Embryonic Hemoglobins Research Articles

Induced Pluripotent Stem Cells and Erythrocyte Production

Abstract SCI-38Induced pluripotent stem cells (iPSCs) are somatic cells that have been turned into embryonic-like stem cells by forced expression of factors critical for establishing pluripotency. Because iPSCs can be differentiated into any type of cell in the human body, including hematopoietic cells, they are seen as a logical alternative source of red blood cells (RBCs) for transfusion. In addition, the unlimited expansion potential of iPSCs makes it easy to adopt iPSC technology for RBC biomanufacturing. iPSCs can be generated from any type of donor, including O/Rh-negative universal donors and donors with very rare blood phenotypes, which makes it possible to generate blood products to accommodate virtually all patient groups. We have developed an approach for generating large quantities of RBCs from iPSCs by inducing them to differentiate into CD34+CD43+ hematopoietic progenitors in coculture with OP9 stromal cells, followed by selective expansion of erythroid cells in serum-free media with erythropoiesis-supporting cytokines. Erythroid cultures produced by this approach consist of leukocyte-free populations of CD235a+ RBCs with robust expansion potential and long (up to 90 days) life spans. In these cultures, up to 1.8×105 RBCs can be generated from a single iPSC. Similar to embryonic stem cells, iPSC-derived RBCs express predominantly embryonic and fetal hemoglobin, with very little adult hemoglobin. It is already feasible to adopt iPSC technologies for producing cGMP-grade RBCs using defined animal-product-free differentiation conditions. However, the induction of the complete switch from embryonic to fetal and adult hemoglobin, as well as the terminal maturation and enucleation of iPSC-derived erythroid cells, remains a significant challenge. We recently identified at least three distinct waves of hematopoietic progenitors with erythroid potential in iPSC differentiation cultures. The characterization of erythroid cells produced from these waves of hematopoiesis may help to define populations with definitive erythroid potential and facilitate the production of erythrocytes from iPSCs. Additional critical steps toward translating iPSC-based RBC technologies to the clinic include the development of bioreactor-based-technology for further scaling-up of cell production, and evaluation of the therapeutic potential and safety of human pluripotent stem cell-derived blood cells in animal models. Overall, the manufacturing of RBCs provides several advantages. It can improve the continuity of the blood supply, minimize/eliminate the risk of infection transmission, reduce the incidence of hemolytic and nonhemolytic transfusion reactions, and provide an opportunity to generate RBCs that fit specific clinical needs by using genetically engineered iPSCs or iPSCs with rare blood groups. Disclosures:Slukvin:CDI: Consultancy, Equity Ownership; Cynata: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Anemia in Infancy

1. Jennifer Cobelli Kett, MD 1. Children’s National Medical Center Washington, DC 1. Anemia and Pallor . Kolb EA, Levy AS. In: McInerny TK, Adam HM, Campbell DE, Kamat DM, Kelleher KJ, eds. American Academy of Pediatrics Textbook of Pediatric Care. Elk Grove Village, IL: American Academy of Pediatrics; 2009:1395–1405. 2. Diseases of the Blood . In: Behrman RE, Kliegman RM, Jenson HB, eds. Nelson Textbook of Pediatrics. 17th ed. Philadelphia, PA: Saunders; 2004:1599–1678. 3. The Blood and Hematopoietic System . In: Martin RJ, Fanaroff AA, Walsh MC, eds. Fanaroff and Martin’s Neonatal-Perinatal Medicine: Diseases of the Fetus and Infant. 8th ed. Philadelphia, PA: Mosby; 2006:1287–1356. Anemia is the reduction in red blood cell (RBC) number, hematocrit, or hemoglobin concentration to a value >2 SDs below the age-specific mean. Anemia in infancy, which may result from increased erythrocyte loss or inadequate RBC production, raises unique considerations. Understanding the development of the hematopoietic system may be helpful in the evaluation of neonates with anemia. Erythropoiesis begins in the yolk sac at 2 weeks’ gestation, generating cells that express embryonic hemoglobin. At 6 weeks’ gestation, the liver becomes the predominant site of RBC production, and the cells produced primarily express fetal hemoglobin. Not until 6 months’ gestation does the bone marrow become the major site of hematopoiesis. Throughout fetal life, erythrocytes decrease in size and increase in number: hematocrit increases from 30% to 40% during the second trimester to 50% to 63% at term. In late gestation and after birth, RBCs gradually switch from the production of fetal hemoglobin to adult hemoglobin. After birth, RBC mass normally declines in response to an increase in the availability of oxygen and downregulation of erythropoietin. RBC count decreases until oxygen delivery is inadequate for metabolic demand and erythropoietin production is stimulated again. In healthy term infants, the RBC nadir, a physiologic response to postnatal life and not a hematologic disorder, typically occurs at 8 to 12 weeks of life and at a hemoglobin level of 9 to 11 g/dL. Preterm infants also experience a decrease in hemoglobin concentration after birth, with a decline that typically is more abrupt and more profound than in term infants, reaching hemoglobin levels of …

A New Case of KLF1 G973A Mutation and Congenital Dyserythropoeitic Anemia (CDA)- Further Definition of Emerging New Syndrome and Possible Association with Gonadal Dysgenesis

