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Reactivation of fetal hemoglobin expression alleviates the symptoms associated with ß-globinopathies, severe hereditary diseases with significant global health implications due to their high morbidity and mortality rates. The symptoms emerge following the postnatal transition from fetal-to-adult hemoglobin expression. Extensive research has focused on inducing the expression of the fetal γ-globin subunit to reverse this switch and ameliorate these symptoms. Despite decades of research, only one compound, hydroxyurea, found its way to the clinic as an inducer of fetal hemoglobin. Unfortunately, its efficacy varies among patients, highlighting the need for more effective treatments. Erythroid cell lines have been instrumental in the pursuit of both pharmacological and genetic ways to reverse the postnatal hemoglobin switch. Here, we describe the first endogenously tagged fetal hemoglobin reporter cell line based on the adult erythroid progenitor cell line HUDEP2. Utilizing CRISPR-Cas9-mediated knock-in, a bioluminescent tag was integrated at the HBG1 gene. Subsequent extensive characterization confirmed that the resulting reporter cell line closely mirrors the HUDEP2 characteristics and that the cells report fetal hemoglobin induction with high sensitivity and specificity. This novel reporter cell line is therefore highly suitable for evaluating genetic and pharmacologic strategies to induce fetal hemoglobin. Furthermore, it provides an assay compatible with high-throughput drug screening, exemplified by the identification of a cluster of known fetal hemoglobin inducers in a pilot study. This new tool is made available to the research community, with the aspiration that it will accelerate the search for safer and more effective strategies to reverse the hemoglobin switch.
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Differentiation of induced pluripotent stem cells (iPSCs) into hematopoietic lineages offers great therapeutic potential. During embryogenesis, hemogenic endothelium (HE) gives rise to hematopoietic stem and progenitor cells through the endothelial-to-hematopoietic transition (EHT). Understanding this process using iPSCs is key to generating functional hematopoietic stem cells (HSCs), a currently unmet challenge. In this study, we examined the role of the transcriptional factor GFI1B and its co-factor LSD1/KDM1A in EHT. To this end, we employed patient-derived iPSC lines with a dominant negative dysfunctional GFI1BQ287* and irreversible pharmacological LSD1/KDM1A inhibition in healthy iPSC lines. The formation of HE remained unaffected; however, hematopoietic output was severely reduced in both conditions. Single-cell RNA sequencing (scRNAseq) performed on the CD144+/CD31+ population derived from healthy iPSCs revealed similar expression dynamics of genes associated with in vivo EHT. Interestingly, LSD1/KDM1A inhibition in healthy lines before EHT resulted in a complete absence of hematopoietic output. However, uncommitted HE cells did not display GFI1B expression, suggesting a timed transcriptional program. To test this hypothesis, we ectopically expressed GFI1B in uncommitted HE cells, leading to downregulation of endothelial genes and upregulation of hematopoietic genes, including GATA2, KIT, RUNX1, and SPI1. Thus, we demonstrate that LSD1/KDM1A and GFI1B can function at distinct temporal points in different cellular subsets during EHT. Although GFI1B is not detected in uncommitted HE cells, its ectopic expression allows for partial hematopoietic specification. These data indicate that precisely timed expression of specific transcriptional regulators during EHT is crucial to the eventual outcome of EHT.
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The transcription factor Growth Factor Independence 1B (GFI1B) recruits Lysine Specific Demethylase 1 A (LSD1/KDM1A) to stimulate gene programs relevant for megakaryocyte and platelet biology. Inherited pathogenic GFI1B variants result in thrombocytopenia and bleeding propensities with varying intensity. Whether these affect similar gene programs is unknow. Here we studied transcriptomic effects of four patient-derived GFI1B variants (GFI1BT174N,H181Y,R184P,Q287*) in MEG01 megakaryoblasts. Compared to normal GFI1B, each variant affected different gene programs with GFI1BQ287* uniquely failing to repress myeloid traits. In line with this, single cell RNA-sequencing of induced pluripotent stem cell (iPSC)-derived megakaryocytes revealed a 4.5-fold decrease in the megakaryocyte/myeloid cell ratio in GFI1BQ287* versus normal conditions. Inhibiting the GFI1B-LSD1 interaction with small molecule GSK-LSD1 resulted in activation of myeloid genes in normal iPSC-derived megakaryocytes similar to what was observed for GFI1BQ287* iPSC-derived megakaryocytes. Thus, GFI1B and LSD1 facilitate gene programs relevant for megakaryopoiesis while simultaneously repressing programs that induce myeloid differentiation.
