High performance liquid chromatography of rat globin chains of fetal liver during the switch from embryonic to adult hemoglobin

A reversed-phase high performance liquid chromatography (HPLC) method for the direct separation of globin chains from cell lysates of peripheral blood and fetal liver from rat fetuses is described. Partial amino acid analysis of the globins eluted from the HPLC columns as well as comparison with the known elution positions of the adult globin chains in carboxymethyl cellulose chromatography indicated that alpha-chains are eluted first, followed by adult beta chains. The last chains to be eluted are considered as embryonic globins because of their absence in adult rats and their rapid disappearance from the liver after the 14th day of gestation. Liver erythroid cells isolated from rat fetuses on day 14 of gestation mainly synthesized alpha-chains and embryonic globin chains, whereas cells prepared from 16-day old fetuses synthesized almost exclusively alpha-chains and adult beta chains. When the fetal rat liver cells were cultured for 20h with erythropoietin there was a significant stimulation in the synthesis of alpha-globins and adult beta chains but not on the synthesis of embryonic globin chains. It is concluded that HPLC can be useful for the study of rat globin chain synthesis during fetal development, because it separates the globin chains in the three groups of globins, namely alpha, beta and embryonic chains which are important in the switch occurring in the liver.     

Hemoglobins in Human Fetuses: Evidence for Adult Hemoglobin Production After the 11th Gestational Week

Hemoglobin patterns were examinedin human fetuses in order to determinethe onset of synthesis of adult hemoglobin in fetal red cells. In fetusesyounger than 11 gestational wk, hemoglobin A could not be detected withelectrophoretic techniques. A smallamount of adult hemoblobin was constantly present, however, in hemolysates from fetuses older than 11 gestational wk, and it had the structuralproperties of adult hemoglobin A.Heterozygous fetuses had hemoglobinsC and A or S and A as early as the11th and 13th gestational wk. The findings are compatible with onset of betahemoglobin gene action as early asthe 11th gestational wk and synthesisof approximately 5% of adult hemoglobin by the end of the first andthroughout the second trimester ofgestation.

Submitted on July 28, 1971 Revised on October 8, 1971 Accepted on October 22, 1971     

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

The regulation of fetal hemoglobin in adult erythroid cells was investigated with bone marrow cultures. Fetal hemoglobin (Hb F) was identified in individual erythroid colonies with fluorescent antibodies against Hb F and synthesis of gamma chains was determined with analyses of radioactive globins. The appearance of fetal hemoglobin in erythroid colonies was clonal. All the cells of the Hb F synthesizing colonies contained fetal hemoglobin. The frequency of erythroid colonies showing Hb F was higher than expected compared to the frequency of Hb F containing cells in the blood. Production of Hb F in culture, as shown by analysis of the radioactive globins, was 5 to 14 times higher than baseline Hb F synthesis. These results suggest that the ability for gamma chain synthesis in erythroid cells is determined at or above the level of the precursor cell from which the erythroid colonies, in vitro, derive (probably an erythropoietin responsive stem cell), and that stimulation of fetal hemoglobin synthesis in adult erythroid cells is possible.     

Fetal and embryonic hemoglobins in erythroblasts of chromosomally normal and abnormal fetuses at 10-40 weeks of gestation