Abstract▪2101▪This icon denotes a clinically relevant abstractArnaud et ala recently reported on two children with an as yet unclassified CDA, characterized by hemolytic anemia with high fetal hemoglobin (HbF), presence of embryonic hemoglobin Portland (HbPort) and absence on red blood cells (RBC) of CD44, an adhesion molecule, aquaporin 1 (AQP1) and the linked In(Lu) and Colton blood groups. The underlying molecular defect involves the erythroid transcription factor KLF1 with a mutation in zinc finger 2 at position 973 of Guanine to Adenine. The G973A mutation results in substitution of glutamate to lysine at position 325 in the transcribed protein. The CDA results from a dominant negative effect of KLF1 E325K on the gamma-globin repressor BCL11A, leading to the persistence of fetal erythropoiesis, high Hb F and HbPort. The absence of CD44, AQP1 and deficiency of certain cytoskeletal proteins in the RBC indicate that KLF1 is necessary for the terminal differentiation of RBC. The presence of CD44 on the leucocytes in these cases would appear to suggest an erythroid restrictive effect of the mutation. Of note, the child in whom the E325K mutation was first identified, a 46XY male, was described to have micropenis and hypospadias; the hemolytic anemia was severe and required splenectomy. The original case was that of phenotypic female with 46XX and the clinical course was much milder.We have identified a new case (a caucasian child) with the KLF1 E325K mutation. Comparison with the clinical findings in the two published cases indicates that KLF1 E325K mutation may cause profound developmental disturbances in organs beyond erythroid development, particularly of the gonads. Our patient, now 5 yrs old, was followed with an undiagnosed hemolytic anemia, which started with fetal hydrops at 23 weeks gestation, requiring intrauterine transfusions. Because of the presence of spherocytes on the blood smear and history of renal stones, a mutant band 3 was suspected; however direct nucleotide sequence analysis of the promoter and coding region exons of the band 3 gene from amplified genomic DNA of the proband was normal. Splenectomy was done at 4-years-of-age and the emerging picture was one of extreme normoblastemia, with the presence of both spherocytes and massively enlarged erythrocytes. Osmotic gradient ektacytometry showed reduced erythrocyte deformability with a population of swollen erythrocytes. Hemoglobin analyses revealed a HbF of31% and, HgPort (isoelectric focusing done by James Hoyer at Mayo Laboratories). Flow cytometry demonstrated that CD44 was not expressed on patient's erythrocytes but was expressed on lymphocytes. AQP1 was absent on western blots of RBC ghosts. Nucleotide sequence analysis of the KLF1 gene identified the KLF1 E325K mutation in lymphocyte DNA from the patient, but not in her parents. In addition to the severe anemia, the child who is 46XY, has female external genitalia. Ultrasonography demonstrated a vagina and uterus. Surgically excised streak gonads showed no cellular elements. Array comparative genomic hybridization studies did not reveal any copy number variations in male sex determining genes, but identified polymorphic deletion of estrogen metabolizing UGT2B17.Together these 3 cases suggest that KLF1 E325K may cause more severe anemia and gonadal dysgenesis in 46XY fetuses while the disease is considerably milder in 46XX individuals. Both of the 46 XY cases also had fetal hydrops and were salvaged with transfusions in utero, suggesting that KLF1 mutations may cause early loss of male fetuses. Given that KFL1 interacts with more than 1000 proteins,b,c it is likely that KLF1 is important in the developmental regulation of non erythroid genes including male sex determining genes. Disclosures:No relevant conflicts of interest to declare.

Congenital dyserythropoietic anemias

Congenital dyserythropoietic anemias (CDAs) are rare hereditary disorders characterized by ineffective erythropoiesis and by distinct morphological abnormalities of erythroblasts in the bone marrow. Characteristic morphological aberrations were the cornerstone of diagnosis, but following the identification of several causative genes, the molecular approach could represent a rapid tool for the identification of these conditions. This review presents advances in diagnosis and classification of CDAs. The classification of CDAs has long been based on morphological features. Now, the discovery of some of the responsible genes allows reconsideration of part of the classification. The first CDA partly accounted for genetically has been CDA 1, through the discovery in 2002 of the gene responsible, CDAN1, encoding codanin-1. Recently, the dramatic identification of the genes responsible for CDA II, SEC23B, and for a hitherto unnamed CDA, KLF1, took place. SEC23B encodes SEC23B which is a component of the coated vesicles transiting from the endoplasmic reticulum to the cis compartment of the Golgi apparatus. A unique mutation in KLF1, which encodes the erythroid transcription factor KLF1, causes major ultrastructural abnormalities, the persistence of embryonic and fetal hemoglobins, and the absence of some red cell membrane proteins. Studies of genotype-phenotype relationship, as has already been done for CDA II, will allow a more accurate prognosis. Identification of the responsible genes has opened new vistas for research on CDAs.

MicroRNA-15a and -16-1 act via MYB to elevate fetal hemoglobin expression in human trisomy 13

Many human aneuploidy syndromes have unique phenotypic consequences, but in most instances it is unclear whether these phenotypes are attributable to alterations in the dosage of specific genes. In human trisomy 13, there is delayed switching and persistence of fetal hemoglobin (HbF) and elevation of embryonic hemoglobin in newborns. Using partial trisomy cases, we mapped this trait to chromosomal band 13q14; by examining the genes in this region, two microRNAs, miR-15a and -16-1, appear as top candidates for the elevated HbF levels. Indeed, increased expression of these microRNAs in primary human erythroid progenitor cells results in elevated fetal and embryonic hemoglobin gene expression. Moreover, we show that a direct target of these microRNAs, MYB, plays an important role in silencing the fetal and embryonic hemoglobin genes. Thus we demonstrate how the developmental regulation of a clinically important human trait can be better understood through the genetic and functional study of aneuploidy syndromes and suggest that miR-15a, -16-1, and MYB may be important therapeutic targets to increase HbF levels in patients with sickle cell disease and β-thalassemia.