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Hematopoyesis , Megacariocitos , Humanos , Megacariocitos/metabolismo , Diferenciación Celular/genética , Hematopoyesis/genética , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Regulación de la Expresión Génica , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismoRESUMEN
The Gárdos channel (KCNN4) and Piezo1 are the best-known ion channels in the red blood cell (RBC) membrane. Nevertheless, the quantitative electrophysiological behavior of RBCs and its heterogeneity are still not completely understood. Here, we use state-of-the-art biochemical methods to probe for the abundance of the channels in RBCs. Furthermore, we utilize automated patch clamp, based on planar chips, to compare the activity of the two channels in reticulocytes and mature RBCs. In addition to this characterization, we performed membrane potential measurements to demonstrate the effect of channel activity and interplay on the RBC properties. Both the Gárdos channel and Piezo1, albeit their average copy number of activatable channels per cell is in the single-digit range, can be detected through transcriptome analysis of reticulocytes. Proteomics analysis of reticulocytes and mature RBCs could only detect Piezo1 but not the Gárdos channel. Furthermore, they can be reliably measured in the whole-cell configuration of the patch clamp method. While for the Gárdos channel, the activity in terms of ion currents is higher in reticulocytes compared to mature RBCs, for Piezo1, the tendency is the opposite. While the interplay between Piezo1 and Gárdos channel cannot be followed using the patch clamp measurements, it could be proved based on membrane potential measurements in populations of intact RBCs. We discuss the Gárdos channel and Piezo1 abundance, interdependencies and interactions in the context of their proposed physiological and pathophysiological functions, which are the passing of small constrictions, e.g., in the spleen, and their active participation in blood clot formation and thrombosis.
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Eritrocitos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio , Reticulocitos , Transporte Biológico , Calcio/metabolismo , Eritrocitos/metabolismo , Reticulocitos/metabolismo , Humanos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Canales Iónicos/metabolismoRESUMEN
Hermansky-Pudlak syndrome type 2 (HPS2) is a rare autosomal recessive disorder, caused by mutations in the AP3B1 gene, encoding the ß3A subunit of the adapter protein complex 3. This results in mis-sorting of proteins within the cell. A clinical feature of HPS2 is severe neutropenia. Current HPS2 animal models do not recapitulate the human disease. Hence, we used induced pluripotent stem cells (iPSCs) of an HPS2 patient to study granulopoiesis. Development into CD15POS cells was reduced, but HPS2-derived CD15POS cells differentiated into segmented CD11b+CD16hi neutrophils. These HPS2 neutrophils phenocopied their circulating counterparts showing increased CD63 expression, impaired degranulation capacity, and intact NADPH oxidase activity. Most noticeable was the decrease in neutrophil yield during the final days of HPS2 iPSC cultures. Although neutrophil viability was normal, CD15NEG macrophages were readily phagocytosing neutrophils, contributing to the limited neutrophil output in HPS2. In this iPSC model, HPS2 neutrophil development is affected by a slower rate of development and by macrophage-mediated clearance during neutrophil maturation.