BACKGROUND AND OBJECTIVES: During fetal development a change in erythropoiesis from hepatic to medullary site occurs. In chromosomally abnormal fetuses this change is delayed. Hemoglobin production also undergoes developmental switches from embryonic to fetal hemoglobins in the first trimester of pregnancy. The aim of study was to determine the proportion of embryonic and fetal hemoglobins in fetal erythroblasts of chromosomally normal and abnormal fetuses at 10-40 weeks of gestation. DESIGN AND METHODS: Fetal blood was obtained from 93 chromosomally normal and 19 abnormal fetuses at 10-40 weeks of gestation. Fetal erythroblasts were isolated by triple density gradient centrifugation and magnetic cell sorting with CD71 antibody. Fluorescent antibodies were used to immuno-stain for zeta (zeta), epsilon (epsilon) and gamma (gamma) hemoglobin chains. RESULTS: The percentages of the positively stained cells were calculated. In chromosomally normal fetuses the percentage of erythroblasts expressing the zeta chain was 25% at 10 weeks but this decreased exponentially with gestation to less than 1% by 17 weeks. Similarly, the percentage of cells expressing the epsilon chain decreased from 97% at 10 weeks to less than 1% by 25 weeks. In contrast, expression of the gamma chain increased from about 30% at 10 weeks to 90% by 16 weeks and decreased thereafter to 60% at 40 weeks. In the abnormal fetuses, the percentage of erythroblasts expressing the zeta chain and the epsilon chain decreased to less than 1% by 23 and 28 weeks respectively, while maximum expression of the gamma chain was at about 22 weeks. INTERPRETATION AND CONCLUSIONS: In the chromosomally abnormal group the pattern of change in the expression of the various hemoglobin chains during gestation was similar to that in the normal fetuses but was delayed by three to six weeks. These findings suggest that in fetuses with chromosomal abnormalities there is a developmental delay in the switch from embryonic to fetal hemoglobin chains.     

Adult hemoglobins are synthesized in murine fetal hepatic erythropoietic cells

The hemoglobins present in murine fetal hepatic erythroblasts on days 12-15 of gestation were studied by biochemical and immunocytologic techniques. In addition, fetal hepatic hemopoietic progenitor cells obtained from normal and mutant f/f mouse fetuses on days 11-13 of gestation were cultured in vitro with added erythropoietin and adult spleen cell conditioned medium to form large erythroid colonies. In all instances, adult hemoglobin synthesis was detected in the fetal hepatic erythroblasts and in the erythroid cell cultures in vitro. The tumor promoter, 12-O-tetradecanoylphorbol 13-acetate, enhanced the fetal hepatic erythroid colony growth in vitro, but did not alter the hemoglobin phenotypic expression.     

Embryonic erythroid differentiation in the human leukemic cell line K562.

K562 human leukemia cells synthesize embryonic hemoglobins after culture in the presence of hemin. We have rigorously identified these hemoglobins by globin chain analysis and peptide mapping. No adult hemoglobin could be detected, and beta-globin synthesis was less than 2 ppm of total protein synthesis. Persistent embryonic globin gene expression is known to occur as a consequence of globin gene deletions. However, restriction endonuclease mapping showed that the globin gene complexes in K562 cells are indistinguishable from normal. Hemin increased the rate of embryonic globin synthesis. The pattern of hemoglobin synthesis proved to be stable when cells from different laboratories were compared. One line, however, synthesized large amounts of Hb X and very little Hb Portland in response to hemin. Hb X has been previously detected in human embryos; we show here that it has the composition epsilon 2 gamma 2 and is diagnostic of imbalanced chain synthesis or "zeta thalassemia." We have identified several agents that induce hemoglobin synthesis in K562 cells. Different inducers induced different patterns of embryonic hemoglobin synthesis but never any adult hemoglobin synthesis.     

