Search

Unlimited self renewal capacity and differentiation potential make human pluripotent stem cells (PSC) a promising source for the ex vivo manufacture of red blood cells (RBCs) for safe transfusion. Current methods to induce erythropoiesis from PSC suffer from low yields of RBCs, most of which are immature and contain embryonic and fetal rather than adult hemoglobins. We have previously shown that homodimerization of the intracellular component of MPL (ic-MPL) induces erythropoiesis from human cord blood progenitors. The goal of this study was to investigate the potential of ic-MPL dimerization to induce erythropoiesis from human embryonic stem cells (hESCs) and to identify the signaling pathways activated by this strategy. We present here the evidence that ic-MPL dimerization induces erythropoietin (EPO)-independent erythroid differentiation from hESC by inducing the generation of erythroid progenitors and by promoting more efficient erythroid maturation with increased RBC enucleation as well as increased gamma:epsilon globin ratio and production of beta-globin protein. ic-MPL dimerization is significantly more potent than EPO in inducing erythropoiesis, and its effect is additive to EPO. Signaling studies show that dimerization of ic-MPL, unlike stimulation of the wild type MPL receptor, activates AKT in the absence of JAK2/STAT5 signaling. AKT activation upregulates GATA-1 and FOXO3 transcriptional pathways with resulting inhibition of apoptosis, modulation of cell cycle, and enhanced maturation of erythroid cells. These findings open up potential new targets for the generation of therapeutically relevant RBC products from hPSC.

Condensin is a central regulator of mitotic genome structure with mutants showing poorly condensed chromosomes and profound segregation defects. Here, we identify NCT, a complex comprising the Nrc1 BET-family tandem bromodomain protein (SPAC631.02), casein kinase II (CKII), and several TAFs, as a regulator of condensin function. We show that NCT and condensin bind similar genomic regions but only briefly colocalize during the periods of chromosome condensation and decondensation. This pattern of NCT binding at the core centromere, the region of maximal condensin enrichment, tracks the abundance of acetylated histone H4, as regulated by the Hat1-Mis16 acetyltransferase complex and recognized by the first Nrc1 bromodomain. Strikingly, mutants in NCT or Hat1-Mis16 restore the formation of segregation-competent chromosomes in cells containing defective condensin. These results are consistent with a model where NCT targets CKII to chromatin in a cell-cycle-directed manner in order to modulate the activity of condensin during chromosome condensation and decondensation.

Individuals with β-thalassemia develop progressive systemic iron overload, resulting in high morbidity and mortality. These complications are caused by labile plasma iron, which is taken up by parenchymal cells in a dysregulated manner; in contrast, erythropoiesis depends on transferrin-bound iron uptake via the transferrin receptor. We hypothesized that the ineffective erythropoiesis and anemia observed in β-thalassemia might be ameliorated by increasing the amount of circulating transferrin. We tested the ability of transferrin injections to modulate iron metabolism and erythropoiesis in [Hbb.sup.th1/th1] mice, an experimental model of β-thalassemia. Injected transferrin reversed or markedly improved the thalassemia phenotype in these mice. Specifically, transferrin injections normalized labile plasma iron concentrations, increased hepcidin expression, normalized red blood cell survival and increased hemoglobin production; this treatment concomitantly decreased reticulocytosis, erythropoietin abundance and splenomegaly. These results indicate that transferrin is a limiting factor contributing to anemia in these mice and suggest that transferrin therapy might be beneficial in human β-thalassemia., β-thalassemias are caused by mutations in the gene encoding β-globin, resulting in reduced or absent β-chain synthesis (1). A relative excess of α-globin chain synthesis leads to increased erythroid precursor [...]

Linker histone H1 plays an important role in chromatin folding in vitro and in order to study the role of H1 in vivo, mouse embryonic stem cells null for three H1 genes were derived and were found to have 50% of the normal level of H1. Results indicate that linker histones can participate in epigenetic regulation of gene expression by contributing to the maintenance or establishment of specific DNA methylation patterns.

Mammalian [beta]-globin loci contain multiple [beta]-like genes that are expressed at different times during development. The murine [beta]-globin locus contains two genes expressed during the embryo stage, Ey and/[beta]h1, and two genes expressed at both the fetal and postnatal stages,[beta]-major and [beta]-minor. Studies of transgenic human [beta]-like globin loci in mice have suggested that expression of one gene at the locus will suppress expression of other genes at the locus. To test this hypothesis we produced mouse lines with deletions of either the Ey or [beta]h1 promoter in the endogenous murine [beta]-globin locus. Promoter deletion eliminated expression of the mutant gene but did not affect expression of the remaining embryonic gene or the fetal/adult [beta]-globin genes on the mutant allele. These results demonstrate a lack of competitive effects between individual mouse embryonic [beta]-globin gene promoters and other genes in the locus. The implication of these findings for models of [beta]-globin gene expression are discussed.

Achieving long-term pancellular expression of a transferred gene at therapeutic level in a given hematopoietic lineage remains an important goal of gene therapy. Advances have recently been made in the genetic correction of the hemoglobinopathies by means of lentiviral vectors and large locus control region (LCR) derivatives. However, panerythroid [beta] globin gene expression has not yet been achieved in [beta] thalassemic mice because of incomplete transduction of the hematopoietic stem cell compartment and position effect variegation of proviruses integrated at a single copy per genome. Hefe, we report the permanent, panerythroid correction of severe [beta] thalassemia in mice, resulting from a homozygous deletion of the [beta] major globin gene, by transplantation of syngeneic bone marrow transduced with an HIV-l-derived [beta] globin gene/LCR] lentiviral vector also containing the Rev responsive element and the central polypurine tract/DNA flap. The viral titers produced were high enough to achieve transduction of virtually all of the hematopoietic stem cells in the graft with an average of three integrated proviral copies per genome in all transplanted mice; the transduction was sustained for >7 months in both primary and secondary transplants, at which time [approximately equal to] 95% of the red blood cells in all mice contained human [beta] globin contributing to 32 [+ or -] 4% of all [beta]-like globin chains. Hematological parameters approached complete phenotypic correction, as assessed by hemoglobin levels and reticulocyte and red blood cell counts. All circulating red blood cells became and remained normocytic and normochromic, and their density was normalized. Free [alpha] globin chains were completely cleared from red blood cell membranes, splenomegaly abated, and iron deposit was almost eliminated in liver sections. These findings indicate that virtually complete transduction of the hematopoietic stem cell compartment can be achieved by high-titer lentiviral vectors and that position effect variegation can be mitigated by multiple events of proviral integration to yield balanced, panerythroid expression. These results provide a solid foundation for the initiation of human clinical trials in [beta] thalassemia patients.