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Síndrome de Hermanski-Pudlak , Animales , Humanos , Síndrome de Hermanski-Pudlak/genética , Síndrome de Hermanski-Pudlak/metabolismo , Neutrófilos/metabolismo , Mutación , Macrófagos/metabolismoRESUMEN
E-cadherin is a crucial regulator of epithelial cell-to-cell adhesion and an established tumor suppressor. Aside epithelia, E-cadherin expression marks the erythroid cell lineage during human but not mouse hematopoiesis. However, the role of E-cadherin in human erythropoiesis remains unknown. Because rat erythropoiesis was postulated to reflect human erythropoiesis more closely than mouse erythropoiesis, we investigated E-cadherin expression in rat erythroid progenitors. E-cadherin expression is conserved within the erythroid lineage between rat and human. In response to anemia, erythroblasts in rat bone marrow (BM) upregulate E-cadherin as well as its binding partner ß-catenin. CRISPR/Cas9-mediated knock out of E-cadherin revealed that E-cadherin expression is required to stabilize ß-catenin in human and rat erythroblasts. Suppression of ß-catenin degradation by glycogen synthase kinase 3ß (GSK3ß) inhibitor CHIR99021 also enhances ß-catenin stability in human erythroblasts but hampers erythroblast differentiation and survival. In contrast, direct activation of ß-catenin signaling, using an inducible, stable ß-catenin variant, does not perturb maturation or survival of human erythroblasts but rather enhances their differentiation. Although human erythroblasts do not respond to Wnt ligands and direct GSK3ß inhibition even reduces their survival, we postulate that ß-catenin stability and signaling is mostly controlled by E-cadherin in human and rat erythroblasts. In response to anemia, E-cadherin-driven upregulation and subsequent activation of ß-catenin signaling may stimulate erythroblast differentiation to support stress erythropoiesis in the BM. Overall, we uncover E-cadherin/ß-catenin expression to mark stress erythropoiesis in rat BM. This may provide further understanding of the underlying molecular regulation of stress erythropoiesis in the BM, which is currently poorly understood.
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Anemia , beta Catenina , Humanos , Ratas , Ratones , Animales , beta Catenina/metabolismo , Eritropoyesis/fisiología , Glucógeno Sintasa Quinasa 3 beta , Cadherinas/genética , Cadherinas/metabolismoRESUMEN
Iron, supplemented as iron-loaded transferrin (holotransferrin), is an essential nutrient in mammalian cell cultures, particularly for erythroid cultures. The high cost of human transferrin represents a challenge for large scale production of red blood cells (RBCs) and for cell therapies in general. We evaluated the use of deferiprone, a cell membrane-permeable drug for iron chelation therapy, as an iron carrier for erythroid cultures. Iron-loaded deferiprone (Def3·Fe3+, at 52 µmol/L) could eliminate the need for holotransferrin supplementation during in vitro expansion and differentiation of erythroblast cultures to produce large numbers of enucleated RBC. Only the first stage, when hematopoietic stem cells committed to erythroblasts, required holotransferrin supplementation. RBCs cultured in presence of Def3·Fe3+ or holotransferrin (1000 µg/mL) were similar with respect to differentiation kinetics, expression of cell-surface markers CD235a and CD49d, hemoglobin content, and oxygen association/dissociation. Replacement of holotransferrin supplementation by Def3·Fe3+ was also successful in cultures of myeloid cell lines (MOLM13, NB4, EOL1, K562, HL60, ML2). Thus, iron-loaded deferiprone can partially replace holotransferrin as a supplement in chemically defined cell culture medium. This holds promise for a significant decrease in medium cost and improved economic perspectives of the large scale production of red blood cells for transfusion purposes.
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Eritrocitos , Hierro , Animales , Humanos , Hierro/metabolismo , Deferiprona/farmacología , Eritrocitos/metabolismo , Quelantes del Hierro/uso terapéutico , Hemoglobinas/metabolismo , Células Cultivadas , Mamíferos/metabolismoRESUMEN
Ribonucleoproteins (RNPs) are frequently applied for therapeutic gene editing as well as fundamental research because the method is fast, viral free, and shows fewest off target effects. We evaluated various parameters to genetically engineer human hematopoietic stem and progenitor cells (HSPCs) using Streptococcus pyogenes Cas9 (spCas9) RNPs, and achieve gene editing efficiencies up to 80%. We find that guide RNA (gRNA) design is critical to achieve high gene editing efficiencies. However, finding effective gRNAs for HSPCs can be challenging, while the contribution of numerous in silico models is unclear. By screening more than 120 gRNAs, our data demonstrate that in silico gRNA prediction models are ineffective. In this study, we established a time- and cost-efficient in vitro transcribed gRNA screening model in K562 cells that predicts effective gRNAs for HSPCs. RNP based screening thus outperforms in silico modeling and we report that gene editing is equally efficient in distinct CD34+ HSPC subpopulations. Furthermore, no effects on cell proliferation, differentiation, or in vitro hematopoietic lineage commitment were observed. Finally, no upregulation of p21 expression was found, suggesting unperturbed HSPC homeostasis.