Hemoglobin synthesis in cultures of hepatic erythroid cells from the human fetus

A recent theory of the control of human fetal hemoglobin synthesis, based on studies in cultured adult marrow, proposes that the phenotypic expression of fetal hemoglobin is largely dependent on the level of differentiation of the parental stem cells; that is, the earlier the progenitor, the greater the ability of its progeny to express fetal hemoglobin [Papayannopoulou, Th., Brice, M. & Stamatoyannopoulos, G. (1977) Proc. Natl. Acad. Sci. USA 74, 2923-2927]. To test this relationship with fetal tissue, we have studied hemoglobin synthesis in cultured human fetal liver, comparing gamma chain synthesis in the descendants of the early progenitors ("bursts") with that in the descendants of the later progenitors ("colonies"). Cells from the livers of midtrimester fetuses were cultured in methylcellulose with erythropoietin. The beta/(beta + gamma) globin synthetic ratio on days 5 to 7, when colonies predominated, was 0.09-0.11, a value characteristic of fetal reticulocytes, and on days 11 and 12, when bursts predominated, was 0.15-0.17. Thus, in fetal liver, the descendants of the earlier progenitor, the burst-forming unit, may be making more beta chains rather than more gamma chains, compared to descendants of the later progenitor, the colonyforming unit. Our data on fetal liver, taken together with the data on adult marrow by others, suggest that the erythroid colonies express the gene characteristic of the age of the organism to a greater degree than bursts, which express beta and gamma genes less specifically. Thus, the capacity for highly selective gene expression characteristic of differentiated cells appears to be less well developed in the burst-forming unit than in the colony-forming unit.     

Fetal and embryonic hemoglobins in erythroblasts from fetal blood and fetal cells enriched from maternal blood in fetal anemia.

BACKGROUND AND OBJECTIVES: To determine whether there is a delay or reversal in switch mechanisms from embryonic (e and z) to fetal (g) hemoglobins accompanying the erythroblastosis of anemic fetuses and whether an increased erythroblast count in fetal blood is associated with an increase in feto-maternal cell trafficking. DESIGN AND METHODS: Fetal and maternal blood samples were obtained from 10 cases with rhesus isoimmunization and 2 cases with maternal Parvo-B19 virus at 19-33 weeks' gestation. Blood samples were also taken as controls from 61 fetuses and 86 mothers. Fetal erythroblasts were isolated by triple density gradient centrifugation and magnetic cell sorting with CD71 antibody. Fluorescent antibodies were used to immuno-stain for zeta (z), epsilon (e) and gamma (g) hemoglobin chains. In the maternal samples, fluorescence in situ hybridization (FISH) for X and Y chromosomes was also carried out to confirm the presence and proportion of the enriched fetal cells from maternal blood. RESULTS: In both fetal and maternal blood the percentage of erythroblasts positive for g-globin chain was significantly higher in the anemic fetuses compared to the controls (fetal blood, p<0.001, R=0.91; maternal blood, p<0.001, R=0.56), but there was no significant difference in expression of the e and z-chains. The percentage of cells with Y-signals was also higher in the maternal samples of anemic fetuses compared to normal controls (p<0.001, R=0.56). INTERPRETATION AND CONCLUSIONS: These findings suggest that the erythroblastosis of anemic fetuses is not accompanied by a delay or a reversal in switch from embryonic to fetal hemoglobin chains. Severe fetal anemia is associated with an increase in feto-maternal cell trafficking.     

Synthesis of adult-type hemoglobin in human erythremia cell line

KMOE -2/05 cells, derived from a patient with acute erythremia, became benzidine-positive after the addition of cytosine arabinoside (CA). Radioimmunoassays using antihuman hemoglobin antibodies revealed an elevated amount of hemoglobin in the CA-exposed cells over that in the control cells (without CA). Isoelectric focusing of the CA-exposed cell lysate formed benzidine-positive foci in the positions of human adult Hb (HbA) and human fetal Hb (HbF). To determine the types of globin synthesized in the CA-exposed cells, globin chains internally labeled with [3H] leucine were purified by carboxy-methyl (CM)-Sephadex column chromatography, immunoadsorption by Sepharose-coupled antihuman Hb antibodies and Sephadex G-100. The labeled globin chains were finally separated by CM-cellulose chromatography in urea. Two distinct peaks of radioactivity were shown in exactly the same fractions as carrier human globin alpha- and beta-chains. These observations indicate that these KMOE -2/05 cells synthesize HbA.     

