Antigenic characterization of chick erythrocytes and erythropoietic precursors: identification of several definitive populations during embryogenesis.
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Biomedical subjects
Publications and source records attributed to V Nigon.
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The enumeration of erythropoietic colony-forming cells in vitro has allowed us to complete previous data on changes in the various erythroid cell populations during chick embryo-genesis. Erythrocytic colony-forming units in culture (CFU-cE) which are sensitive to avian erythropoietin appear in the blastoderm as soon as the 24th hour of development. They represent most likely precursors of the megalocytic erythropoiesis, and do not seem to derive from stem cells common with normocytic erythropoiesis. Data concerning vitelline normocytic erythropoiesis were analysed in a kinetic model based on stochastic change of the stem cells. From this model it appears that 17-20 cell divisions are required for differentiation of erythrocytes from stem cells.
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1. During multiplication of irradiated cells, a segregation may take place between bleached cells, whose progeny is unable to green, and green ones. Some of the green cells give progenies exclusively made of green cells; the progeny of others is partly composed of bleached cells. 2. If one assumes that greening results from the activity of functional units endowed with genetic continuity (Plastidial Segregating Units = PSU), segregation of these units seems to occur according to a model involving random sorting out during the three first divisions. During the following divisions, functional units seem to multiply faster than those impaired by irradiation. 3. The greening rate of colonies issued from irradiated cells seems to be conditioned mostly by the number of functional PSU remaining in the mother cell of the colony.
Complementary DNA (cDNA) was prepared with viral RNA-dependent DNA polymerase using human globin messenger RNA (mRNA) as template. By selective hydridization to globin mRNA from beta-thalassaemics a probe which was greater than 85% complementary to alpha-globin mRNA was purified. This was hybridized in cDNA excess to human genomic DNA, and the rate and extent of hybridization confirmed that there are two genes for alpha-globin per haploid genome. Cellular DNA was also prepared from peripheral blood from cases expressing the alpha-globin chain mutant Hb J Mexico to varying extents. This DNA was identical in hybridization behaviour to normal DNA demonstrating that the imbalanced mutant chain synthesis seen physiologically is not due to a gene deletion.
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beta-Thalassemia is a major public health problem in Algeria. During a survey, a family including two cases of betaO-thalassemia was studied. The family study indicated that two of the affected siblings had homozygous beta-thalassemia; there were also both normal and heterozygous siblings, and both parents had beta-thalassemia trait. In the two cases of betaO-thalassemia there was no hemoglobin A in the peripheral blood, and no beta-globin chain synthesis in whole cell incubations. Hybridization of purified complementary DNA specific for alpha- and beta-globin messenger RNAs demonstrated less than 1% mRNAbeta relative to mRNAalpha in circulating reticulocytes, and for one case in total RNA from bone marrow. There is no apparent beta-globin gene deletion as determined by hybridization in globin cDNAbeta sequence excess. Therefore the Algerian cases studied are similar in molecular pathology to some Southern Italian and Asian cases described previously, and differ from other Italian and Chinese betaO-thalassemias, in which hybridizable mRNAbeta has been demonstrated, and from deltabetaO-thalassemia, which is caused by a gene deletion.
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1. When injected into irradiated chickens, haemopoietic stem cells give rise to well-defined erythrocytic colonies in the host marrow. Such stem cells (CFU-M = Colony Forming Unit in Marrow) have been found in different tissue of the chicke embryo (yolk sac, blood, marrow). Analysis of the properties of CFU-M reveals that they represent two classes of stem cells: pluripotent stem cells mainly in adult marrow and erythrocytic-committed stem cells present in yolk sac. 2. Yolk sac contains the main pool of CFU-M during the major part of embryonic life. In the blood of 6-day-old embryo, there are three or four times more CFU-Ms than in the yolk sac; they are no longer detected in the blood after the 16th day of incubation. During development of the marrow, stem cells are actively differentiating and their total number remains the same from 16 days to hatching.
Irradiated chicken are injected with haemopoietic tissues from adult or 11-day-old embryos. Development of stem cells gives rise to well-defined erythrocytic colonies on the surface of the tibial marrow. Erythropoietic production appears to be similar from adult marrow and embryonic blood stem cells; production from injected vitelline stem cells seems to be between 3 and 4 times higher than that of adult marrow stem cells. Results are discussed on the basis of two hypotheses: -existence of an extramedullary erythropoietic site in the host after vitelline cells grafting; -development of vitelline stem cells in the host marrow with kinetic patterns different from those of grafted adult marrow or embryonic blood stem cells. Anyway, the 11-day-old embryo appears to contain at least two types of blood stem cells with distinctive properties. Developmental origin, relationship and future of these different stem cells remain to be analysed.
Between the 18th and 20th day of incubation, the proportion of haemoglobin F (Hb F) synthesis decreases in the embryo bone marrow from the value found in the yolk sac (10% of the total haemoglobin synthesis) to the value found in the bone marrow of young chicks (2%). A similar drop in Hb F synthesis is observed with a delay of 48 hours in the blood. Two membrane antigens each specific of either embryo or adult erythrocytes have been detected. The study by immunofluorescence of their cellular distribution shows that 3 erythrocyte populations are successively produced: a population E bearing the embryonic antigen only, a population EA bearing both antigens and a population A bearing the adult antigen only. The presence of a relatively high proportion of Hb F in population E is strongly suggested by the similar kinetics of Hb F and E cells disappearance within the first posthatching month.
Haemopoietic stem cells from donors of different ages (embryos, adults) have been grafted into irradiated hosts. Cell multiplication kinetics, foetal haemoglobin production, age-specific antigen production were examined during two weeks after grafting. The results obtained tend to show that the erythrocyte characteristics are, for a large part, already determined in a stem cell type endowed with a large proliferative capacity.