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Edición Génica , ARN Guía de Kinetoplastida , Humanos , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Edición Génica/métodos , Sistemas CRISPR-Cas/genética , Streptococcus pyogenes/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Células Madre Hematopoyéticas/metabolismoRESUMEN
Transfusion of donor-derived red blood cells (RBCs) is the most common form of cell therapy. Production of transfusion-ready cultured RBCs (cRBCs) is a promising replacement for the current, fully donor-dependent therapy. A single transfusion unit, however, contains 2 × 1012 RBC, which requires large scale production. Here, we report on the scale-up of cRBC production from static cultures of erythroblasts to 3 L stirred tank bioreactors, and identify the effect of operating conditions on the efficiency of the process. Oxygen requirement of proliferating erythroblasts (0.55-2.01 pg/cell/h) required sparging of air to maintain the dissolved oxygen concentration at the tested setpoint (2.88 mg O2 /L). Erythroblasts could be cultured at dissolved oxygen concentrations as low as 0.7 O2 mg/ml without negative impact on proliferation, viability or differentiation dynamics. Stirring speeds of up to 600 rpm supported erythroblast proliferation, while 1800 rpm led to a transient halt in growth and accelerated differentiation followed by a recovery after 5 days of culture. Erythroblasts differentiated in bioreactors, with final enucleation levels and hemoglobin content similar to parallel cultures under static conditions.
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Reactores Biológicos , Eritroblastos , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Hemoglobinas , OxígenoRESUMEN
BACKGROUND: Treatment choices for individual patients with an inborn bleeding disorder are increasingly challenging due to increasing options and rising costs for society. We have initiated an integrated interdisciplinary national research programme. OBJECTIVES: The SYMPHONY consortium strives to orchestrate personalized treatment in patients with an inborn bleeding disorder, by unravelling the mechanisms behind inter-individual variations of bleeding phenotype. PATIENTS: The SYMPHONY consortium will investigate patients with an inborn bleeding disorder, both diagnosed and not yet diagnosed. RESULTS: Research questions are categorized under the themes: 1) Diagnosis; 2) Treatment; and 3) Fundamental research and consist of workpackages addressing specific domains. Importantly, collaborations between patients and talented researchers from different areas of expertise promise to augment the impact of the SYMPHONY consortium, leading to unique interactions and intellectual property. CONCLUSIONS: SYMPHONY will perform research on all aspects of care, treatment individualization in patients with inborn bleeding disorders as well as diagnostic innovations and results of molecular genetics and cellular model technology with regard to the hemostatic process. We believe that these research investments will lead to health care innovations with long-term clinical and societal impact. This consortium has been made possible by a governmental, competitive grant from the Netherlands Organization for Scientific Research (NWO) within the framework of the NWA-ORC Call grant agreement NWA.1160.18.038.
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Hypoxia is associated with increased erythropoietin (EPO) release to drive erythropoiesis. At high altitude, EPO levels first increase and then decrease, although erythropoiesis remains elevated at a stable level. The roles of hypoxia and related EPO adjustments are not fully understood, which has contributed to the formulation of the theory of neocytolysis. We aimed to evaluate the role of oxygen exclusively on erythropoiesis, comparing in vitro erythroid differentiation performed at atmospheric oxygen, a lower oxygen concentration (three percent oxygen) and with cultures of erythroid precursors isolated from peripheral blood after a 19-day sojourn at high altitude (3450 m). Results highlight an accelerated erythroid maturation at low oxygen and more concave morphology of reticulocytes. No differences in deformability were observed in the formed reticulocytes in the tested conditions. Moreover, hematopoietic stem and progenitor cells isolated from blood affected by hypoxia at high altitude did not result in different erythroid development, suggesting no retention of a high-altitude signature but rather an immediate adaptation to oxygen concentration. This adaptation was observed during in vitro erythropoiesis at three percent oxygen by a significantly increased glycolytic metabolic profile. These hypoxia-induced effects on in vitro erythropoiesis fail to provide an intrinsic explanation of the concept of neocytolysis.
