Loyda N. Vida

Biologic properties and enucleation of red blood cells from human embryonic stem cells

Human erythropoiesis is a complex multistep process that involves the differentiation of early erythroid progenitors to mature erythrocytes. Here we show that it is feasible to differentiate and mature human embryonic stem cells (hESCs) into functional oxygen-carrying erythrocytes on a large scale (10(10)-10(11) cells/6-well plate hESCs). We also show for the first time that the oxygen equilibrium curves of the hESC-derived cells are comparable with normal red blood cells and respond to changes in pH and 2,3-diphosphoglyerate. Although these cells mainly expressed fetal and embryonic globins, they also possessed the capacity to express the adult beta-globin chain on further maturation in vitro. Polymerase chain reaction and globin chain specific immunofluorescent analysis showed that the cells increased expression of beta-globin (from 0% to > 16%) after in vitro culture. Importantly, the cells underwent multiple maturation events, including a progressive decrease in size, increase in glycophorin A expression, and chromatin and nuclear condensation. This process resulted in extrusion of the pycnotic nuclei in up to more than 60% of the cells generating red blood cells with a diameter of approximately 6 to 8 mum. The results show that it is feasible to differentiate and mature hESCs into functional oxygen-carrying erythrocytes on a large scale.

Hematopoietic Progenitor Cells Derived from Embryonic Stem Cells: Analysis of Gene Expression

Rhesus monkey embryonic stem (ES) cells undergo differentiation in vitro to generate hematopoietic progenitor cells. Our previous studies demonstrated a high degree of similarity in the expression of genes associated with hematopoietic differentiation, homing, and engraftment in CD34+ and CD34+CD38− cells from rhesus monkey ES cells and from fresh or cultured bone marrow (BM). In the present study, we compared the expression patterns of cyclins, cyclin‐dependent kinases (CDKs) and CDK inhibitors (CDIs) in these cells. The expression of genes for cyclins, CDKs, and CDIs was similar among the hematopoietic progenitor cells of different origins, with only minor differences. Differentially expressed genes were also analyzed in CD34+ lineage‐negative cells derived from mouse ES cells and from BM. No difference or totally divergent results were obtained with the latter system, suggesting that this variation may be species specific. We observed, however, that CD34+ and CD34+CD38− cells derived from ES cells expressed embryonic ε and ζ as well as α, β, and γ globin genes, whereas no expression of embryonic globins could be detected in the cell preparations from BM. Moreover, erythroblast‐enriched CD34− cells derived from 4‐ or 5‐week ES cell differentiation cultures also expressed embryonic, fetal, and adult globin genes, with greater β gene expression, but otherwise were identical to those of the more primitive CD34+ cells derived from 2‐week ES cultures. These latter observations may reflect the presence of heterogeneous cell populations within the cell fractions that were compared, or they may represent variability among ES‐cell‐derived hematopoietic stem cells.

Comparative gene expression in hematopoietic progenitor cells derived from embryonic stem cells.

OBJECTIVE The aim of this study was to characterize at the molecular level the hematopoietic progenitor cells derived from rhesus monkey embryonic stem (ES) cell differentiation. MATERIALS AND METHODS We purified CD34(+) and CD34(+)CD38(-) cells from rhesus monkey ES cell cultures and examined the expression of a variety of genes associated with hematopoietic development, by semiquantitative polymerase chain reaction analysis. For comparison, we examined cell preparations from fresh or cultured rhesus monkey bone marrow (BM) and from mouse ES cells and BM. RESULTS We observed a high degree of similarity in the expression patterns of these genes, with only a few exceptions. Most notably, the message of the flt3 gene was undetectable in rhesus monkey ES cell-derived CD34(+) and CD34(+)CD38(-) cells, whereas substantial flt3 expression was observed in the corresponding cells from fresh BM and in CD34(+) cells from cultured BM. The integrin alphaL and interleukin-6 (IL-6) receptor genes also were expressed in CD34(+)CD38(-) cells from BM, but there was little or no expression of these genes in CD34(+)CD38(-) cells derived from ES cells. Parallel analyses, using CD34(+)Lin(-) cells derived from murine ES cell cultures, showed no apparent expression of flt3, integrin alphaL, or IL-6 receptor, whereas corresponding cell preparations isolated from mouse BM expressed high levels of all of these genes. CONCLUSIONS ES cell-derived hematopoietic progenitors, both from the rhesus monkey and from the mouse, exhibited the same alterations in gene expression compared with BM-derived cells from these animals. These observations could reflect the presence of different subpopulations in the cell fractions that were compared, or they may represent altered biologic properties of ES cell-derived hematopoietic stem cells.

