Yolk sac-derived primitive erythroblasts enucleate during mammalian embryogenesis

The enucleated definitive erythrocytes of mammals are unique in the animal kingdom. The observation that yolk sac-derived primitive erythroid cells in mammals circulate as nucleated cells has led to the conjecture that they are related to the red cells of fish, amphibians, and birds that remain nucleated throughout their life span. In mice, primitive red cells express both embryonic and adult hemoglobins, whereas definitive erythroblasts accumulate only adult hemoglobins. We investigated the terminal differentiation of murine primitive red cells with use of antibodies raised to embryonic beta H1-globin. Primitive erythroblasts progressively enucleate between embryonic days 12.5 and 16.5, generating mature primitive erythrocytes that are similar in size to their nucleated counterparts. These enucleated primitive erythrocytes circulate as late as 5 days after birth. The enucleation of primitive red cells in the mouse embryo has not previously been well recognized because it coincides with the emergence of exponentially expanding numbers of definitive erythrocytes from the fetal liver. Our studies establish a new paradigm in the understanding of primitive erythropoiesis and support the concept that primitive erythropoiesis in mice shares many similarities with definitive erythropoiesis of mammals.     

Anaemia and its association with month and blood phenotype in blood donors in Fako division,Cameroon

Background

Mammalian erythropoiesis can be divided into two distinct types, primitive and definitive, in which new cells are derived from the yolk sac and hematopoietic stem cells, respectively. Primitive erythropoiesis occurs within a restricted period during embryogenesis. Primitive erythrocytes remain nucleated, and their hemoglobins are different from those in definitive erythrocytes. Embryonic type hemoglobin is expressed in adult animals under genetically abnormal condition, but its later expression has not been reported in genetically normal adult animals, even under anemic conditions. We previously reported that injecting animals with nitrogen-containing bisphosphonate (NBP) decreased erythropoiesis in bone marrow (BM). Here, we induced severe anemia in a mouse model by injecting NBP injection in combination with phenylhydrazine (PHZ), and then we analyzed erythropoiesis and the levels of different types of hemoglobin.

Methods

Splenectomized mice were treated with NBP to inhibit erythropoiesis in BM, and with PHZ to induce hemolytic anemia. We analyzed hematopoietic sites and peripheral blood using morphological and molecular biological methods.

Results

Combined treatment of splenectomized mice with NBP and PHZ induced critical anemia compared to treatment with PHZ alone, and numerous nucleated erythrocytes appeared in the peripheral blood. In the BM, immature CD71-positive erythroblasts were increased, and extramedullary erythropoiesis occurred in the liver. Furthermore, embryonic type globin mRNA was detected in both the BM and the liver. In peripheral blood, spots that did not correspond to control hemoglobin were observed in 2D electrophoresis. ChIP analyses showed that KLF1 and KLF2 bind to the promoter regions of β-like globin. Wine-colored capsuled structures were unexpectedly observed in the abdominal cavity, and active erythropoiesis was also observed in these structures.

Conclusion

These results indicate that primitive erythropoiesis occurs in adult mice to rescue critical anemia because primitive erythropoiesis does not require macrophages as stroma whereas macrophages play a pivotal role in definitive erythropoiesis even outside the medulla. The cells expressing embryonic hemoglobin in this study were similar to primitive erythrocytes, indicating the possibility that yolk sac-derived primitive erythroid cells may persist into adulthood in mice.

     

Globin switches in yolk sac-like primitive and fetal-like definitive red blood cells produced from human embryonic stem cells