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Eritropoyesis , Eritropoyetina , Aclimatación , Eritropoyetina/metabolismo , Eritropoyetina/farmacología , Humanos , Hipoxia , Oxígeno/metabolismoRESUMEN
Haploinsufficiency for the erythroid-specific transcription factor KLF1 is associated with hereditary persistence of fetal hemoglobin (HPFH). Increased HbF ameliorates the symptoms of ß-hemoglobinopathies and downregulation of KLF1 activity has been proposed as a potential therapeutic strategy. However, the feasibility of this approach has been challenged by the observation that KLF1 haploinsufficient individuals with the same KLF1 variant, within the same family, display a wide range of HbF levels. This phenotypic variability is not readily explained by co-inheritance of known HbF-modulating variants in the HBB, HBS1L-MYB and/or BCL11A loci. We studied cultured erythroid progenitors obtained from Maltese individuals in which KLF1 p.K288X carriers display HbF levels ranging between 1.3 and 12.3% of total Hb. Using a combination of gene expression analysis, chromatin accessibility assays and promoter activity tests we find that variation in expression of the wildtype KLF1 allele may explain a significant part of the variability in HbF levels observed in KLF1 haploinsufficiency. Our results have general bearing on the variable penetrance of haploinsufficiency phenotypes and on conflicting interpretations of pathogenicity of variants in other transcriptional regulators such as EP300, GATA2 and RUNX1.
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Epigénesis Genética , Epigenoma , Epigenómica , Eritroblastos/metabolismo , Haploinsuficiencia , Hemoglobinopatías/genética , Factores de Transcripción de Tipo Kruppel/genética , Células Cultivadas , Secuenciación de Inmunoprecipitación de Cromatina , Eritroblastos/patología , Eritropoyesis/genética , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Predisposición Genética a la Enfermedad , Hemoglobinopatías/sangre , Hemoglobinopatías/diagnóstico , Humanos , Factores de Transcripción de Tipo Kruppel/metabolismo , Malta , Penetrancia , Fenotipo , Cultivo Primario de Células , RNA-SeqRESUMEN
In 2016, the European Hematology Association (EHA) published the EHA Roadmap for European Hematology Research 1 aiming to highlight achievements in the diagnostics and treatment of blood disorders, and to better inform European policy makers and other stakeholders about the urgent clinical and scientific needs and priorities in the field of hematology. Each section was coordinated by 1-2 section editors who were leading international experts in the field. In the 5 years that have followed, advances in the field of hematology have been plentiful. As such, EHA is pleased to present an updated Research Roadmap, now including eleven sections, each of which will be published separately. The updated EHA Research Roadmap identifies the most urgent priorities in hematology research and clinical science, therefore supporting a more informed, focused, and ideally a more funded future for European hematology research. The 11 EHA Research Roadmap sections include Normal Hematopoiesis; Malignant Lymphoid Diseases; Malignant Myeloid Diseases; Anemias and Related Diseases; Platelet Disorders; Blood Coagulation and Hemostatic Disorders; Transfusion Medicine; Infections in Hematology; Hematopoietic Stem Cell Transplantation; CAR-T and Other Cell-based Immune Therapies; and Gene Therapy.