SIRT1 Deficiency Compromises Mouse Embryonic Stem Cell Differentiation, and Embryonic and Adult Hematopoiesis In the Mouse.

AbstractAbstract 1612SIRT1 is a conserved NAD-dependent deacetylase capable of deacetylating a number of protein substrates including, but not limited to, p53 and FOXO transcription factors. SIRT1 plays an important role in a variety of biological processes including stress resistance, metabolism, differentiation and aging (Rodgers et al, Nature, 2005; 434:113). SIRT1 is expressed at high levels in mouse embryos. A role for SIRT1 in mouse (m) embryonic stem cell (ESC) maintenance and differentiation is only beginning to be elucidated (Han et al, Cell Stem Cell, 2008; 2:241, Calvanese V et al, PNAS, 2010; 10713736). Here we focus on a role for SIRT1 in differentiation of mESCs into hematopoietic progenitors (HPCs), and in embryonic and adult hematopoiesis. We hypothesized that SIRT1 is involved in hematopoietic commitment within the mouse. We initially assessed the ability of WT and SIRT1-/- mESC to give rise to blast colony forming cells (BL-CFC), a transient population that is present in EBs between day 2.5 and day 3.5 of differentiation and represents the in vitro equivalent of the hemangioblast and as such, the earliest commitment step in the differentiation of mesoderm to the hematopoietic and endothelial lineages. SIRT1-/- ESCs exhibited markedly delayed formation of BL-CFC. The emergence of the Flk-1+/c-Kit- cell population pattern was also delayed, consistent with the delayed pattern of BL-CFC development in SIRT1-/- EBs. This observed delay appears to result from a slower differentiation of the SIRT1-/- ESCs as the kinetics of decline in secondary EB potential, an indication of undifferentiated ES cells, is delayed compared to that of SIRT1+/+ ES cells. When analyzed for hematopoietic and endothelial potential of individual blast colony, replated SIRT1-/- BL-CFC presented limited hematopoietic potential, whereas endothelial potential was essentially unaltered. Next, the ability of SIRT1-/- ESCs to form primitive and definitive hematopoietic cells was evaluated and we found that primitive erythroid progenitors formed from SIRT1-/- R1 cells were not only delayed but greatly decreased. Moreover, after differentiation of SIRT1 -/- mESC there were also significant decreases in granulocyte-macrophage (CFU-GM), and multipotential (CFU-GEMM) progenitors. Differences in primitive and definitive erythroid progenitors were confirmed by gene analysis of βH1 globin (embryonic hemoglobin), a marker for primitive erythroid cells, and βmajor globin (adult hemoglobin). The above delay defects were associated with delayed ability to switch off Oct4, Nanog and Fgf5, decreased β-H1 Globin, β-major globin, Scl gene expression and reduced activation of the Erk1/2 pathway upon SIRT1-/- ESC commitment. Reintroduction of WT SIRT1 into SIRT1-/- cells partially rescued the primitive erythroid progenitor formation of SIRT1-/- cells and the expression of hemoglobin genes, Hbb-bh1 and Hbb-b1, suggesting that the defect of hematopoietic commitment is due to deletion of SIRT1, and not to genetic drifting of SIRT1-/- cells. To confirm SIRT1 effects, we assessed embryonic and adult hematopoiesis in SIRT1+/+, +/− and -/- mice. Yolk sacs from SIRT1 mutant embryos generated fewer primitive erythorid precursors compared to wild-type and heterozygous mice. Moreover, knockout of SIRT1 decreased primary bone marrow HPCs in 5 week and 12 month old mice, effects especially notable at lower (5%) O2 tension. Taken together, these results demonstrate that SIRT1 plays a role in mouse embryonic and adult stem cell differentiation. Disclosures:No relevant conflicts of interest to declare.

The Hox cofactors Meis1 and Pbx act upstream of gata1 to regulate primitive hematopoiesis

During vertebrate development, the initial wave of hematopoiesis produces cells that help to shape the developing circulatory system and oxygenate the early embryo. The differentiation of primitive erythroid and myeloid cells occurs within a short transitory period, and is subject to precise molecular regulation by a hierarchical cascade of transcription factors. The TALE-class homeodomain transcription factors Meis and Pbx function to regulate embryonic hematopoiesis, but it is not known where Meis and Pbx proteins participate in the hematopoietic transcription factor cascade. To address these questions, we have ablated Meis1 and Pbx proteins in zebrafish, and characterized their molecular effects on known markers of primitive hematopoiesis. Embryos lacking Meis1 and Pbx exhibit a severe reduction in the expression of gata1, the earliest marker of erythroid cell fate, and fail to produce visible circulating blood cells. Concomitant with a loss of gata1, Meis1- and Pbx-depleted embryos exhibit downregulated embryonic hemoglobin (hbae3) expression, and possess increased numbers of pu.1-positive myeloid cells. gata1-overexpression rescues hbae3 expression in Pbx-depleted; meis1-morphant embryos, placing Pbx and Meis1 upstream of gata1 in the erythropoietic transcription factor hierarchy. Our study conclusively demonstrates that Meis1 and Pbx act to specify the erythropoietic cell lineage and inhibit myelopoiesis.

