Generation of an immortalised erythroid cell line from haematopoietic stem cells of a haemoglobin E/?-thalassemia patient.
ABSTRACT: The ?-thalassemia syndromes are the most prevalent genetic disorder globally, characterised by reduced or absent ?-globin chain synthesis. HbE/?-thalassemia is a subtype of ?-thalassemia with extremely high frequency in Asia. Studying molecular defects behind ?-thalassemia is severely impeded by paucity of material from patients and lack of suitable cell lines. Approaches to derive erythroid cells from induced pluripotent stem cells (iPSCs) created from patients are confounded by poor levels of erythroid cell expansion, aberrant or incomplete erythroid differentiation and foetal/embryonic rather than adult globin expression. In this study we generate an immortalised erythroid cell line from peripheral blood stem cells of a HbE/?-thalassemia patient. Morphological analysis shows the cells are proerythroblasts with some early basophilic erythroblasts, with no change in morphology over time in culture. The line differentiates along the erythroid pathway to orthochromatic erythroblasts and reticulocytes. Importantly, unlike iPSCs, the line maintains the haemoglobin profile of the patient's red blood cells. This is the first human cellular model for ?-thalassemia providing a sustainable source of disease cells for studying underlying disease mechanisms and for use as drug screening platform, particularly for reagents designed to increase foetal haemoglobin expression as we have additionally demonstrated with hydroxyurea.
Project description:Fetal hemoglobin (HbF; ?2?2) is a potent genetic modifier of the severity of ?-thalassemia and sickle cell anemia. Differences in the levels of HbF that persist into adulthood affect the severity of sickle cell disease and the ?-thalassemia syndromes. Sry type HMG box (SOX6) is a potent silencer of HbF. Here, we reactivated ?-globin expression by downregulating SOX6 to alleviate anemia in the ?-thalassemia patients.SOX6 was downregulated by lentiviral RNAi (RNA interference) in K562 cell line and an in vitro culture model of human erythropoiesis in which erythroblasts are derived from the normal donor mononuclear cells (MNC) or ?-thalassemia major MNC. The expression of ?-globin was analyzed by qPCR (quantitative real-time PCR) and WB (western blot).Our data showed that downregulation of SOX6 induces ?-globin production in K562 cell line and human erythrocytes from normal donors and ?-thalassemia major donors, without altering erythroid maturation.This is the first report on ?-globin induction by downregulation of SOX6 in human erythroblasts derived from ?-thalassemia major.
Project description:Robust ?-globin expression in erythroid cells derived from induced pluripotent stem cells (iPSCs) increases the resolution with which red blood cell disorders such as sickle cell disease and ? thalassemia can be modeled in vitro. To better quantify efforts in augmenting ?-globin expression, we report the creation of a ?-globin reporter iPSC line that allows for the mapping of ?-globin expression throughout human erythropoietic development in real time at single-cell resolution. Coupling this tool with single-cell RNA sequencing (scRNAseq) identified features that distinguish ?-globin-expressing cells and allowed for the dissection of the developmental and maturational statuses of iPSC-derived erythroid lineage cells. Coexpression of embryonic, fetal, and adult globins in individual cells indicated that these cells correspond to a yolk sac erythromyeloid progenitor program of hematopoietic development, representing the onset of definitive erythropoiesis. Within this developmental program, scRNAseq analysis identified a gradient of erythroid maturation, with ?-globin-expressing cells showing increased maturation. Compared with other cells, ?-globin-expressing cells showed a reduction in transcripts coding for ribosomal proteins, increased expression of members of the ubiquitin-proteasome system recently identified to be involved in remodeling of the erythroid proteome, and upregulation of genes involved in the dynamic translational control of red blood cell maturation. These findings emphasize that definitively patterned iPSC-derived erythroblasts resemble their postnatal counterparts in terms of gene expression and essential biological processes, confirming their potential for disease modeling and regenerative medicine applications.
Project description:<h4>Background</h4>Thalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matched donors and the risk of post-transplant complications. Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems. We now report genetic correction of the beta hemoglobin (HBB) gene in iPSCs derived from a patient with a double heterozygote for hemoglobin E and ?-thalassemia (HbE/?-thalassemia), the most common thalassemia syndrome in Thailand and Southeast Asia.<h4>Methods</h4>We used the CRISPR/Cas9 system to target the hemoglobin E mutation from one allele of the HBB gene by homology-directed repair with a single-stranded DNA oligonucleotide template. DNA sequences of the corrected iPSCs were validated by Sanger sequencing. The corrected clones were differentiated into hematopoietic progenitor and erythroid cells to confirm their multilineage differentiation potential and hemoglobin expression.<h4>Results</h4>The hemoglobin E mutation of HbE/?-thalassemia iPSCs was seamlessly corrected by the CRISPR/Cas9 system. The corrected clones were differentiated into hematopoietic progenitor cells under feeder-free and OP9 coculture systems. These progenitor cells were further expanded in erythroid liquid culture system and developed into erythroid cells that expressed mature HBB gene and HBB protein.<h4>Conclusions</h4>Our study provides a strategy to correct hemoglobin E mutation in one step and these corrected iPSCs can be differentiated into hematopoietic stem cells to be used for autologous transplantation in patients with HbE/?-thalassemia in the future.
