The Formation of Foetal and Adult Haemoglobin in Cell Cultures of Neonatal Calf Marrow

Gabuzda, Thomas G.; Silver, Ruth; Chui, Lily Cheung; Lewis, Hugh B.

doi:10.1111/j.1365-2141.1970.tb01646.x

Cited by 20 publications

(7 citation statements)

References 23 publications

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“…Increased synthesis of Hb F (20) or persistence of y chain synthesis (21) in intro has been reported but was not confirmed (22). The discrepancy between the present and previous (22) data may reflect differences in the experimental system.…”

Section: Resultscontrasting

confidence: 87%

“…The discrepancy between the present and previous (22) data may reflect differences in the experimental system. Previous work has utilized suspension bone marrow culture systems in which erythroid cell proliferation is limited and globin chain synthesis progressively declines (20)(21)(22). In contrast, the culture system used in this study permits …”

Section: Resultsmentioning

confidence: 99%

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Stimulation of fetal hemoglobin synthesis in bone marrow cultures from adult individuals.

Papayannopoulou¹,

Brice²,

Stamatoyannopoulos³

1976

Proc. Natl. Acad. Sci. U.S.A.

115

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The replacement of fetal by adult hemoglobin after birth is not complete, since small amounts of fetal hemoglobin are consistently present in the blood of normal adults (6-9). It is, therefore, of interest to learn how continued synthesis of fetal hemoglobin in the adult is regulated and whether it can be further stimulated. We examined this in vitro, with a bone marrow culture system which permits selective proliferation of erythroid cells (10). We report here data which suggest appearance of increased and clonal synthesis of fetal hemoglobin in the adult erythroid cells, in vitro. METHODS Bone Marrow Cultures. Bone marrow aspirates from healthy volunteers without a hemoglobinopathy and from a patient homozygous for hemoglobin S were used; on a few occasions ribs surgically removed from patients were the source of marrow cells. Cells were washed in medium (Microbiological Associates, Bethesda, Md.) containing 2% (vol/vol) fetal calf serum, penicillin (50 units/ml) and streptomycin (50 ,gg/ml); the buffy coat layers were removed, and resuspended in fresh media. Antibodies and Immunofluorescent Labeling. Antibodies against hemoglobin F (anti-Hb F) or A (anti-Hb A) were raised in rabbits (anti-Hb F) or horses (anti-Hb A) and purified by affinity chromatography. The purification procedure, the conjugation with fluorescein isothiocyanate (FITC), and evidence of specificity of the anti-Hb F antibodies have been described (9). Peripheral blood smears were fixed and labeled with anti-Hb F antibodies conjugated with FITC (anti-Hb F-FITC), as previously described (9); smears prepared from bone marrow cell suspensions were treated similarly. Flattened plasma clots were fixed for 12 min in an acetone-methanol (9:1 vol/vol) solution, rinsed for 2 min in phosphate-buffered saline at pH 7.0 (9), rinsed in distilled water for another 2 min, and dried. They were labeled with anti-Hb F-FITC in the same fashion as the smears, except that the whole area of the clot was covered with the fluorescent antibody. After incubation for 1 hr in a humidified chamber at 370, the slides with clots were rinsed in phosphate-buffered saline, then in water, and then dried. They were mounted with clear serum, and viewed in the fluorescent microscope (Zeiss, Universal fluorescence microscope with reflected light excitation) under 150-times magnification. Freshly thawed antibody solutions were used for each experiment and conditions of fixation, staining, and viewing were kept constant. Evaluation of fluorescent labeling was best when plasma clots contained fewer erythroid colonies: cultures inoculated with 5 X 104 nucleated cells per clot were optimal. The reaction of the fixed plasma clots with the fluorescent antihemoglobin antibodies preserved the morphological characteristics of the cells and permitted identification and quantitative estimates of total erythroid colonies under fluorescent light. Erythroid cells and colonies were classified into categories according to intensity of fluorescent labeling. Fluorescence of moderate to bright intens...

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Section: Resultscontrasting

confidence: 87%

Section: Resultsmentioning

confidence: 99%

Stimulation of fetal hemoglobin synthesis in bone marrow cultures from adult individuals.

Papayannopoulou¹,

Brice²,

Stamatoyannopoulos³

1976

Proc. Natl. Acad. Sci. U.S.A.

115

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“…This effect does not appear to be erythropoietin dependent since as long as intermediate normoblasts remained in the control cultures, they showed an equally low labelling index. Thus, although erythropoietin not only induces stem cell differentiation but also has been shown to affect erythroid cell maturation (Winkert et al, 1960;Brecher & Stohlman, 1961;Gordon et a!, 1962;Gallagher et al, 1963;Fisher et al, 196.4, the suggestion that high doses of erythropoietin can result in skipped mitotic divisions (Stohlman et al, 1964) does not appear to be the cause of the reduced normoblast proliferation observed here. Necheles et a1 (1968) were also unable to demonstrate such an effect in human bone marrow cultures.…”

Section: Discussionmentioning

confidence: 58%

Erythroid Cell Proliferation in Human Bone Marrow Suspension Cultures

Wood

1974

Br J Haematol

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Summary. A simple suspension type culture system has been described for the in vitro maintenance of erythroid cells obtained from adult human bone marrow. In the presence of erythropoietin, normoblasts of all stages of maturation were still present after more than 10 days in culture. Following an initial decline, the numbers of basophilic normoblasts showed an absolute increase, reaching a maximum after about 5 days. Evidence of continued DNA synthesis in these cells, the observation of mitotic figures and the tracing of [3H]thymidine label from early normoblasts to intermediate and late normoblasts in culture further suggests that these cells arise by differentiation from unrecognizable erythroid precursor cells. Despite an apparent reduction in proliferation during the maturation of these cells, this system offers a method for investigating various aspects of erythroid cell differentiation and development.