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Each day, about 1012 erythrocytes and platelets are released into the bloodstream. This substantial output from hematopoietic stem cells is tightly regulated by transcriptional and epigenetic factors. Whether and how circular RNAs (circRNAs) contribute to the differentiation and/or identity of hematopoietic cells is to date not known. We recently reported that erythrocytes and platelets contain the highest levels and numbers of circRNAs among hematopoietic cells. Here, we provide the first detailed analysis of circRNA expression during erythroid and megakaryoid differentiation. CircRNA expression not only significantly increased upon enucleation, but also had limited overlap between progenitor cells and mature cells, suggesting that circRNA expression stems from regulated processes rather than resulting from mere accumulation. To study circRNA function in hematopoiesis, we first compared the expression levels of circRNAs with the translation efficiency of their mRNA counterpart. We found that only one out of 2531 (0.04%) circRNAs associated with mRNA-translation regulation. Furthermore, irrespective of thousands of identified putative open reading frames, deep ribosome-footprinting sequencing, and mass spectrometry analysis provided little evidence for translation of endogenously expressed circRNAs. In conclusion, circRNAs alter their expression profile during terminal hematopoietic differentiation, yet their contribution to regulate cellular processes remains enigmatic.
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Hematopoyesis , ARN Circular/metabolismo , ARN Mensajero/genética , Células Cultivadas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Biosíntesis de Proteínas , ARN Circular/genética , ARN Mensajero/metabolismo , TranscriptomaRESUMEN
Beta-hemoglobinopathies become prominent after birth due to a switch from γ-globin to the mutated ß-globin. Haploinsufficiency for the erythroid specific indispensable transcription factor Krueppel-like factor 1 (KLF1) is associated with high persistence of fetal hemoglobin (HPFH). The In(Lu) phenotype, characterized by low to undetectable Lutheran blood group expression is caused by mutations within KLF1 gene. Here we screened a blood donor cohort of 55 Lutheran weak or negative donors for KLF1 variants and evaluated their effect on KLF1 target gene expression. To discriminate between weak and negative Lutheran expression, a flow cytometry (FCM) assay was developed to detect Lu antigen expression. The Lu(a-b-) (negative) donor group, showing a significant decreased CD44 (Indian blood group) expression, also showed increased HbF and HbA2 levels, with one individual expressing HbF as high as 5%. KLF1 exons and promoter sequencing revealed variants in 80% of the Lutheran negative donors. Thirteen different variants plus one high frequency SNP (c.304 T > C) were identified of which 6 were novel. In primary erythroblasts, knockdown of endogenous KLF1 resulted in decreased CD44, Lu and increased HbF expression, while KLF1 over-expressing cells were comparable to wild type (WT). In line with the pleiotropic effects of KLF1 during erythropoiesis, distinct KLF1 mutants expressed in erythroblasts display different abilities to rescue CD44 and Lu expression and/or to affect fetal (HbF) or adult (HbA) hemoglobin expression. With this study we identified novel KLF1 variants to be include into blood group typing analysis. In addition, we provide further insights into the regulation of genes by KLF1.
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Moléculas de Adhesión Celular/genética , Hemoglobina Fetal/análisis , Factores de Transcripción de Tipo Kruppel/genética , Sistema del Grupo Sanguíneo Lutheran/genética , gamma-Globinas/análisis , Células Cultivadas , Células Eritroides/citología , Células Eritroides/metabolismo , Eritropoyesis , Hemoglobina Fetal/genética , Humanos , Mutación , Polimorfismo de Nucleótido Simple , gamma-Globinas/genéticaRESUMEN
BACKGROUND: The therapeutic use of [131I]meta-iodobenzylguanidine ([131I]MIBG) is often accompanied by hematological toxicity, primarily consisting of severe and persistent thrombocytopenia. We hypothesize that this is caused by selective uptake of MIBG via the serotonin transporter (SERT) located on platelets and megakaryocytes. In this study, we have investigated whether in vitro cultured human megakaryocytes are capable of selective plasma membrane transport of MIBG and whether pharmacological intervention with selective serotonin reuptake inhibitors (SSRIs) may prevent this radiotoxic MIBG uptake. METHODS: Peripheral blood CD34+ cells were differentiated to human megakaryocytic cells using a standardized culture protocol. Prior to [3H]serotonin and [125I]MIBG uptake experiments, the differentiation status of megakaryocyte cultures was assessed by flow cytometry. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to assess SERT and NET (norepinephrine transporter) mRNA expression. On day 10 of differentiation, [3H]serotonin and [125I]MIBG uptake assays were conducted. Part of the samples were co-incubated with the SSRI citalopram to assess SERT-specific uptake. HEK293 cells transfected with SERT, NET, and empty vector served as controls. RESULTS: In vitro cultured human megakaryocytes are capable of selective plasma membrane transport of MIBG. After 10 days of differentiation, megakaryocytic cell culture batches from three different hematopoietic stem and progenitor cell donors showed on average 9.2 ± 2.4 nmol of MIBG uptake per milligram protein per hour after incubation with 10-7 M MIBG (range: 6.6 ± 1.0 to 11.2 ± 1.0 nmol/mg/h). Co-incubation with the SSRI citalopram led to a significant reduction (30.1%-41.5%) in MIBG uptake, implying SERT-specific uptake of MIBG. A strong correlation between the number of mature megakaryocytes and SERT-specific MIBG uptake was observed. CONCLUSION: Our study demonstrates that human megakaryocytes cultured in vitro are capable of MIBG uptake. Moreover, the SSRI citalopram selectively inhibits MIBG uptake via the serotonin transporter. The concomitant administration of citalopram to neuroblastoma patients during [131I]MIBG therapy might be a promising strategy to prevent the onset of thrombocytopenia.