Primitive erythropoiesis in the mammalian embryo

Erythropoiesis in adult mammals is characterized by the progressive maturation of hematopoietic stem cells to lineage-specific progenitors, to morphologically identifiable precursors which enucleate to form mature erythrocytes. In contrast, primitive erythropoiesis is characterized by the appearance within the yolk sac of a transient, lineage-restricted progenitor population which generates a wave of erythroid precursors. These precursors undergo progressive maturation in the bloodstream, characterized by nuclear condensation and embryonic hemoglobin accumulation. This process is dependent on erythropoietin signaling through its cognate receptor, as well as the function of several erythroid-specific transcription factors, including GATA1 and EKLF. Targeted disruption of genes in the mouse that result in failure of the emergence or maturation of the primitive erythroid lineage leads to early fetal death, indicating that the primitive erythroid lineage is necessary for survival of the mammalian embryo. While it was thought for over a century that primitive erythroid cells were uniquely nucleated mammalian red cells, it is now recognized that they, like their definitive erythroid counterparts, enucleate to form reticulocytes and pyrenocytes. This surprising finding indicates that the primitive erythroid lineage is indeed mammalian, rather than non-mammalian, in character.

SIRT1 Is Required for Mouse Embroyonic Stem Cell Commitment to Hematopoietic Cell Differentiation.

Abstract 811SIRT1 is a conserved NAD-dependent deacetylase that catalyzes deacetylation of acetyl-lysine residues of proteins such as histone and p53. SIRT1 plays an important role in a variety of biological processes including stress resistance, metabolism, differentiation and aging (Rodgers et al, Nature, 2005; 434:113). A role for SIRT1 in mouse (m) embryonic stem cell (ESC) maintenance is only beginning to be elucidated (Han et al, Cell Stem Cell, 2008; 2:241). Here we focus on a role for SIRT1 in differentiation of mESCs into hematopoietic cells. We hypothesized that SIRT1 is involved in mESC commitment to hematopoietic cell differentiation. We first confirmed that SIRT1 is highly expressed in the R1 mESC line. Hemoglobinized embryoid bodies (EBs) formed from SIRT1−/− R1 ESC were greatly decreased in number upon removal of LIF compared with that of wildtype parental (+/+) R1 cells, as assessed by primary differentiation assay. Differences in hemoglobinized cells were confirmed by gene analysis of βH1 globin (embryonic hemoglobin), markers for primitive erythroid cells. Next, the ability of SIRT1−/− ESCs to form primitive and definitive hematopoietic cells was evaluated and we found that primitive erythroid progenitors formed from SIRT1−/− R1 cells were greatly decreased. Moreover, after differentiation of SIRT1 −/− mESC there were also significant decreases in definitive erythroid (BFU-E), granulocyte-macrophage (CFU-GM), and multipotential (CFU-GEMM) progenitors. We next explored hematopoietic differentiation in EBs from SIRT1−/− cells by flow cytometry analysis of expression of surface antigens. CD41 defines the onset of primitive and definitive hematopoiesis in the murine embryo (Ferkowicz et al, Development, 2003; 130(18): 4393-403). There were much fewer CD41+ cells in SIRT1 d7 EBs compared with those in WT d7 EBs. To further investigate a role for SIRT1 in hematopoietic differentiation, SIRT1−/− ESCs were tested in an alternative in vitro hematopoietic system involving use of the OP9 stromal cell line and after ectopically-inducing HOXB4 to expand hematopoietic cell differentiation. Unlike WT cells, cells from day 6 EBs of SIRT1−/− ESCs did not differentiate into hematopoietic clusters, instead forming mesoderm-like colonies. This suggested that the defect in differentiation of SIRT1−/− ESCs into hematopoietic cells is before the onset of primitive erythropoiesis. Vascular endothelial growth factor (VEGF)-responsive blast cell colonies are known to contain both endothelial and hematopoietic precursors. This blast-colony-forming cell (BL-CFC) represents a transient population that is present in EBs between day 2.5 and day 3.5 of differentiation and represents the in vitro equivalent of the hemangioblast and as such, the earliest commitment step in the differentiation of mesoderm to the hematopoietic and endothelial lineages. We therefore assessed the ability of WT and SIRT1−/− cells to give rise to BL-CFC. When compared, SIRT1−/− cells generated significantly reduced number of blast colonies, while more colonies of tightly associated cells were observed than with WT cells in the BL-CFC clonogenic assay. Differentiation of mESC towards mesoderm and hemangioblasts (Blast-colony-forming) within the EBs was assessed by measuring brachyury and flk-1 expression respectively. There was no difference in expression of brachyury. However, flk-1 expression was remarkably reduced in SIRT1−/− EBs compared to +/+ EBs. These results indicated that SIRT1−/− cells differentiate properly into mesoderm, while they have a defect in differentiation into blast colonies. Reintroduction of WT SIRT1 into SIRT1−/− cells rescued the hemoglobinized EB formation of SIRT1−/− cells, suggesting that the defect of hematopoietic commitment is due to deletion of SIRT1, and not to genetic drifting of SIRT1−/− cells. Taken together, these results demonstrate that SIRT1 plays a role in hemangioblast development and the earliest stages of hematopoietic cell commitment. Disclosures:No relevant conflicts of interest to declare.

Both Primitive and Definitive Hematopoiesis Arise From Precursors with Endothelial Potential in the Zebrafish.