We have previously shown that coculture of human embryonic stem cells (hESCs) for 14 days with immortalized fetal hepatocytes yields CD34(+) cells that can be expanded in serum-free liquid culture into large numbers of megaloblastic nucleated erythroblasts resembling yolk sac-derived cells. We show here that these primitive erythroblasts undergo a switch in hemoglobin (Hb) composition during late terminal erythroid maturation with the basophilic erythroblasts expressing predominantly Hb Gower I (zeta(2)epsilon(2)) and the orthochromatic erythroblasts hemoglobin Gower II (alpha(2)epsilon(2)). This suggests that the switch from Hb Gower I to Hb Gower II, the first hemoglobin switch in humans is a maturation switch not a lineage switch. We also show that extending the coculture of the hESCs with immortalized fetal hepatocytes to 35 days yields CD34(+) cells that differentiate into more developmentally mature, fetal liver-like erythroblasts, that are smaller, express mostly fetal hemoglobin, and can enucleate. We conclude that hESC-derived erythropoiesis closely mimics early human development because the first 2 human hemoglobin switches are recapitulated, and because yolk sac-like and fetal liver-like cells are sequentially produced. Development of a method that yields erythroid cells with an adult phenotype remains necessary, because the most mature cells that can be produced with current systems express less than 2% adult beta-globin mRNA.     

Molecular and cell biologic aspects of erythropoiesis in long-term bone marrow cultures

In long-term marrow cultures, proliferation and differentiation of hemopoietic stem cells occurs for several months. Normally, only the most primitive erythroid progenitor cells are produced (the BFU-E). Following treatment with anemic mouse serum (AMS) or normal mouse serum plus erythropoietin, the BFU-E mature into CFU-E, which then go to produce mature nonnucleated red cells. This development is associated with the production of adult type hemoglobin. Furthermore, erythropoiesis and granulopoiesis occur in association with discrete cellular elements of the adherent cell layer in the long-term culture. Following treatment with AMS, erythropoiesis is enhanced while granulopoiesis is depressed, with no apparent competition at the stem cell or progenitor cell level.     

Canine cyclic haematopoiesis: the effect of endotoxin on erythropoiesis

We have examined the effects of chronic endotoxin treatment on erythropoiesis in six grey collies with cyclic haematopoiesis. Blood reticulocytes, bone marrow erythroid colony (EC) forming cells, serum iron and erythropoietin (ESF) values showed regular periodic fluctuations in untreated grey collies. Daily endotoxin injections eliminated the cyclic fluctuations of reticulocytes and EC. The mean serum iron values were increased and recurrent hypoferraemia eliminated, while the mean serum ESF values were reduced. The cyclic fluctuations of serum ESF values were no longer apparent in the endotoxin treated grey collies. Tritiated thymidine suicide of the marrow EC forming cells failed to show cyclic changes either in untreated or endotoxin treated dogs. The ESF sensitivity of EC in the grey collie was unchanged during endotoxin treatment and was not different from normal dogs. Endotoxin appears to alter periodic erythropoiesis by stabilizing the flux of cells into the committed erythroid precursor cell pool from a more primitive stem cell compartment.     

Functional requirements for phenotypic correction of murine beta-thalassemia: implications for human gene therapy

As initial human gene therapy trials for beta-thalassemia are contemplated, 2 critical questions important to trial design and planning have emerged. First, what proportion of genetically corrected hematopoietic stem cells (HSCs) will be needed to achieve a therapeutic benefit? Second, what level of expression of a transferred globin gene will be required to improve beta-thalassemic erythropoiesis? These questions were directly addressed by means of a murine model of severe beta-thalassemia. Generation of beta-thalassemic mice chimeric for a minority proportion of genetically normal HSCs demonstrated that normal HSC chimerism levels as low as 10% to 20% resulted in significant increases in hemoglobin (Hb) level and diminished extramedullary erythropoiesis. A large majority of the peripheral red cells in these mice were derived from the small minority of normal HSCs. In a separate set of independent experiments, beta-thalassemic mice were bred with transgenic mice that expressed different levels of human globins. Human gamma-globin messenger RNA (mRNA) expression at 7% of the level of total endogenous alpha-globin mRNA in thalassemic erythroid cells resulted in improved red cell morphology, a greater than 2-g/dL increase in Hb, and diminished reticulocytosis and extramedullary erythropoiesis. Furthermore, gamma-globin mRNA expression at 13% resulted in a 3-g/dL increase in Hb and nearly complete correction of red cell morphology and other indices of inefficient erythropoiesis. These data indicate that a significant therapeutic benefit could be achieved with expression of a transferred globin gene at about 15% of the level of total alpha-globin mRNA in patients with severe beta-thalassemia in whom 20% of erythroid precursors express the vector genome.     