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“…Sci. USA 70 (1973) of studies with neonatal calf marrow in vitro (25), the authors suggested that an increased concentration of erythropoietin stimulates selective synthesis of HbA over HbF. A switch in sheep marrow from HbA to HbC in cultures maintained for 48 hr has also been reported by Adamson and Stamatoyannopoulos (15).…”

Section: Methodsmentioning

confidence: 86%

“…In erythropoietin-containing cultures, the amount of HbC detected increased severalfold between 24 and 72 hr in culture, while the HbA content often decreased in the same time period; in cultures without erythropoietin present, synthesis of HbC was markedly less than in cultures containing erythropoietin (data not shown). Data are presented in tabular form in Table 2 (17), and in marrow cultures of rabbits (3,22), rats (23,24), calves (25), and humans (26). In a report The data (from goat no.…”

Section: Methodsmentioning

confidence: 99%

Hemogloblin Switching in Sheep and Goats: Erythropoietin-Dependent Synthesis of Hemoglobin C in Goat Bone-Marrow Cultures

Barker

Adams

Nienhuis³

et al. 1973

Proc. Natl. Acad. Sci. U.S.A.

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The anemia-induced switch from hemo. In eukaryotes, the molecular mechanisms involved in gene activation and in the subsequent production of specific cell proteins are largely unknown. To study gene activation in mammals, a cell population is needed that can be stimulated to transcribe the messenger RNA for a particular protein in response to a defined stimulus. We have studied the erythropoietin-dependent synthesis of hemoglobin C (HbC) in goat (and sheep) bone-marrow cultures for this purpose. Erythropoietin, a glycoprotein hormone, appears to stimulate precursor blood cells in the bone marrow to differentiate into cells that produce hemoglobin (1-3). The type of hemoglobin produced by these cells is determined by the genetic makeup of the animal and by its stage of development. For example, sheep and goats homozygous for hemoglobin A (HbA) pass through a series of developmental stages during which they produce HbF (a2Y2), then HbC (aZ2c), andfinally-the adult HbA (aZ82A) (4-9). However, in response to severe anemia, an adult goat making HbA completely switches from the synthesis of HbA to HbC (4). There is a lag of 3 days between the induction of anemia and the detection of Oc globin synthesis in the marrow (10), suggesting that flc globin is produced by a newly stimulated population of precursor cells. As the anemia is relieved, HbA again is synthesized. The switch from MA to tic synthesis can be elicited in normal, nonanemic animals by injection of partially purified erythropoietin preparations (10-13).We have shown (14) that sheep and goats that are synthesizing HbA have only the mRNA for MA (and a) globin in their erythroid cells, while animals synthesizing HbC have only the mRNA for flc (and a) METHODSSource and Preparation of Cells. Goats used in this study were over 6 months old and had only HbA detectable in their peripheral blood, as determined by thin-layer electrophoresis on cellulose acetate (14). The animals were anesthesized by intravenous injection of sodium pentobarbital (60 mg/ml, 0.5 ml/kg) and exsanguinated through a femoral artery. The femurs and humeri were removed, their proximal and distal ends were cut off to expose the marrow cavity, and the marrow was extruded by flushing medium (Eagle's minimum essential medium for suspension culture; Microbiological Associates, Bethesda, Md.) through the bones. The cells were separated by being expelled from a 50-ml syringe through a. 20-gauge needle, were washed once with Eagle's medium, and then were resuspended in NCTC-109 medium (Microbiological Assoc.) and disaggregated by expulsion through 'successively smaller-bore syringe needles down to 25-gauge. The concentration of nucleated cells was adjusted to 0.5 to 1 X 101 cells.per ml by the addition of appropriate volumes of a culture medium (16) containing 5 ml of fetal-bovine serum, 2.5 ml of 7.5% NaHCO3, 1 ml of 200 mM iglutamine, 2 ml of penicillinstreptomycin (5000 units of each per ml), 86.7 ml of NCTC-109 medium (all of the preceding components were obtained from Microbiological ...

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The Formation of Foetal and Adult Haemoglobin in Cell Cultures of Neonatal Calf Marrow

Cited by 20 publications

References 23 publications

Stimulation of fetal hemoglobin synthesis in bone marrow cultures from adult individuals.

Stimulation of fetal hemoglobin synthesis in bone marrow cultures from adult individuals.

Erythroid Cell Proliferation in Human Bone Marrow Suspension Cultures

Hemogloblin Switching in Sheep and Goats: Erythropoietin-Dependent Synthesis of Hemoglobin C in Goat Bone-Marrow Cultures

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