Project description:?-thalassemia major (?-TM) is an inherited haemoglobinopathy caused by a quantitative defect in the synthesis of ?-globin chains of haemoglobin, leading to the accumulation of free ?-globin chains that aggregate and cause ineffective erythropoiesis. We have previously demonstrated that terminal erythroid maturation requires a transient activation of caspase-3 and that the chaperone Heat Shock Protein 70 (HSP70) accumulates in the nucleus to protect GATA-1 transcription factor from caspase-3 cleavage. This nuclear accumulation of HSP70 is inhibited in human ?-TM erythroblasts due to HSP70 sequestration in the cytoplasm by free ?-globin chains, resulting in maturation arrest and apoptosis. Likewise, terminal maturation can be restored by transduction of a nuclear-targeted HSP70 mutant. Here we demonstrate that in normal erythroid progenitors, HSP70 localization is regulated by the exportin-1 (XPO1), and that treatment of ?-thalassemic erythroblasts with an XPO1 inhibitor increased the amount of nuclear HSP70, rescued GATA-1 expression and improved terminal differentiation, thus representing a new therapeutic option to ameliorate ineffective erythropoiesis of ?-TM.
Project description:The pathophysiology of ineffective erythropoiesis in ?-thalassemia is poorly understood. We report that RAP-011, an activin receptor IIA (ActRIIA) ligand trap, improved ineffective erythropoiesis, corrected anemia and limited iron overload in a mouse model of ?-thalassemia intermedia. Expression of growth differentiation factor 11 (GDF11), an ActRIIA ligand, was increased in splenic erythroblasts from thalassemic mice and in erythroblasts and sera from subjects with ?-thalassemia. Inactivation of GDF11 decreased oxidative stress and the amount of ?-globin membrane precipitates, resulting in increased terminal erythroid differentiation. Abnormal GDF11 expression was dependent on reactive oxygen species, suggesting the existence of an autocrine amplification loop in ?-thalassemia. GDF11 inactivation also corrected the abnormal ratio of immature/mature erythroblasts by inducing apoptosis of immature erythroblasts through the Fas-Fas ligand pathway. Taken together, these observations suggest that ActRIIA ligand traps may have therapeutic relevance in ?-thalassemia by suppressing the deleterious effects of GDF11, a cytokine which blocks terminal erythroid maturation through an autocrine amplification loop involving oxidative stress and ?-globin precipitation.
Project description:The therapeutic use of patient-specific induced pluripotent stem cells (iPSCs) is emerging as a potential treatment of ?-thalassemia. Ideally, patient-specific iPSCs would be genetically corrected by various approaches to treat ?-thalassemia including lentiviral gene transfer, lentivirus-delivered shRNA, and gene editing. These corrected iPSCs would be subsequently differentiated into hematopoietic stem cells and transplanted back into the same patient. In this article, we present a proof of principle study for disease modeling and screening using iPSCs to test the potential use of the modified U7 small nuclear (sn) RNA to correct a splice defect in IVS2-654 ?-thalassemia. In this case, the aberration results from a mutation in the human ?-globin intron 2 causing an aberrant splicing of ?-globin pre-mRNA and preventing synthesis of functional ?-globin protein. The iPSCs (derived from mesenchymal stromal cells from a patient with IVS2-654 ?-thalassemia/hemoglobin (Hb) E) were transduced with a lentivirus carrying a modified U7 snRNA targeting an IVS2-654 ?-globin pre-mRNA in order to restore the correct splicing. Erythroblasts differentiated from the transduced iPSCs expressed high level of correctly spliced ?-globin mRNA suggesting that the modified U7 snRNA was expressed and mediated splicing correction of IVS2-654 ?-globin pre-mRNA in these cells. Moreover, a less active apoptosis cascade process was observed in the corrected cells at transcription level. This study demonstrated the potential use of a genetically modified U7 snRNA with patient-specific iPSCs for the partial restoration of the aberrant splicing process of ?-thalassemia. Stem Cells Translational Medicine 2017;6:1059-1069.