A soluble adenosine triphosphate-dependent proteolytic system in human peripheral red blood cells

A soluble adenosine triphosphate (ATP)-dependent proteolytic system has been detected in human peripheral blood erythroid cells. Hemolysates prepared from reticulocyte-rich blood of subjects with autoimmune hemolytic anemia, treated pernicious anemia, and iron deficiency anemia or from pools of red blood cells enriched for reticulocytes by density gradient centrifugation were tested against a radioactive casein standard. Up to 57% of the casein was rendered trichloroacetic acid (TCA) soluble after incubation with such hemolysates for 60 minutes in the presence of 1.0 mmol/L ATP. In the absence of ATP or in hemolysates prepared from reticulocyte-poor blood as little as 6% to 10% of the casein was hydrolyzed. The proteolytic activity was found in the 100,000-g supernatant of active hemolysates and was blocked by hemin, N- ethylmaleimide, and sodium vanadate and thus resembles a previously described activity in rabbit reticulocytes. In the presence of ATP, similar lysates prepared from rabbit reticulocytes preferentially hydrolyzed the abnormal human hemoglobins Leiden and Gun Hill compared with hemoglobin A. These results suggest that there is an active ATP- dependent proteolytic system in young human erythroid cells that can degrade certain abnormal globin chains; the enzymatic activity is lost in the transition from reticulocyte to erythrocyte.     

Fetal erythropoiesis and hemoglobin ontogeny in tail-short (Ts/+) mutant mice.

Mutant Ts/+ fetuses are developmentally retarded as compared to normal +/+ littermates. Mutant fetuses have less total hemoglobin than do normal fetuses of the same gestational age. However, when compared to +/+ fetuses of similar body weight, Ts/+ fetuses have the appropriate amount of total hemoglobin, suggesting that the apparent anemia observed in mutant fetuses is most likely the result of delay in growth and development. Changes in proportions of embryonic hemoglobins during fetal development are similar in Ts/+ and +/+ fetuses at day 12 and later of gestation. Moreover, adult hemoglobin is detected in circulating primitive nucleated erythrocytes in the developmentally retarded Ts/+ mutant fetuses at about the same chronologic age as their +/+ normal littermates.     

Gamma-interferon alters globin gene expression in neonatal and adult erythroid cells

Interferons have the ability to enhance or diminish the expression of specific genes and have been shown to affect the proliferation of certain cells. Here, the effect of gamma-interferon on fetal hemoglobin synthesis by purified cord blood, fetal liver, and adult bone marrow erythroid progenitors was studied with a radioligand assay to measure hemoglobin production by BFU-E-derived erythroblasts. Coculture with recombinant gamma-interferon resulted in a significant and dose-dependent decrease in fetal hemoglobin production by neonatal and adult, but not fetal, BFU-E-derived erythroblasts. Accumulation of fetal hemoglobin by cord blood BFU-E-derived erythroblasts decreased up to 38.1% of control cultures (erythropoietin only). Synthesis of both G gamma/A gamma globin was decreased, since the G gamma/A gamma ratio was unchanged. Picograms fetal hemoglobin per cell was decreased by gamma-interferon addition, but picograms total hemoglobin was unchanged, demonstrating that a reciprocal increase in beta-globin production occurred in cultures treated with gamma-interferon. No toxic effect of gamma-interferon on colony growth was noted. The addition of gamma-interferon to cultures resulted in a decrease in the percentage of HbF produced by adult BFU-E-derived cells to 45.6% of control. Fetal hemoglobin production by cord blood, fetal liver, and adult bone marrow erythroid progenitors, was not significantly affected by the addition of recombinant GM-CSF, recombinant interleukin 1 (IL-1), recombinant IL-2, or recombinant alpha-interferon. Although fetal progenitor cells appear unable to alter their fetal hemoglobin program in response to any of the growth factors added here, the interaction of neonatal and adult erythroid progenitors with gamma-interferon results in an altered expression of globin genes. This supports the concept that developmental globin gene switching can be regulated by environmental factors.     