Long-term monoclonal reconstitution of erythropoiesis in genetically anemic W/Wv mice by injection of 5-fluorouracil-treated bone marrow cells of Pgk-1b/Pgk-1a mice

The spleen colony-forming assay does not represent the number of hematopoietic stem cells with extensive self-maintaining capacity because five to 50 spleen colony-forming units (CFU-S) are necessary to rescue a genetically anemic (WB X C57BL/6)F1-W/Wv(WBB6F1-W/Wv) mouse. We investigated which is more important for the reconstitution of erythropoiesis, the transplantation of multiple CFU-S or that of a single stem cell with extensive self-maintaining potential. The electrophoretic pattern of hemoglobin was used as a marker of reconstitution and that of phosphoglycerate kinase (PGK), an X chromosome-linked enzyme, as a tool for estimating the number of stem cells. For this purpose, we developed the C57BL/6 congeneic strain with the Pgk-1a gene. Bone marrow cells were harvested after injection of 5- fluorouracil from C57BL/6-Pgk-1b/Pgk-1a female mice in which each stem cell had either A-type PGK or B-type PGK due to the random inactivation of one or two X chromosomes. When a relatively small number of bone marrow cells (ie, 10(3) or 3 X 10(3] were injected into 200-rad- irradiated WBB6F1-W/Wv mice, the hemoglobin pattern changed from the recipient type (Hbbd/Hbbs) to the donor type (Hbbs/Hbbs) in seven of 150 mice for at least 8 weeks. Erythrocytes of all these WBB6F1-W/Wv mice showed either A-type PGK alone or B-type PGK alone during the time of reconstitution, which suggests that a single stem cell with extensive self-maintaining potential may sustain the whole erythropoiesis of a mouse for at least 8 weeks.     

Alterations in erythropoiesis in TGF-beta 1-treated mice.

Chronic treatment of mice with transforming growth factor beta 1 (TGF-beta 1) resulted in a dose-dependent inhibition of erythropoiesis. Following 14 daily s.c. injections of 5 or 25 micrograms of TGF-beta 1, a significant degree of anemia was observed. In addition, erythroid progenitor cells were present in reduced numbers in the bone marrow and spleen. Pluripotent stem cells were present in normal numbers in the bone marrow of mice treated with 25 micrograms of TGF-beta 1. However, significantly elevated levels were present in the peripheral blood. Adequate levels of erythropoietin were present in TGF-beta 1-treated mice. Following suspension of treatment with TGF-beta 1, erythropoiesis was restored, and TGF-beta-treated mice were able to compensate the anemia. One week following treatment, only mice treated with 25 micrograms of TGF-beta 1 continued to show evidence of anemia. However, in contrast to 1 day following treatment, these mice had levels of reticulocytes that were significantly above control values. In addition, erythroid progenitor cells had returned to normal levels in the bone marrow and were present in elevated levels in the spleen in both groups of TGF-beta 1 treated mice. The results provide evidence that the anemia associated with sustained TGF-beta 1 treatment is the result, in part, of a reversible inhibition of the maturation of erythroid progenitor cells.     

Lymphoid differentiation of the hematopoietic stem cell that reconstitutes total erythropoiesis of a genetically anemic W/Wv mouse

We investigated whether the stem cell that reconstitutes total erythropoiesis of a WBB6F1-W/Wv mouse differentiates into lymphoid lineage. The electrophoretic pattern of hemoglobin was used as a marker of the reconstitution; 3-phosphoglycerate kinase (PGK), an X chromosome-linked enzyme was used as a tool for estimating clonality. We injected 10(5) bone marrow cells of 5-FU treated C57BL/6-Pgk-1b/Pgk-1a female mice, in which each stem cell had either A-type PGK or B-type PGK due to random inactivation of one of two X chromosomes, into genetically anemic (WB x C57BL/6)F1-W/Wv (hereafter WBB6F1-W/Wv) mice that contained only B-type PGK. The recipient WBB6F1-W/Wv mice, in which erythropoiesis was reconstituted with donor cells for a long term, were killed and the PGK patterns of bone marrows, thymus, lymph nodes, and Peyer's patches were examined. A considerable amount of A-type PGK was detected in the lymphoid organs of the WBB6F1-W/Wv mice in which erythrocytes showed only A-type PGK when killed. In contrast, A-type PGK was scarcely detectable in the lymphoid organs of the WBB6F1-W/Wv mice in which erythrocytes showed only B-type PGK when killed. The present results suggest that the hematopoietic stem cells estimated by the erythropoiesis reconstituting assay differentiate into lymphoid lineage and that the long-term erythropoiesis reconstitution assay is useful for detecting the true primitive hematopoietic stem cells.     

