat5 is essential for the high erythropoietic rate during fetal development. Stat5a-/-5b-/- embryos are severely anemic; erythroid progenitors are present in low numbers, show higher levels of apoptosis, and are less responsive to Epo. These findings are explained by a crucial role for Stat5 in EpoR's antiapoptotic signaling: it mediates the immediate-early induction of Bcl-X(L) in erythroid cells through direct binding to the Bcl-X promoter.     

Chronic psychological stress activates BMP4‐dependent extramedullary erythropoiesis

Psychological stress affects different physiological processes including haematopoiesis. However, erythropoietic effects of chronic psychological stress remain largely unknown. The adult spleen contains a distinct microenvironment favourable for rapid expansion of erythroid progenitors in response to stressful stimuli, and emerging evidence suggests that inappropriate activation of stress erythropoiesis may predispose to leukaemic transformation. We used a mouse model to study the influence of chronic psychological stress on erythropoiesis in the spleen and to investigate potential mediators of observed effects. Adult mice were subjected to 2 hrs daily restraint stress for 7 or 14 consecutive days. Our results showed that chronic exposure to restraint stress decreased the concentration of haemoglobin in the blood, elevated circulating levels of erythropoietin and corticosterone, and resulted in markedly increased number of erythroid progenitors and precursors in the spleen. Western blot analysis revealed significantly decreased expression of both erythropoietin receptor and glucocorticoid receptor in the spleen of restrained mice. Furthermore, chronic stress enhanced the expression of stem cell factor receptor in the red pulp. Moreover, chronically stressed animals exhibited significantly increased expression of bone morphogenetic protein 4 (BMP4) in the red pulp as well as substantially enhanced mRNA expression levels of its receptors in the spleen. These findings demonstrate for the first time that chronic psychological stress activates BMP4‐dependent extramedullary erythropoiesis and leads to the prolonged activation of stress erythropoiesis pathways. Prolonged activation of these pathways along with an excessive production of immature erythroid cells may predispose chronically stressed subjects to a higher risk of leukaemic transformation.     

Macrophages Support Splenic Erythropoiesis in 4T1 Tumor-Bearing Mice

Anemia is a common complication of cancer; a role of spleen in tumor-stress erythropoiesis has been suggested. However, the molecular mechanisms involved in the splenic erythropoiesis following tumor maintenance remain poorly understood. Here we show that tumor development blocks medullar erythropoiesis by granulocyte colony-stimulating factor (G-CSF) and then causes anemia in murine 4T1 breast tumor-bearing mice. Meanwhile, tumor-stress promotes splenic erythropoiesis. Splenectomy worsened tumor-induced anemia, and reduced tumor volume and tumor weight, indicating the essential role of spleen in tumor-stress erythropoiesis and tumor growth. Tumor progression of these mice led to increased amounts of bone morphogenetic protein 4 (BMP4) in spleen. The in vivo role of macrophages in splenic erythropoiesis under tumor-stress conditions was investigated. Macrophage depletion by injecting liposomal clodronate decreased the expression of BMP4, inhibited splenic erythropoiesis, aggravated the tumor-induced anemia and suppressed tumor growth. Our results provide insight that macrophages and BMP4 are positive regulators of splenic erythropoiesis in tumor pathological situations. These findings reveal that during the tumor-stress period, the microenvironment of the spleen is undergoing changes, which contributes to adopt a stress erythropoietic fate and supports the expansion and differentiation of stress erythroid progenitors, thereby replenishing red blood cells and promoting tumor growth.     

Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells

We describe here the large-scale ex vivo production of mature human red blood cells (RBCs) from hematopoietic stem cells of diverse origins. By mimicking the marrow microenvironment through the application of cytokines and coculture on stromal cells, we coupled substantial amplification of CD34(+) stem cells (up to 1.95 x 10(6)-fold) with 100% terminal differentiation into fully mature, functional RBCs. These cells survived in nonobese diabetic/severe combined immunodeficient mice, as do native RBCs. Our system for producing 'cultured RBCs' lends itself to a fundamental analysis of erythropoiesis and provides a simple in vitro model for studying important human viral or parasitic infections that target erythroid cells. Further development of large-scale production of cultured RBCs will have implications for gene therapy, blood transfusion and tropical medicine.     

Alpha4beta1 integrin and erythropoietin mediate temporally distinct steps in erythropoiesis: integrins in red cell development

Erythropoietin (Epo) is essential for the terminal proliferation and differentiation of erythroid progenitor cells. Fibronectin is an important part of the erythroid niche, but its precise role in erythropoiesis is unknown. By culturing fetal liver erythroid progenitors, we show that fibronectin and Epo regulate erythroid proliferation in temporally distinct steps: an early Epo-dependent phase is followed by a fibronectin-dependent phase. In each phase, Epo and fibronectin promote expansion by preventing apoptosis partly through bcl-xL. We show that alpha(4), alpha(5), and beta(1) are the principal integrins expressed on erythroid progenitors; their down-regulation during erythropoiesis parallels the loss of cell adhesion to fibronectin. Culturing erythroid progenitors on recombinant fibronectin fragments revealed that only substrates that engage alpha(4)beta(1)-integrin support normal proliferation. Collectively, these data suggest a two-phase model for growth factor and extracellular matrix regulation of erythropoiesis, with an early Epo-dependent, integrin-independent phase followed by an Epo-independent, alpha(4)beta(1)-integrin-dependent phase.     