Abstract 697Development of the blood system is complex, with multiple waves of hematopoietic precursors arising in different embryonic locations. Monopotent, or primitive, precursors first give rise to embryonic macrophages or erythrocytes. Multipotent, or definitive, precursors are subsequently generated to produce the adult-type lineages. In both the zebrafish and mouse, the first definitive precursors are committed erythromyeloid progenitors (EMPs) that lack lymphoid potential. We have previously shown that zebrafish EMPs arise in the posterior blood island independently from hematopoietic stem cells (HSCs) that arise along the ventral side of the aorta. By using the CRE/LOX technology, recent reports have nicely showed that vascular endothelial cells on the ventral side of the aorta were giving rise to HSCs in the mouse embryo.Here we show similar results in the zebrafish, that HSCs are also born from hemogenic endothelial cells (ECs). We used the kdrl/flk1:CRE transgenic line. Unlike in mammals, the zebrafish flk1 promoter used here is not pan-mesodermic but restricted to ECs and their precursors, so we could specifically trace the progeny of ECs in the zebrafish by crossing this CRE line to a switch-reporter line. This latter consists of the b-actin promoter driving the expression of DsRED after a STOP cassette is excised by the CRE recombinase. Double transgenic adults were sacrificed at 3 months, and in all animals (n=30), 100% of adult leukocytes were switched (DsRED+). Thus, zebrafish HSCs originate from ECs in the embryo. To corroborate these findings, we could isolate cells during their transition from EC to hematopoietic cells using the flk1:RFP and cmyb:GFP reporter lines, and show that, during the transition, the endothelial genetic program is shut down, while hematopoietic genes are turned on. Moreover, by using the transparency of the zebrafish embryo, we could directly image the birth of HSCs from the ventral floor of the aorta.As stated above, three other hematopoietic lineages arise during embryogenesis: primitive macrophages/erythrocytes and definitive EMPs. Whether or not these other lineages arise from hemogenic ECs is still unknown. In order to address this question, we generated triple transgenic animals (flk1:CRE; b-actin:switch; lineage:GFP), where the lineage specific promoter was either lysozymeC or cd41, in order to mark primitive macrophages (lysC+ at 24hpf) and EMPs (cd41+ at 26-30hpf), respectively. In all combinations studied, GFP-positive cells were always also expressing DsRED from the switch reporter, revealing that primitive macrophages and EMPs were also derived from flk1+ cells in the embryo. Primitive erythrocytes could not be traced as well, since the b-actin promoter driving the switch reporter is silenced during erythroid commitment. However, flk1:eGFP+ cells could be sorted as early as 8-10 somites during zebrafish development, and we could show that only this fraction in the embryo was expressing the transcripts for gata1 and hbae3/hbae1 (embryonic hemoglobins). Thus we conclude that primitive erythroblasts originate from a flk1-expressing subset of cells in the embryo.As previously described, HSCs do arise from hemogenic ECs in the aorta, which we can now directly visualize. Primitive macrophages and erythrocytes also derive from flk1-expressing cells in the early embryo, which are likely to be hemangioblasts since vasculogenesis has not occurred yet. Finally, our data do not allow to precisely define the origin of EMPs, which could be attributed to either a caudal hemangioblast or a hemogenic endothelium. Altogether, these results should give new insights on the generation of blood cells in the vertebrate embryo. Disclosures:No relevant conflicts of interest to declare.

Energetic Differences at the Subunit Interfaces of Normal Human Hemoglobins Correlate with Their Developmental Profile

A previously unrecognized function of normal human hemoglobins occurring during protein assembly is described, i.e. self-regulation of subunit pairings and their durations arising from the variable strengths of their subunit interactions. Although many mutant human hemoglobins are known to have altered subunit interface strengths, those of the normal embryonic, fetal, and adult human hemoglobins have not been considered to differ significantly. However, in a comprehensive study of both types of subunit interfaces of seven of the eight normal oxy human hemoglobins, we found that the strengths, i.e., the free energies of the tetramer-dimer interfaces, contrary to previous reports, differ by 3 orders of magnitude and display an undulating profile similar to the transitions ("switches") of various globin subunit types over time. The dimer interface strengths are also variable and correlate linearly with their developmental profile. Embryonic hemoglobins are the weakest; fetal hemoglobin is of intermediate strength, and adult hemoglobins are the strongest. The pattern also correlates generally with their different O(2) affinities and responses to allosteric regulatory molecules. Acetylation of fetal hemoglobin weakens its unusually strong subunit interactions and occurs progressively as its level of expression diminishes and adult hemoglobin A formation begins; a causal relationship is suggested. The relative contributions of globin gene order and competition among subunits due to differences in their interface strengths were found to be complementary and establish a connection among genetics, thermodynamics, and development.

Inactivation of G-protein-coupled Receptor 48 (Gpr48/Lgr4) Impairs Definitive Erythropoiesis at Midgestation through Down-regulation of the ATF4 Signaling Pathway

G-protein-coupled receptors (GPCRs), one of the most versatile groups of cell surface receptors, can recognize specific ligands from neural, hormonal, and paracrine organs and regulate cell growth, proliferation, and differentiation. Gpr48/LGR4 is a recently identified orphan GPCR with unknown functions. To reveal the functions of Gpr48 in vivo, we generated Gpr48-/- mice and found that Gpr48-/- fetuses displayed transient anemia during midgestation and abnormal definitive erythropoiesis. The dramatic decrease of definitive erythroid precursors (Ter119pos population) in Gpr48-/- fetal liver at E13.5 was confirmed by histological analysis and blood smear assays. Real-time PCR analyses showed that in Gpr48-/- mice both adult hemoglobin alpha and beta chains were decreased while embryonic hemoglobin chains (zeta, betaH1, and epsilony) were increased, providing another evidence for the impairment of definitive erythropoiesis. Furthermore, proliferation was suppressed in Gpr48-/- fetal liver with decreased c-Myc and cyclin D1 expression, whereas apoptosis was unaffected. ATF4, a key transcription factor in erythropoiesis, was down-regulated in Gpr48-/- fetal livers during midgestation stage through the cAMP-PKA-CREB pathway, suggesting that Gpr48 regulated definitive erythropoiesis through ATF4-mediated definitive erythropoiesis.