Ontogeny of erythropoiesis

PURPOSE OF REVIEW: The present study review examines the current understanding of the ontogeny of erythropoiesis with a focus on the emergence of the embryonic (primitive) erythroid lineage and on the similarities and differences between the primitive and the fetal/adult (definitive) forms of erythroid cell maturation. RECENT FINDINGS: Primitive erythroid precursors in the mouse embryo and cultured in vitro from human embryonic stem cells undergo 'maturational' globin switching as they differentiate terminally. The appearance of a transient population of primitive 'pyrenocytes' (extruded nuclei) in the fetal bloodstream indicates that primitive erythroblasts enucleate by nuclear extrusion. In-vitro differentiation of human embryonic stem cells recapitulates hematopoietic ontogeny reminiscent of the murine yolk sac, including overlapping waves of hemangioblast, primitive, erythroid, and definitive erythroid progenitors. Definitive erythroid potential in zebrafish embryos, like that in mice, initially arises prior to, and independent of, hematopoietic stem cell emergence in the region of the aorta. Maturation of definitive erythroid cells within macrophage islands promotes erythroblast-erythroblast and erythroblast-stromal interactions that regulate red cell output. SUMMARY: The study of embryonic development in several different model systems, as well as in cultured human embryonic stem cells, continues to provide important insights into the ontogeny of erythropoiesis. Contrasting the similarities and differences between primitive and definitive erythropoiesis will lead to an improved understanding of erythroblast maturation and the terminal steps of erythroid differentiation.     

Differentiation of chimeric bone marrow in vivo reveals genotype-restricted contributions to hematopoiesis

Bone marrow transplantation is of increasing utility in cancer treatment and is an important component of gene therapy protocols. Understanding the functional identities of progenitor cells involved in repopulation is important for the optimal application of this procedure. We have simultaneously used two types of genetic markers to study engraftment of mice after irradiation. The first involves intrinsic genetic differences, including a cellular marker, between two mouse strains used to construct chimeric mice by aggregating embryos. To produce a second marking system, bone marrow from these allophenic mice was subsequently infected with retrovirus. Individual progenitor cells, including primitive lympho-hematopoietic stem cells, participating in repopulation were identified by virtue of their uniquely marked clonal progeny. In this way numbers and genotypic identities of clones contributing to repopulation were determined. Engraftment could be divided into two distinguishable temporal phases. The first comprised roughly the first 3-4 months following transplant and was characterized by numerous clones, many of which apparently had limited lineage potencies. The subsequent phase was characterized by few, often a single, clones represented in all lympho-hematopoietic tissues. These findings are consistent with the notion that different classes of progenitor cells are differentially responsible for temporal progression. More differentiated, perhaps lineage restricted, progenitors transiently dominate the first few months before the emergence of pluripotent stem cell clones. Senescence of progenitors of the first phase may reflect their limited lifespans. A clear genotypic difference was obvious in engraftment. Cells of one strain, DBA/2, completely dominated the first temporal phase, whereas the C57BL/6 partner strain dominated the second phase. The genotype-restricted dominance of different stages of repopulation suggests important differences in the organization and regulation of stem and progenitor cell populations. Inherent differences in seeding, proliferation, and differentiation of progenitors of the two inbred strains may account for the differences. This in vivo model of competitive repopulation provides the opportunity to explore potentially important loci in the process of engraftment. We propose that DBA/2 progenitor cells, due to a proliferative and/or numerical advantage, account for their superiority immediately after engraftment. C57BL/6 stem cells, with long-term repopulating potential, predominate later, perhaps because of subtle numerical or proliferative advantages.     