Project description:In ?-thalassemia, accumulated free ?-globin forms intracellular precipitates that impair erythroid cell maturation and viability. Protein quality control systems mitigate ?-thalassemia pathophysiology by degrading toxic free ?-globin, although the associated mechanisms are poorly understood. We show that loss of the autophagy-activating Unc-51-like kinase 1 (Ulk1) gene in ?-thalassemic mice reduces autophagic clearance of ?-globin in red blood cell precursors and exacerbates disease phenotypes, whereas inactivation of the canonical autophagy-related 5 (Atg5) gene has relatively minor effects. Systemic treatment with the mTORC1 inhibitor rapamycin reduces ?-globin precipitates and lessens pathologies in ?-thalassemic mice via an ULK1-dependent pathway. Similarly, rapamycin reduces free ?-globin accumulation in erythroblasts derived from CD34+ cells of ?-thalassemic individuals. Our findings define a drug-regulatable pathway for ameliorating ?-thalassemia.
Project description:Fetal globin genes are transcriptionally silenced during embryogenesis through hemoglobin switching. Strategies to derepress fetal globin expression in the adult could alleviate symptoms in sickle cell disease and ?-thalassemia. We identified a zinc-finger protein, pogo transposable element with zinc-finger domain (POGZ), expressed in hematopoietic progenitor cells. Targeted deletion of Pogz in adult hematopoietic cells in vivo results in persistence of embryonic ?-like globin expression without affecting erythroid development. POGZ binds to the Bcl11a promoter and erythroid-specific intragenic regulatory regions. Pogz+/- mice show elevated embryonic ?-like globin expression, suggesting that partial reduction of Pogz expression results in persistence of embryonic ?-like globin expression. Knockdown of POGZ in primary human CD34+ progenitor cell-derived erythroblasts reduces BCL11A expression, a known repressor of embryonic ?-like globin expression, and increases fetal hemoglobin expression. These findings are significant, since new therapeutic targets and strategies are needed to treat ?-globin disorders.
Project description:Heme-regulated eIF2? kinase (Hri) is necessary for balanced synthesis of heme and globin. In addition, Hri deficiency exacerbates the phenotypic severity of ?-thalassemia intermedia in mice. Activation of Hri during heme deficiency and in ?-thalassemia increases eIF2? phosphorylation and inhibits globin translation. Under endoplasmic reticulum stress and nutrient starvation, eIF2? phosphorylation also induces the Atf4 signaling pathway to mitigate stress. Although the function of Hri in regulating globin translation is well established, its role in Atf4 signaling in erythroid precursors is not known. Here, we report the role of the Hri-activated Atf4 signaling pathway in reducing oxidative stress and in promoting erythroid differentiation during erythropoiesis. On acute oxidative stress, Hri(-/-) erythroblasts suffered from increased levels of reactive oxygen species (ROS) and apoptosis. During chronic iron deficiency in vivo, Hri is necessary both to reduce oxidative stress and to promote erythroid differentiation. Hri(-/-) mice developed ineffective erythropoiesis during iron deficiency with inhibition of differentiation at the basophilic erythroblast stage. This inhibition is recapitulated during ex vivo differentiation of Hri(-/-) fetal liver erythroid progenitors. Importantly, the Hri-eIF2?P-Atf4 pathway was activated and required for erythroid differentiation. We further demonstrate the potential of modulating Hri-eIF2?P-Atf4 signaling with chemical compounds as pharmaceutical therapies for ?-thalassemia.
Project description:A cytosine to thymine mutation at nucleotide 654 of human ?-globin intron 2 (?IVS2-654) is one of the most common mutations causing ?-thalassaemia in Chinese and Southeast Asians. This mutation results in aberrant ?-globin pre-mRNA splicing and prevents synthesis of ?-globin protein. Splicing correction using synthetic splice-switching oligonucleotides (SSOs) has been shown to restore expression of the ?-globin protein, but to maintain therapeutically relevant levels of ?-globin it would require lifelong administration. Here, we demonstrate long-term splicing correction using U7 snRNA lentiviral vectors engineered to target several pre-mRNA splicing elements on the ?IVS2-654-globin pre-mRNA such as cryptic 3' splice site, aberrant 5' splice site, cryptic branch point and an exonic splicing enhancer. A double-target engineered U7 snRNAs targeted to the cryptic branch point and an exonic splicing enhancer, U7.BP?+?623, was the most effective in a model cell line, HeLa IVS2-654. Moreover, the therapeutic potential of the vector was demonstrated in erythroid progenitor cells derived from ?IVS2-654-thalassaemia/HbE patients, which showed restoration of correctly spliced ?-globin mRNA and led to haemoglobin A synthesis, and consequently improved thalassaemic erythroid cell pathology. These results demonstrate proof of concept of using the engineered U7 snRNA lentiviral vector for treatment of ?-thalassaemia.