Adult hemoglobins are synthesized in erythroid colonies in vitro derived from murine circulating hemopoietic progenitor cells during embryonic development.

Circulating peripheral blood cells and disaggregated yolk sac cells were obtained from normal mouse embryos as early as day 9 of gestation, prior to the formation of the fetal liver. These were cultured in vitro in plasma clots or methylcellulose, in the presence of either embryonic fluid or adult spleen cell-conditioned medium, with or without added erythropoietin. Large erythroid colonies were observed by the sixth day of culture. In all instances, these erythroid colonies synthesized adult hemoglobins. These results indicate that erythroid progenitor cells committed to adult hemoglobin synthesis are present in early embryonic circulation.     

Recombinant human hemoglobins designed for gene therapy of sickle cell disease.

Two human hemoglobins designed to inhibit the polymerization of sickle hemoglobin (Hb S; alpha 2 beta S2) have been produced. Mutations that disrupt the ability of Hb S to form polymers were introduced into the normal human beta-globin gene by site-specific mutagenesis. These mutations affect the axial and lateral contacts in the sickle fiber. The recombinant hemoglobin designated anti-sickling hemoglobin 1 (Hb AS1) contains the mutations beta 22 glutamic acid to alanine and beta 80 asparagine to lysine. Hb AS2 has the same beta 22 glutamic acid to alanine mutation combined with beta 87 threonine to glutamine. Human alpha- and beta AS-globin genes were separately fused downstream of beta-globin locus control region sequences and these constructs were coinjected into fertilized mouse eggs. Transgenic mouse lines that synthesize high levels of each anti-sickling hemoglobin were established and anti-sickling hemoglobins were purified from hemolysates and characterized. Both AS hemoglobins bind oxygen cooperatively and the oxygen affinities of these molecules are in the normal range. Delay time experiments demonstrate that Hb AS2 is a potent inhibitor of Hb S polymerization; therefore, locus control region beta AS2-globin gene constructs may be suitable for future gene therapy of sickle cell disease.     

Specific radioimmunochemical identification and quantitation of hemoglobins a2 and f

Hyperimmune antisera to chromatographically purified hemoglobins F and A₂, were produced in rabbits and made specific for the immunogen by adsorption with normal human hemoglobin A conjugated to cyanogen bromide-activated agarose. A radioimmunoassay was established that permitted identification and quantitation of each of these two minor hemoglobins in hemolysates containing other hemoglobin components. The quantities of hemoglobins A₂, and/or F present in hemolysates of individuals with β-thalassemia, sickle cell anemia, Hb-C disease, and other hematological disorders were determined immunochemically, and the results were compared to values obtained by microcolumn chromatography for the measurement of Hb-A₂ or with the alkali denaturation technique in quantitating Hb-F. The immunoassay procedure has a greater sensitivity than other commonly employed techniques and can detect as little as 0.05 μg of these hemoglobins.     

Hemopoietic stem cells in murine embryonic yolk sac and peripheral blood.

Disaggregated embryonic yolk sac cells and circulating peripheral blood cells were obtained from normal murine day 9 embryos, prior to the formation of the fetal liver. These cells were microinjected transplacentally into days 11-15 W mutant anemic fetuses, when the fetal liver was the major hemopoietic organ. In a small proportion of the recipient animals examined after birth, long-term repopulation by the embryonic donor hemopoietic cells was observed. The donor hemopoietic stem cells proliferated and differentiated in the hosts as evidenced by the presence of donor hemoglobins in the growing recipient host animals. Some mothers of the pups were also repopulated by the donor stem cells. These results provide direct evidence that, during early murine embryogenesis, there are functional hemopoietic stem cells which are capable of colonizing the adult hemopoietic organs and probably the fetal liver and spleen to initiate hemopoiesis in these tissues.     