Successful treatment of murine beta-thalassemia intermedia by transfer of the human beta-globin gene

The beta-thalassemias are caused by more than 200 mutations that reduce or abolish beta-globin production. The severity of the resulting anemia can lead to lifelong transfusion dependency. A genetic treatment based on globin gene transfer would require that transgene expression be erythroid specific, elevated, and sustained over time. We report here that long-term synthesis of chimeric hemoglobin (mualpha(2):hubeta(A)(2)) could be achieved in mice with beta-thalassemia intermedia following engraftment with bone marrow cells transduced with a lentiviral vector encoding the human beta-globin gene. In the absence of any posttransduction selection, the treated chimeras exhibit durably increased hemoglobin levels without diminution over 40 weeks. Ineffective erythropoiesis and extramedullary hematopoiesis (EMH) regress, as reflected by normalization of spleen size, architecture, hematopoietic colony formation, and disappearance of liver EMH. These findings establish that a sustained increase of 3 to 4 g/dL hemoglobin is sufficient to correct ineffective erythropoiesis. Hepatic iron accumulation is markedly decreased in 1-year-old chimeras, indicating persistent protection from secondary organ damage. These results demonstrate for the first time that viral-mediated globin gene transfer in hematopoietic stem cells effectively treats a severe hemoglobin disorder.     

Lifelong hematopoiesis in both reconstituted and sublethally irradiated mice is provided by multiple sequentially recruited stem cells

OBJECTIVE: To evaluate the dynamics of stem cell production to hematopoiesis, the number of active stem cell clones and the lifespan of individual clones were studied. MATERIALS AND METHODS: The clonal contribution of primitive hematopoietic stem cells (HSC) responsible for long-term hematopoiesis was determined using two approaches. In one model, irradiated female mice were reconstituted with retrovirally marked male hematopoietic cells. In the second model, mice were irradiated sublethally without hematopoietic cell transplantation. In both models, bone marrow cells were serially sampled from the same mouse throughout a 12- to 20-month period and injected into irradiated recipients for analysis of day 10 colony-forming unit-spleen (CFU-S). The donor origin of CFU-S was determined by the presence of retrovirally marked cells or cells with chromosomal aberrations. RESULTS: The results of the two essentially different models show that 1) hematopoiesis is mainly the product of small clones of hematopoietic cells; 2) the lifespan of the majority of clones is only 1 to 2 months; 3) the clones usually function locally; and 4) the vast majority of the clones replace one another sequentially. Primitive HSCs capable of producing long-lived clones (about 10% among all clones), which exist during the entire life of a mouse, were detected by the radiation-marker technique only. CONCLUSION: Multiple short-living clones (at least on the level of CFU-S production) comprise the vast majority of the active stem cells in transplanted recipients or after endogenous recovery from sublethal irradiation.     

A new method for isolation of reticulocytes: positive selection of human reticulocytes by immunomagnetic separation

A method for isolating pure reticulocytes from leukocyte-depleted blood of normal persons is presented. The separation was achieved using an immunomagnetic technique. A monoclonal mouse antibody against human transferrin receptor was bound to magnetic beads conjugated with sheep antimouse antibody. The recovery of reticulocytes from peripheral blood was 15% to 42%. Blood used for isolation of reticulocytes could be stored for 4 days at 22 degrees C without altering the yield of reticulocytes. At 37 degrees C incubation, the reticulocytes matured rapidly and the transferrin receptor was found to have a half-life of 16 hours. The activity of several enzymes and the amount of creatine and hemoglobin A1C were measured both in the reticulocytes and peripheral blood. Of the enzymes, porphobilinogen deaminase had the best discriminatory power with a ratio of 8.8 between reticulocytes and peripheral red blood cells. The ratio for creatine was 16.7. The ability to isolate pure human reticulocytes, released after normal erythropoiesis, will offer new possibilities in the study of these cells.     