Efficient Erythroid Differentiation of a PGD-Derived Human Pluripotent Stem Cell Line Affected with Sickle Cell Hemoglobinopathy

The technology of preimplantation genetic diagnosis (PGD) screens IVF-derived cleavage-stage embryos or oocytes from parents with inherited disorders, and is routinely used to avoid births with severe genetic disorders. More than one hundred testable genetic conditions, including severe hematologic diseases such as beta thalassemia, sickle cell anemia, and Fanconi anemia can be PCR-screened from either a micro-manipulated blastomere, or a pre-fertilization ovarian polar body. Derivation of human embryonic stem cell (hESC) lines from diseased IVF blastocysts has recently been reported, and these PGD-hESC are untested yet potentially valuable tools for investigating cellular and molecular events of human embryogenesis in diseased states. For example, a great deal of interest has recently been generated in treating hematologic diseases with genetically-corrected hematopoietic stem cells (HSC) derived from patient-specific pluripotent stem cells. Generation of hematopoietic progenitors from PGD-hESC affected with genetic syndromes may thus provide novel opportunities for testing cell-based and gene therapeutic strategies. An important candidate for such cell-based therapy includes sickle cell disease (SSD) hemoglobinopathy, a classic inherited single gene disorder resulting from the substitution of glutamic acid by valine at position 6 of the beta chain of hemoglobin. Human pluripotent stem cells derived from PGD-selected blastocysts or induced pluripotency (iPS; e.g., using patient's somatic cells), will serve as critical reagents for testing such therapeutic strategies. In these studies, we report the characterization and erythropoietic differentiation of a novel PGD-hESC line affected with SSD hemoglobinopathy. The sickle point mutation was confirmed in this PGD-hESC line with direct genomic sequencing of the beta globin locus. This hESC line possesses typical pluripotency characteristics and forms multilineage teratomas in vivo. SSD-hESC can be efficiently differentiated to the hematopoietic lineage under serum-free conditions, and gave rise to robust primitive and definitive erythropoieses. The expression of embryonic, fetal and adult globin genes in SSD PGD-hESC derived erythroid cells was confirmed by qRT-PCR, intracytoplasmic FACS, and in situ immunostaining of PGD-hESC teratoma sections. Moreover, we defined culture conditions for massive, long-term liquid culture expansion of sickle affected erythroid progenitors that remained in an undifferentiated erythroblastic phenotype for at least two months. These sickle erythroblasts were continuously maintained as a primary cell line that could be frozen and thawed without loss of viability. In vitro-expanded sickle erythroblasts expressed CD71+CD36+ and CD71+CD235a+ phenotypes, and underwent developmentally appropriate embryonic, fetal, and adult hemoglobin switching over a period of several months. Moreover, hESC-derived erythroblasts were readily infected with Plasmodium falciparum malaria parasites, thus demonstrating their potential utility in studying the effects of this important pathogen in normal and diseased erythropoiesis. These data demonstrate the utility of using patient-specific, hemoglobinopathic hESC for generating significant numbers of erythroid progenitors for molecular, developmental, gene therapeutic, pharmacologic, and microbiological studies. We are currently conducting a comparative erythropoietic differentiation study using SSD PGD-hESC vs. SSD iPS that were generated from somatic fibroblasts using defined pluripotency factors.

Structural Analysis of a Methyl Cytosine-Binding Protein Complex That Regulates Expression of Fetal/Embryonic Globin Genes

DNA methylation is one of the major epigenetic mechanisms for regulating gene expression in eukaryotic organisms. Regions of increased CpG content (CpG islands) are often associated with gene promoters and can recruit regulatory complexes that specifically recognize methyl cytosine and down-regulate expression of the associated gene. One such regulatory complex, MeCPC, has recently been purified from a primary cell source and characterized as binding to the methylated ρ-globin promoter in chickens [Kransdorf et al. Blood 2006; 108:2836–45]. This complex contains the chicken homologues of MBD2, MTA1, HDAC2, p66, and Mi2. Methylation of the ρ-globin promoter and binding of the MeCPC promotes the switch to β-globin during development while siRNA knock-down of cMBD2 leads to a 25-fold increase in ρ-globin expression. Likewise, knockout of MBD2 results in a ~20 fold upregulation of the human gamma globin gene in β.YAC transgenic mice [Rupon et al. PNAS 2006; 103:6617–22]. These observations suggest that disruption of the MeCPC would increase fetal hemoglobin expression; a therapeutically beneficial effect for both sickle cell anemia and β-thalassemia. This possibility is further supported by the observation that DNA methylation inhibitors such as 5-azacitidine can increase the expression of γ-globin in patients. In order to determine the molecular details of the interactions that form the MeCPC, we are pursuing atomic resolution structural analysis of MBD2 and its binding partners. The solution structure of the methyl binding domain from MBD2 bound to a methylated oligonucleotide from the ρ-globin promoter has been determined by NMR. MBD2 recognizes a central methylated CpG in this oligonucleotide through highly conserved arginine and tyrosine amino acids. Interestingly, MBD2 adopts a single orientation on the ρ-globin promoter sequence despite the symmetry of the CpG sequence. This observation indicates that interactions between MBD2 and the nucleotides surrounding the methylated CpG likely orient the protein on this promoter and may help explain why different methyl binding domain proteins are selectively recruited to different promoter regions and CpG islands. This work represents the first atomic resolution structure reported to date of MBD2 bound to a methylated promoter sequence identified as a target in a primary cell source. Additional structural studies reported here build on the observation that MBD2 directly interacts with p66α through two highly conserved regions in p66α and two regions in MBD2 [Brackertz et al. J. Biol. Chem. 2002; 277:40958–66]. The coiled coil domains of each protein have been characterized by gel filtration chromatography and NMR spectroscopy. These studies demonstrate that a specific and stable interaction is formed between the coiled domains from each protein. Intermolecular NOEs have been measured by NMR which highlight the amino residues responsible for direct protein:protein contacts. Ultimately these structural studies will provide a molecular basis for the development of agents that will specifically disrupt the MeCPC complex and increase expression of embryonic and fetal hemoglobin in adult erythrocytes.