Hemoglobin F Koelliker (α2 minus 141 (HC 3) Arg γ2) A Modification of Fetal Hemoglobin

An electrophoretically HbA-like hemoglobin component is produced In increasing amounts during storage in hemolysate preparations from macerated tissue (liver, kidney, spleen) of fetuses. Within twenty four hours after hemolysate preparation the “fast moving” fraction increases up to 40 per cent of total hemoglobin, while the concentration of HbA remains constant (5-7 %) in hemolysates obtained from peripheral blood of the same donor individuals. By structural studies (fingerprint and aminoacid analysis) the HbA-like component was identified as ai. artefact of HbF, characterized by the absence of the C-terminal arginine of the α chains. From experimental data it is concluded, that the break down product results from a digestion of HbF by carboxypeptidase B, the enzyme being released from the macerated tissues. Analogous to a modification of HbA, i.e. Hb Koelliker (α₂ minus 141 Arg β₂), the structure of the degradaeion product of Hbf is α₂ minus 141 Arg γ2 (HbF Koelliker).

These findings should be considered when hemolysate preparations contaminated with tissue cells are used for investigations on developmental hemoglobins especially by electrophoretic or chromatographic methods.     

Studies on abnormal hemoglobins. VIII. The gelling phenomenon of sickle cell hemoglobin: its biologic and diagnostic significance

1. When sufficiently concentrated sickle cell hemoglobin containing solutionsare exposed to a constant stream of CO₂ gas, the hemolysates gel. This gellingphenomenon is indicative of the presence of S hemoglobin and cannot be obtainedwith any other type of human hemoglobin in the absence of S pigment. Thelowest S hemoglobin concentration (Gm. per cent) of a hemolysate at which thegelling phenomenon can still be elicited is designated as its lowest gelling point.

2. A simple apparatus was developed to analyze the gelling phenomenon understandardized conditions. It could be shown that the lowest gelling points ofhemolysates prepared from erythrocytes of the sickle cell trait (containing A +S hemoglobins), of the "C variant" (containing C + S hemoglobins), and fromsickle cell anemia cells (containing S + F hemoglobins) differ distinctly. Furtherexperiments suggest that the presence of A hemoglobin decreases the minimalamount of S pigment required for gel formation, and that type C hemoglobinreduces this amount even further. F hemoglobin seems to exert no significantinfluence on the gelling phenomenon. Serum albumin is also capable of decreasingthe amount of S hemoglobin required for gelation.

3. A sickled erythrocyte is visualized as an S hemoglobin tactoid or gel, specifically influenced by the companion pigment which interacts with the S compound.Thus, in the sickle cell trait, a positive sickling test is not only caused by thepresence of S hemoglobin, but also by its interaction with A hemoglobin. Onlyin the sickle cell anemia cells does sickling seem to depend solely upon the interaction of the S hemoglobin molecules.

4. The readily demonstrable differences of the lowest gelling points of hemolysates prepared from the various types of sickling red cells form the basis of thediagnostic gelling test which distinguishes sharply between sickle cell anemia andsickle cell trait erythrocytes. By this procedure atypical cases of sickle cell disease,for example, those whose erythrocytes contain C hemoglobin, may also bedetected.

Submitted on April 21, 1953 Accepted on May 25, 1953     

An immunoassay to detect human embryonic epsilon globin chains by a murine monoclonal antibody

Human embryonic epsilon globin chains were isolated from lysates of cultured human leukemic K562 cells induced with hemin. Mice were immunized with epsilon globin chains, and a murine hybridoma cell line was established that secreted monoclonal anti-epsilon globin antibody. A sensitive and specific slot blot immunoassay for epsilon globin chains has been developed. epsilon Globin chains were shown to be present in hemolysates from fetuses of 20 weeks' gestation but were not detected in normal cord blood hemolysates. This immunoassay is useful for the study of epsilon globin ontogeny during embryonic and fetal development as well as for searching for inappropriate epsilon globin chain expression in hematologic disorders resulting from genetic abnormalities or neoplastic processes.