Negative autoregulation by Fas stabilizes adult erythropoiesis and accelerates its stress response

Erythropoiesis maintains a stable hematocrit and tissue oxygenation in the basal state, while mounting a stress response that accelerates red cell production in anemia, blood loss or high altitude. Thus, tissue hypoxia increases secretion of the hormone erythropoietin (Epo), stimulating an increase in erythroid progenitors and erythropoietic rate. Several cell divisions must elapse, however, before Epo-responsive progenitors mature into red cells. This inherent delay is expected to reduce the stability of erythropoiesis and to slow its response to stress. Here we identify a mechanism that helps to offset these effects. We recently showed that splenic early erythroblasts, 'EryA', negatively regulate their own survival by co-expressing the death receptor Fas, and its ligand, FasL. Here we studied mice mutant for either Fas or FasL, bred onto an immune-deficient background, in order to avoid an autoimmune syndrome associated with Fas deficiency. Mutant mice had a higher hematocrit, lower serum Epo, and an increased number of splenic erythroid progenitors, suggesting that Fas negatively regulates erythropoiesis at the level of the whole animal. In addition, Fas-mediated autoregulation stabilizes the size of the splenic early erythroblast pool, since mutant mice had a significantly more variable EryA pool than matched control mice. Unexpectedly, in spite of the loss of a negative regulator, the expansion of EryA and ProE progenitors in response to high Epo in vivo, as well as the increase in erythropoietic rate in mice injected with Epo or placed in a hypoxic environment, lagged significantly in the mutant mice. This suggests that Fas-mediated autoregulation accelerates the erythropoietic response to stress. Therefore, Fas-mediated negative autoregulation within splenic erythropoietic tissue optimizes key dynamic features in the operation of the erythropoietic network as a whole, helping to maintain erythroid homeostasis in the basal state, while accelerating the stress response.     

Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of red cell formation is regulated by the hormone erythropoietin (Epo), whose synthesis is triggered by tissue hypoxia. A threat to adequate tissue oxygenation results in a rapid increase in Epo, driving an increase in erythropoietic rate, a process known as the erythropoietic stress response. The resulting increase in the number of circulating red cells improves tissue oxygen delivery. An efficient erythropoietic stress response is therefore critical to the survival and recovery from physiological and pathological conditions such as high altitude, anemia, hemorrhage, chemotherapy or stem cell transplantation. The mouse is a key model for the study of erythropoiesis and its stress response. Mouse definitive (adult-type) erythropoiesis takes place in the fetal liver between embryonic days 12.5 and 15.5, in the neonatal spleen, and in adult spleen and bone marrow. Classical methods of identifying erythroid progenitors in tissue rely on the ability of these cells to give rise to red cell colonies when plated in Epo-containing semi-solid media. Their erythroid precursor progeny are identified based on morphological criteria. Neither of these classical methods allow access to large numbers of differentiation-stage-specific erythroid cells for molecular study. Here we present a flow-cytometric method of identifying and studying differentiation-stage-specific erythroid progenitors and precursors, directly in the context of freshly isolated mouse tissue. The assay relies on the cell-surface markers CD71, Ter119, and on the flow-cytometric ''forward-scatter'' parameter, which is a function of cell size. The CD71/Ter119 assay can be used to study erythroid progenitors during their response to erythropoietic stress in vivo, for example, in anemic mice or mice housed in low oxygen conditions. It may also be used to study erythroid progenitors directly in the tissues of genetically modified adult mice or embryos, in order to assess the specific role of the modified molecular pathway in erythropoiesis.Download video file.^{(92M, mov)}     

Developmental rescue of an embryonic-lethal mutation in the retinoblastoma gene in chimeric mice.

The requirement for a functional retinoblastoma gene, Rb-1, in murine development around days 12-15 of gestation precludes monitoring the effect of loss of Rb-1 function on later stages of development and on tumorigenesis in adult mice. Here we describe the developmental rescue of embryonic stem cells carrying two inactive Rb-1 alleles in chimeric mice. Rb-1- cells contributed substantially to most tissues in adult chimeras, including blood, liver and central nervous system, which were severely affected in pure Rb-1- embryos. The adult chimeric erythroid compartment appeared completely normal, but an increased number of nucleated red cells was observed during fetal liver erythropoiesis in highly chimeric embryos. No ostensive abnormalities were seen in the developing and adult CNS. However, the developing retina of chimeric Rb-1- embryos showed ectopic mitoses and substantial cell degeneration, while the contribution of Rb-1- cells to the adult retina was much reduced. Moreover, the formation of lens fibre cells was severely disturbed. No retinoblastomas developed in any of these mice. Instead, nearly all animals died of pituitary gland tumours which were exclusively derived from Rb-1- cells.