Gfi1.1 regulates hematopoietic lineage differentiation during zebrafish embryogenesis

Growth factor independence 1 (GFI1) is important for maturation of mammalian lymphocytes and neutrophils and maintenance of adult hematopoietic stem cells (HSCs). The role of GFI1 in embryonic hematopoiesis is less well characterized. Through an enhancer trap screen and bioinformatics analysis, we identified a zebrafish homolog of Gfi1 (named gfi1.1) and analyzed its function during embryonic development. Expression of both an endogenous gfi1.1 gene and a GFP reporter gene inserted near its genomic locus was detected in hematopoietic cells of zebrafish embryos. Morpholino (MO) knockdown of gfi1.1 reduced expression of scl, lmo2, c-myb, mpo, rag1, gata1 and hemoglobin alpha embryonic-1 (hbae1), as well as the total amount of embryonic hemoglobin, but increased expression of pu.1 and l-plastin. Under the same conditions, MO injection did not affect the markers involved in vascular and pronephric development. Conversely, overexpression of gfi1.1 via mRNA injection enhanced expression of gata1 but inhibited expression of pu.1. These findings suggest that Gfi1.1 plays a critical role in regulating the balance of embryonic erythroid and myeloid lineage determination, and is also required for the differentiation of lymphocytes and granulocytes during zebrafish embryogenesis.

Inactivation of G‐protein coupled receptor 48 impairs definitive erythropoiesis at midgestation through downregulation of ATF4 pathway

G protein‐coupled receptors (GPCRs), one of the most versatile groups of cell surface receptors, can recognize specific ligands from neural, hormonal, and paracrine organs and regulate cell growth, proliferation and differentiation. Gpr48/LGR4 is a recently identified orphan GPCR with unknown functions. To reveal the functions of Gpr48 in vivo, we generated Gpr48−/− mice and found that Gpr48−/− fetuses displayed transient anemia during midgestation and abnormal definitive erythropoiesis. The dramatic decrease of definitive erythroid precursors (Ter119pos population) in Gpr48−/− fetal liver at E13.5 was confirmed by histological analysis and blood smear assays. Real‐time PCR analyses showed that in Gpr48−/− mice both adult hemoglobin α and β chains were decreased while embryonic hemoglobin chains (ξ, βH1 and Єy) were increased, providing another evidence for the impairment of definitive erythropoiesis. Proliferation but not apoptosis was deficient in Gpr48−/−fetal liver, associated with the down‐regulation of c‐Myc and cyclin D1 expression levels. Furthermore, ATF4, a key transcription factor in erythropoiesis, was downregulated in Gpr48−/− fetal livers during midgestation stage through cAMP‐PKA‐CREB pathway, suggesting that Gpr48 regulated definitive erthropoiesis through ATF4‐mediated definitive erythropoiesis.

Analysis of Functional Groups of Differentially Expressed Genes in the Peripheral Blood of Patients with Cervical Cancer Undergoing Concurrent Chemoradiation Treatment

Chao, A., Wang, T. H., Lee, Y. S., Hong, J. H., Tsai, C. N., Chen, C. K., Tsai, C. S., Chao, A. S. and Lai, C. H. Analysis of Functional Groups Differentially Expressed Genes in the Peripheral Blood of Patients with Cervical Cancer Undergoing Concurrent Chemoradiation Treatment. Radiat. Res. 169, 76-86 (2008). We prospectively investigated the gene expression profiles of cervical cancer patients undergoing concurrent chemoradiation treatment. Up-regulated genes associated with anemia were analyzed. Peripheral blood of 20 patients (bulky stage IB-IVA cervical squamous cell carcinomas) undergoing concurrent chemoradiation treatment at four times was collected. Total RNA extracted by the PAXgene Blood RNA System was analyzed with microarrays and MetaCoretrade mark functional network analyses. Fifty-three genes were significantly differentially expressed during concurrent chemoradiation treatment. Fetal and embryonic hemoglobin genes were up-regulated when patients had been severely myelosuppressed. Twenty-eight genes correlated significantly with the hemoglobin genes are involved in responses to hypoxia and oxygenation, TGF-beta signaling, cell cycle suppression, G-protein signaling, and transcriptional regulation. c-Myc has the highest rank in transcriptional co-regulation. In addition, IGKV1D-13 was significantly down-regulated in patients with severe hematological toxicity. These approaches identified biological processes in peripheral blood modulated by concurrent chemoradiation treatment and subsequent anemia.

Multiple Defects of Both Primitive and Definitive Erythrocytes in EKLF-Deficient Mice.