Juvenile myelomonocytic leukemia (JMML) with the hematologic phenotype of severe β thalassemia

A 3‐year‐old Filipino‐American child with recurrent fever, splenomegaly, anemia, and thrombocytopenia, was found to have a hemoglobin F level of 76.9%. His reticulocyte count was elevated (4.3%), and erythroblasts were present in his peripheral blood. The childs erythrocytes were microcytic (MCV 66.9 fl) but his serum ferritin level was normal. His bone marrow at initial presentation demonstrated normal cellularity without an increase in blast cells. The disease progressed with worsening anemia, leukocytosis, and thrombocytopenia, with increased blasts in his marrow and the appearance of a mediastinal mass. His liver, spleen, and lymph nodes were found to be infiltrated with myeloblasts, supporting a diagnosis of juvenile myelomonocytic leukemia (JMML). Analysis of the childs Hb F showed a Gγ/Aγ ratio of 2.2, which was within the characteristic range for JMML. A globin synthesis study using blood reticulocytes showed an α/non‐α globin synthesis ratio of 2.24, typical of severe homozygous β thalassemia. Southern blot analysis of blood‐leukocyte DNA from the patient and his parents demonstrated no apparent abnormality in the β‐globin gene promoter or coding regions. The elevated level of Hb F in this child with JMML appeared to be part of an acquired Cooleys anemia‐like hematologic phenotype. Am. J. Hematol. 58:67–71, 1998.

Fetal hemoglobin expression in transplant recipients of placental blood hematopoietic progenitor cells.

ABSTRACT: Patients who achieved bone marrow engraftment of cord blood-derived progenitor cells provided an opportunity to examine the expression of fetal Hb by neonatal hematopoietic progenitors in a postneonatal host. Cord blood cells from histocompatible siblings were successfully transplanted in two children with the Fanconi anemia syndrome. One of the transplant donors had heterocellular hereditary persistence of fetal Hb, apparently due to γ-globin gene triplication; the other donor was hematologically normal. The Gγ/Aγratio of the patient who received his transplant from the donor with hereditary persistence of fetal Hb was markedly elevated, similar to that of the transplant donors cord blood, and this ratio remained elevated in subsequent months. In the other child, the Gγ/Aγratio immediately after her transplant was typical of the normal newborn, and over the next several months it reverted to the adult pattern. Globin synthesis studies performed shortly after engraftment demonstrated ratios of fetal Hb/adult Hb synthesis in both patients that were typical of those of normal newborns. Over the next several months, both patients converted to the adult pattern. Fetal Hb to adult Hb switching in these patients seemed to follow a temporal sequence intrinsic to the transplanted neonatal progenitor cells, without discernible influence of postneonatal environmental factors. The program for Hb switching seems to be an inherent feature of neonatal hematopoietic progenitor cells.

HB Mizuho [β68(E12)LEU-PRO]. Second Occurrence Identified in A Caucasian Child with Hemolytic Anenia and Dense Erythrocyte Inclusions

Hb Mizuho [beta 68(E12)Leu----Pro] was identified in a child of Italian/Sicilian descent who exhibited severe, transfusion dependent hemolytic anemia which improved following splenectomy. The patients peripheral blood smear, which prior to splenectomy demonstrated coarse erythrocytic basophilic stippling, showed large, dense erythrocytic hemoglobin inclusions following splenectomy. Whole blood oxygen equilibrium results were consistent with the presence of a hemoglobin component exhibiting increased oxygen affinity with decreased cooperativity. The abnormal beta chain was characterized by high performance liquid chromatography analysis of the isopropanol precipitable hemoglobin fraction.