Hematopoiesis following disruption of the Pitx2 homeodomain gene

OBJECTIVE: Study the effect of loss of expression of Pitx2, a homeodomain gene preferentially expressed in murine hematopoietic stem/progenitor cells, on hematopoietic stem cells (HSCs). METHODS: We examined the fetal livers of mouse embryos with homozygous disruption of the Pitx2 gene, using flow cytometry immunophenotyping analysis, as well as immunohistochemistry techniques. We further investigated the role of Pitx2 in HSCs using a chimeric mouse model system. Pitx2 null embryonic stem (ES) cell clones were generated from embryonic day 3.5 blastocysts of Pitx2 null embryos. The Pitx2 null donor ES cell contribution to the adult hematopoietic system was confirmed by identifying donor-specific glucose-phosphate isomerase isotype in the erythrocytes using cellulose acetate eletrophoresis, and by demonstrating donor-specific major histocompatibility complex antigen allotype on the granulocytes/monocytes and T and B lymphocytes of the chimeric mice using flow cytometry analysis. RESULTS: Pitx2 homozygous null fetal livers are decreased in size and overall cellularity. The erythroid cell component of these livers is further reduced as compared to that of their wild-type and heterozygous littermates. Detailed quantitative analysis of the chimeric mice revealed contribution of Pitx2 null ES cells to erythroid, myeloid, lymphoid, and megakaryocytic lineages. The quantitative level of ES cell contribution to the peripheral hematopoietic cells was proportional to the level of general chimerism as determined by coat color. CONCLUSION: Although the fetal livers of Pitx2 null embryos displayed signs of impaired erythropoiesis, Pitx2 gene disrupted HSCs can contribute to hematopoiesis under physiological conditions.     

Decreased Erythropoiesis: The Origin of the BCG Induced Anaemia in Mice

Transient anaemia follows a high dose of viable BCG given intravenously to mice. The anaemia was dependent upon the dose, viability and the route of injection of the BCG. The evidence of an absolute anaemia was based on red cell mass decrease.
Serum iron concentration and red cell life span were unchanged in the first 14 d after BCG infection. The erythropoietin level increased 20-fold. Despite the high level of this hormone and the appearance of increased erythropoiesis in the spleen, the production of erythrocytes was decreased presumably because of a decrease in numbers of erythropoietin-responsive cells.     

Selective erythroid replacement in murine beta-thalassemia using fetal hematopoietic stem cells.

We have explored the application of fetal hematopoietic stem cell (HSC) transplants for cellular replacement in a murine model of beta-thalassemia. Liver-derived HSCs from nonthalassemic syngeneic murine fetal donors were transplanted into nonirradiated neonatal beta-thalassemic recipients. Significant erythrocyte chimerism (9-27%) was demonstrated in the majority of recipients at 1 month and remained stable or increased (up to 55%) during long-term follow-up in almost all cases. Chimeras had improved phenotypes, as evidenced by decreased reticulocyte counts, increased mean erythrocyte deformability, and decreased iron deposits in comparison to controls. To investigate whether the high degree of peripheral blood chimerism was predominantly a feature of erythroid elements or was a general feature of all hematopoietic elements, chimeras were created using donor HSCs "tagged" with a DNA transgene. Whereas donor hemoglobin comprised > 30% of total hemoglobin, nucleated tagged nonerythroid donor cells comprised < 1% of peripheral blood elements. Explanations for the observed selective increase in erythroid chimerism include longer survival of normal donor red cells compared to that of thalassemic red cells and the effective maturation of the donor erythroid elements in the bone marrow in chimeric animals. The latter explanation bears consideration because it is consistent with the process of ineffective erythropoiesis, well documented to occur in thalassemia, in which the majority of thalassemic erythroid cells are destroyed during erythropoiesis prior to release from the bone marrow. Overall, these data demonstrate the potential for significant erythroid chimerism and suggest that fetal HSC transplantation may play a significant role in future treatment.     