Erythroid Krüppel-Like Factor (EKLF) is the founding member of the mammalian Krüppel subfamily of transcription factors with 3 C2H2-type zinc fingers. In mice, loss of EKLF is lethal at day 14 of gestation (dE14), due to severe anemia. To study the physiological properties of the mixed population of circulating primitive and definitive erythrocytes in dE13.5 mice, we developed an osmotic fragility assay that compares the relative amount of embryonic (primitive) and adult (definitive) hemoglobin in the supernatant and pellet of cells exposed to increasing concentrations of NaCl. We found that both primitive and definitive EKLF-deficient cells were resistant to osmotic stress, consistent with the thalassemia-like phenotype of the cells. Flow cytometric analysis of WT dE13.5 fetal liver (FL) cells with TER119 and CD71 identified 5 previously described populations: R1+R2, composed of pro- and basophilic normoblasts, and R3+R4+R5, composed of more mature polychromatic and orthochromatic normoblasts and reticulocytes, respectively. Analysis of dE13.5 EKLF-deficient FL cells showed that R3+R4+R5 were absent, and morphological examination demonstrated that R1 and R2 were homogeneous populations of pro- and basophilic normoblasts. Colony-forming assays revealed an increase in the absolute number of BFU-E and CFU-E in EKLF-deficient R1 and R2 FL cells. Although the EKLF-deficient colonies required 24–72 more hours of culture than WT colonies to mature, they were composed of definitive erythroid cells as they expressed only adult β-globin. EKLF-deficient FL also contained 2-fold more CFU-Meg colonies (p<0.01). From these data we concluded that the lack of R3+R4+R5 cells in EKLF-deficient FL is due to a block in erythroid differentiation. We compared mRNA from sorted R1+R2 WT and EKLF-deficient FL cells by microarray and used Ingenuity Pathways Analysis (IPA) to demonstrate that the levels of mRNAs encoding proteins involved in the cell cycle and DNA replication were significantly altered. Cell cycle analyses showed that EKLF-deficient R1+R2 cells are significantly delayed exiting G0/G1 (p<0.002). A central node in the cell cycle network was E2F2, a transcription factor involved in cell cycle progression and differentiation. E2F2 mRNA was decreased to 7.6±1.6% of WT levels in EKLF-deficient FL, with a comparable reduction in protein. We hypothesized that E2F2 is a direct EKLF target gene, and examined the chromatin of the E2F2 locus in a high throughput DNase-I hypersensitive site (HS) assay. The E2F2 locus is sensitive to DNase I in WT FL cells and resistant to DNase I in EKLF-deficient FL cells. Three EKLF-dependent HS were identified: in the promoter, intron 2, and the 3′ untranslated region. We used chromatin immunoprecipitation to show that EKLF associates with consensus DNA-binding sites in the E2F2 promoter HS. IPA also demonstrated that apoptosis was abnormal in EKLF-deficient R1+R2 cells. Flow cytometry with annexin V staining demonstrated that EKLF-deficient R1+R2 cells were resistant to apoptosis (p<0.01), consistent with the severe anemia, indicating that apoptosis was not contributing to the differentiation block. Our results support a model in which EKLF-deficiency leads to a block in definitive erythroid maturation associated with decreased cell cycling, resulting in the limited production of dehydrated erythrocytes.

Microarray analysis reveals differences in expression of cell surface and extracellular matrix components during development of the trout ovary and testis

Trout normally spawn at 3 years of age, however, a small percentage mature a year early. This provides an opportunity to study reproductive timing and developmental processes. The ovarian and testicular extracellular matrix (ECM) participates in processes such as growth, adhesion, differentiation, cell migration and patterning. The composition of the ECM defines the interactions of specific regulatory ligands with their receptors and modulates and regulates gonadal function. To identify some of the genes involved in these processes, a 16,006-gene salmonid cDNA microarray was used to compare three-year-old normal with two-year-old normal (maturing) and with two-year-old precocious (pre-spawn) ovarian and testicular transcriptomes. We provide evidence for differences in expression of some of the genes during vasculogenesis, angiogenesis, fibrillogenesis and other processes involving ECM remodeling. Sex-specific gene expression differences of ECM components were documented between the trout ovary and testis in each developmental state. Significant differences in the expression of genes involved in translation, transcription, cell-cycling and differentiation were identified. We also report, for the first time, unequivocal evidence for the transcription of high levels of adult and embryonic hemoglobins in the developed ovary; and for the expression of transcripts that encode zona pellucida glycoproteins in both the ovary and testis of trout.

Human embryonic, fetal, and adult hemoglobins have different subunit interface strengths. Correlation with lifespan in the red cell

The different types of naturally occurring, normal human hemoglobins vary in their tetramer-dimer subunit interface strengths (stabilities) by three orders of magnitude in the liganded (CO or oxy) state. The presence of embryonic zeta-subunits leads to an average 20-fold weakening of tetramer-dimer interfaces compared to corresponding hemoglobins containing adult alpha-subunits. The dimer-monomer interfaces of these hemoglobins differ by at least 500-fold in their strengths; such interfaces are weak if they contain zeta-subunits and exchange with added beta-subunits in the form of beta(4) (HbH) significantly faster than do those with alpha-subunits. Subunit exchange occurs at the level of the dimer, although tetramer formation reciprocally influences the amount of dimer available for exchange. Competition between subunit types occurs so that pairs of weak embryonic hemoglobins can exchange subunits to form the stronger fetal and adult hemoglobins. The dimer strengths increase in the order Hb Portland-2 (zeta(2)beta(2)) < Hb Portland-1 (zeta(2)gamma(2)) approximately equal Hb Gower-1 (zeta(2)epsilon(2)) < Hb Gower-2 (alpha(2)epsilon(2)) < HbF(1) < HbF (alpha(2)gamma(2)) < HbA(2) (alpha(2)delta(2)), i.e., from embryonic to fetal to adult types, representing maturation from weaker to stronger monomer-monomer subunit contacts. This increasing order recapitulates the developmental order in which globins are expressed (embryonic --> fetal --> adult), suggesting that the intrinsic binding properties of the subunits themselves regarding the strengths of interfaces they form with competing subunits play an important role in the dynamics of protein assemblies and networks.