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The YPEL family genes are highly conserved across a diverse range of eukaryotic organisms and thus potentially involved in essential cellular processes. Ypel4, one of five YPEL family gene orthologs in mouse and human, is highly and specifically expressed in late terminal erythroid differentiation (TED). In this study, we investigated the role of Ypel4 in murine erythropoiesis, providing for the first time an in-depth description of a Ypel4-null phenotype in vivo. We demonstrated that the Ypel4-null mice displayed a secondary polycythemia with macro- and reticulocytosis. While lack of Ypel4 did not affect steady-state TED in the bone marrow or spleen, the anemia-recovering capacity of Ypel4-null cells was diminished. Furthermore, Ypel4-null red blood cells (RBC) were cleared from the circulation at an increased rate, demonstrating an intrinsic defect of RBCs. Scanning electron micrographs revealed an ovalocytic morphology of Ypel4-null RBCs and functional testing confirmed reduced deformability. Even though Band 3 protein levels were shown to be reduced in Ypel4-null RBC membranes, we could not find support for a physical interaction between YPEL4 and the Band 3 protein. In conclusion, our findings provide crucial insights into the role of Ypel4 in preserving normal red cell membrane integrity.
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Proteínas Portadoras/genética , Membrana Eritrocítica/fisiología , Eritropoyesis/genética , Anemia/metabolismo , Animales , Proteína 1 de Intercambio de Anión de Eritrocito/metabolismo , Proteínas Portadoras/metabolismo , Membrana Eritrocítica/genética , Eritrocitos/metabolismo , Eritrocitos Anormales/metabolismo , Eritropoyesis/fisiología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Policitemia/genética , BazoRESUMEN
Induced pluripotent stem cell (iPSC) line was generated from erythroblasts (EBLs) derived from a patient diagnosed with severe congenital neutropenia, caused by mutations in ELANE (c.614delG). Transgene-free iPSC line was generated using Sendai virus reprogramming. The iPSC line showed normal karyotype, expressed pluripotency associated genes and was capable of in vitro spontaneous differentiation towards the three germ layers. The generated iPSC line can be used to study severe congenital neutropenia and the role of neutrophil elastase protein.
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Células Madre Pluripotentes Inducidas , Diferenciación Celular , Reprogramación Celular , Humanos , Mutación , Virus Sendai/genéticaRESUMEN
Induced pluripotent stem cells (iPSCs) were generated from erythroblasts (EBLs) obtained from a patient diagnosed with Chédiak-Higashi Syndrome (CHS), caused by mutations in LYST (c.4322_4325delAGAG and c.10127A>G). EBLs were reprogrammed with CytoTune-iPS 2.0 Sendai Reprogramming Kit, where the generated iPSCs showed normal karyotype, expression of pluripotency associated markers and in vitro spontaneous differentiation towards the three germ layers. The generated iPSCs can be used to study CHS pathophysiology and the role of LYST in different cell types.