Hematopoiesis in the yolk sac: more than meets the eye

The first blood cells observed in the embryo are large nucleated erythroblasts generated in blood islands of the extraembryonic yolk sac. These unique red cells have been termed primitive because of their resemblance to nucleated erythroblasts of nonmammalian species. It is now widely assumed that hematopoiesis in the yolk sac is "primitive" and that "definitive" hematopoiesis has its origins in the aorta/gonad/mesonephros (AGM) region. Recent studies of yolk sac hematopoiesis have challenged several aspects of this paradigm. First, primitive erythropoiesis in mammals shares many features with definitive erythropoiesis, including progressive erythroblast maturation leading to the circulation of enucleated erythrocytes. Second, the emergence of primitive erythroid progenitors in the yolk sac prior to somitogenesis may be associated with the macrophage and megakaryocyte lineages, raising the possibility that "primitive" hematopoiesis may be multilineage in nature. Third, a second wave of hematopoietic progenitors emerge from the yolk sac during early somitogenesis that consists of multiple myeloid lineages that are temporally and spatially associated with definitive erythroid progenitors. These "definitive" hematopoietic progenitors expand in numbers in the yolk sac and are thought to seed the fetal liver and generate the first definitive blood cells that rapidly emerge from the liver. Recent findings support a model of hematopoietic ontogeny in which the conceptus' first maturing blood cells and committed progenitors are provided by the yolk sac, allowing survival until AGM-derived hematopoietic stem cells can emerge, seed the liver and differentiate into mature blood cells.     

Fetal hemoglobin and potassium in isolated transferrin receptor- positive dense sickle reticulocytes

A subset of sickle cells have an increased density at the reticulocyte stage of development, indicating that they are either abnormally dense upon release from the bone marrow or become dense quickly in the circulation. These cells are of interest because they most likely have severely disrupted cation regulation and a short lifespan. Based on the distribution of fetal hemoglobin (HbF) in the density fractions of sickle red blood cells (RBCs) and in vitro studies of cellular K+ loss, it seems likely that HbF content is an important in vivo determinant of dense cell formation. In this study, we tested the hypothesis that young, dense cells have low HbF content. Sickle RBCs were first separated into light and dense fractions. Reticulocytes were isolated from unfractionated cells and from each density fraction with an immunomagnetic technique directed against transferrin receptors (TfR) and assayed for the percentage of HbF and K+/Hb ratio. TfR+ reticulocytes isolated from unfractionated cells had a much lower HbF content when compared with all the unfractionated RBCs. This is most likely caused by enrichment of F cells because of a longer circulation life span. Heavy TfR+ reticulocytes had a K+/Hb ratio similar to that measured in the entire dense population and contained very low levels of HbF, averaging 2.5% of the level in all RBCs, 11.7% of the level in all TfR+ reticulocytes, and 4.0% of the level in all dense RBCs. These findings suggest that TfR+ dense cells derive predominantly from non-F cells. Furthermore, the amount of HbF in the circulating dense cells suggests that many of these cells do not derive from the TfR+ dense cells.     

The role of the spleen in a lactate dehydrogenase mutant mouse (Ldh-1c/Ldh-1c) with hemolytic anemia

The lactate dehydrogenase mouse mutant Ldh-1c/Ldh-1c is afflicted with a severe hemolytic anemia associated with extreme reticulocytosis (95%) and splenomegaly. Ninety-one percent of the total body colony-forming units--erythroid (CFU-E) have been quantified in the seven- to ten-times enlarged spleens of the mutant mice. Moreover, the splenic fraction of morphologically recognizable erythroid precursors was 134 times normal. From these data it was apparent that the spleen crucially contributes to the maintenance of steady state erythropoiesis in the mutants. On the other hand, an enhanced sequestration of red blood cells in the enlarged spleen may augment the anemia. Splenectomy experiments were performed with LDH mutant and wild type mice in order to investigate the role of the spleen in this particular hemolytic disease. Following splenectomy, the peripheral blood values and the frequency of femoral stem and progenitor cells were determined, and histological investigations were carried out. The life span of the splenectomized mutants was not shortened, in spite of a very low red blood cell count (25% of the untreated mutant value). Compared to the splenic loss only a moderate increase in bone marrow erythropoiesis was observed, such as a 250% increase of CFU-E. It is concluded that the reduction in red blood cell survival due to splenic sequestration in the mutants is of such a magnitude that it counterbalances a significant portion of splenic erythropoiesis.