Sickle Cell Syndromes. I. Hemoglobin SC-α-Thalassemia

Extract: Hematologic and globin synthesis studies were performed in a black American family in which the genes for α-thalassemia and hemoglobins (Hb) S and C were segregating. The following distribution of these abnormalities was found: father, sickle cell trait + α-thalassemia; mother, HbC trait + α-thalassemia, propositus, HbSC + α-thalassemia; older sibling, α-thalassemia trait; and younger sibling, hemoglobin H disease.The child with HbSC-α-thalassemia demonstrated more severe anemia and a more hemolytic picture than is typical of HbSC disease. Her erythrocytes exhibited decreased osmotic fragility in comparison with HbSC erythrocytes, but had an indistinguishable oxygen equilibrium curve and 2,3-diphosphoglycerate (2,3-DPG) level. Erythrocyte sickling in the patient, however, was significantly reduced, with less than 35% sickle forms observed at nearly complete oxygen desaturation.The sibling with hemoglobin H disease exhibited 26% Barts (γ4) hemoglobin at birth, a level comparable with that seen in infants with HbH disease in Far Eastern populations. At age 5 months typical findings of mild hemoglobin H disease appeared, with HbH making up 6.5% of the total hemoglobin.Speculation: The presence of α-thalassemia in the proband of this study appeared to modify her HbSC disease so as to reduce its clinical severity as well as its pathologic potential. The ameliorative effect of α-thalassemia would appear to be related to a reduction in the intracellular hemoglobin concentration in the patients erythrocytes, but other factors may also be responsible for these changes. Eurther study of genetically modified sickle hemoglobinopathy syndromes may ultimately aid in the development of effective means for the treatment of these disorders.

Globin chain synthesis in sickle cell trait under conditions of folate antagonism.

Folic acid deficiency has been reported to produce a substantial change in the ratio of hemoglobin S to hemoglobin A in the blood of individuals having sickle cell trait. Synthesis of the specific <

Hb RAMPA [α95(G2)Pro→Ser (α2)] IN A FAMILY OF EUROPEAN ANCESTRY: DNA ANALYSIS CONFIRMS THE C CG→T CG MUTATION AT CODON 95 OF THE α2-GLOBIN GENE; CLINICAL AND LABORATORY FEATURES

A 53-year-old asymptomatic male of German ancestry was examined because his half-sister had been found to have Hb S trait, according to a state neonatal screening program for hemoglobinopathies. Further investigations showed that a hemoglobin (Hb) variant was present in five members of this kindred, being the proband, his father, his son, one of his daughters, and his half-sister (Fig. 1). The paternal lineage is believed to be derived from Pomerania, i.e., the Baltic region of northeastern Germany. All heterozygotes were apparently well and hematologically normal or nearly normal (Table 1). In each, the variant was approximately 20% of the total Hb. By peptide chromatography and amino acid sequence analysis, it was shown to be Hb Rampa [a95(G2)Pro!Ser (a2)] rather than Hb S [b6(A3)Glu!Val]. Further studies have now characterized the electrophoretic, isoelectrofocusing (IEF) and chromatographic features; the nature of the Hb Rampa mutation, the stability of Hb Rampa, and its effect on oxygen affinity of whole blood.

Sickle Cell Syndromes. III. Silent-Carrier α-Thalassemia in Combination with Hemoglobin S and Hemoglobin C

Summary: Silent carrier α-thalassemia was identified in two individuals, one with sickle-cell trait and the other hemoglobin (Hb) C trait. Both are parents of a child with characteristic hematologic features of the Hb SC-α thalassemia syndrome, including microcytosis and an unbalanced pattern of globin synthesis. In contrast to the typical findings that accompany heterozygous Hb S or Hb C with concomitant α-thalassemia trait, neither of the parents had microcytosis nor a percent of the abnormal hemoglobin in their erythrocytes that was below the normal range. In both, however, globin synthesis of peripheral blood reticulocytes was unbalanced, consistent with mild α-thalassemia. These findings suggest that the α-thalassemia silent carrier may be hematologically indistinguishable from the nonthalassemic individual, even when hemoglobin S or C are present.Speculation: These findings provide additional evidence that the genetic pattern of α-thalassemia in American blacks is probably similar to that previously characterized in Far-Eastern populations, in spite of the clear differences in the clinical and hematologic expression of these disorders in the two populations.