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1.
BMC Bioinformatics ; 22(1): 478, 2021 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-34607573

RESUMO

BACKGROUND: Nonlinear mixed effects models provide a way to mathematically describe experimental data involving a lot of inter-individual heterogeneity. In order to assess their practical identifiability and estimate confidence intervals for their parameters, most mixed effects modelling programs use the Fisher Information Matrix. However, in complex nonlinear models, this approach can mask practical unidentifiabilities. RESULTS: Herein we rather propose a multistart approach, and use it to simplify our model by reducing the number of its parameters, in order to make it identifiable. Our model describes several cell populations involved in the in vitro differentiation of chicken erythroid progenitors grown in the same environment. Inter-individual variability observed in cell population counts is explained by variations of the differentiation and proliferation rates between replicates of the experiment. Alternatively, we test a model with varying initial condition. CONCLUSIONS: We conclude by relating experimental variability to precise and identifiable variations between the replicates of the experiment of some model parameters.


Assuntos
Algoritmos , Eritropoese , Modelos Biológicos , Dinâmica não Linear , Fases de Leitura
2.
Folia Biol (Praha) ; 67(2): 70-75, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34624939

RESUMO

Based on simple microscopic cell morphology in blood and bone marrow smear preparations, it seems to be likely that the cell differentiation and terminal differentiation in human blood cells, and particularly in erythroid or granulocytic lineages, simultaneously reflect ageing of the lineage progenitors and terminal differentiation steps. The terminal differentiation stages of both these lineages actually appear as senescent cells. Abnormal ageing of progenitor cells may represent one of the "dysplastic" phenomena of the premature terminal differentiation state. Such state is characterized by heterochromatin condensation and nucleolar morphology similar to that in fully differentiated terminal cells of granulocytic or erythroid lineages. It should also be mentioned that in some known erythropoietic disorders, less differentiated erythroblasts may lose nuclei similarly as "normal" fully terminally differentiated cells of the erythroid cell lineage. It seems to be clear that cells in both abnormal less differentiated and terminally differentiated stages of erythroid or granulocytic lineages lose the ability to multiply similarly as senescent cells. On the other hand, the background of cell ageing and differentiation is very complicated and requires a different approach than the simple microscopic morphology at the single cell level. However, the morphology and clinical cytology at the single cell level might still contribute with complementary data to more sophisticated complex studies of that topic. In addition, the morphological approach facilitates the study of the main components of single cells in various states, including the differentiation steps or ageing.


Assuntos
Eritroblastos , Eritropoese , Envelhecimento , Diferenciação Celular , Linhagem da Célula , Humanos
3.
Int J Mol Sci ; 22(19)2021 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-34638816

RESUMO

Vertebrates generate mature red blood cells (RBCs) via a highly regulated, multistep process called erythropoiesis. Erythropoiesis involves synthesis of heme and hemoglobin, clearance of the nuclei and other organelles, and remodeling of the plasma membrane, and these processes are exquisitely coordinated by specific regulatory factors including transcriptional factors and signaling molecules. Defects in erythropoiesis can lead to blood disorders such as congenital dyserythropoietic anemias, Diamond-Blackfan anemias, sideroblastic anemias, myelodysplastic syndrome, and porphyria. The molecular mechanisms of erythropoiesis are highly conserved between fish and mammals, and the zebrafish (Danio rerio) has provided a powerful genetic model for studying erythropoiesis. Studies in zebrafish have yielded important insights into RBC development and established a number of models for human blood diseases. Here, we focus on latest discoveries of the molecular processes and mechanisms regulating zebrafish erythropoiesis and summarize newly established zebrafish models of human anemias.


Assuntos
Anemia Aplástica , Desenvolvimento Embrionário , Eritrócitos/metabolismo , Eritropoese , Peixe-Zebra/embriologia , Anemia Aplástica/genética , Anemia Aplástica/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Peixe-Zebra/genética
4.
J Med Case Rep ; 15(1): 463, 2021 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-34538261

RESUMO

BACKGROUND: In ineffective erythropoiesis, hepcidin synthesis is suppressed by erythroid regulators, namely erythroferrone and growth differentiation factor-15. For the first time, the hypothesis that iron overload in megaloblastic anemia may be related to ineffective erythropoiesis is explored by describing the kinetics of hepcidin, erythroferrone, and growth differentiation factor-15 levels in a patient diagnosed with megaloblastic anemia associated with iron overload. CASE PRESENTATION: An 81-year-old Caucasian male was admitted for fatigue. He had type-2 diabetes previously treated with metformin, ischemic cardiac insufficiency, and stage-3 chronic kidney disease. Vitiligo was observed on both hands. Biological tests revealed normocytic non-regenerative anemia associated with hemolysis, thrombocytopenia, and elevated sideremia, ferritin, and transferrin saturation levels. Megaloblastic anemia was confirmed with undetectable blood vitamin B12 and typical cytological findings like hyper-segmented neutrophils in blood and megaloblasts in bone marrow. The patient received vitamin B12 supplementation. At 3 months, biological parameters reached normal values. Hepcidin kinetics from diagnosis to 3 months inversely correlated with those of erythroferrone and growth differentiation factor-15. CONCLUSIONS: This case suggests that iron-overload mechanisms of dyserythropoietic anemias may apply to megaloblastic anemias.


Assuntos
Anemia Megaloblástica , Anemia , Sobrecarga de Ferro , Idoso de 80 Anos ou mais , Anemia Megaloblástica/diagnóstico , Anemia Megaloblástica/tratamento farmacológico , Eritropoese , Humanos , Ferro , Sobrecarga de Ferro/tratamento farmacológico , Masculino
5.
Sci Rep ; 11(1): 18545, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34535746

RESUMO

Emm is a high incidence red cell antigen with eight previously reported Emm- probands. Anti-Emm appears to be naturally occurring yet responsible for a clinically significant acute hemolytic transfusion reaction. Previous work suggests that Emm is located on a GPI-anchored protein, but the antigenic epitope and genetic basis have been elusive. We investigated samples from a South Asian Indian family with two Emm- brothers by whole genome sequencing (WGS). Additionally, samples from four unrelated Emm- individuals were investigated for variants in the candidate gene. Filtering for homozygous variants found in the Emm- brothers and by gnomAD frequency of < 0.001 resulted in 1818 variants with one of high impact; a 2-bp deletion causing a frameshift and premature stop codon in PIGG [NM_001127178.3:c.2624_2625delTA, p.(Leu875*), rs771819481]. PIGG encodes for a transferase, GPI-ethanolaminephosphate transferase II, which adds ethanolamine phosphate (EtNP) to the second mannose in a GPI-anchor. The four additional unrelated Emm- individuals had various PIGG mutations; deletion of Exons 2-3, deletion of Exons 7-9, insertion/deletion (indel) in Exon 3, and new stop codon in Exon 5. The Emm- phenotype is associated with a rare deficiency of PIGG, potentially defining a new Emm blood group system composed of EtNP bound to mannose, part of the GPI-anchor. The results are consistent with the known PI-linked association of the Emm antigen, and may explain the production of the antibody in the absence of RBC transfusion. Any association with neurologic phenotypes requires further research.


Assuntos
Antígenos de Grupos Sanguíneos/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Adulto , Idoso , Eritropoese , Feminino , Mutação da Fase de Leitura , Deleção de Genes , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Linhagem
6.
Int J Mol Sci ; 22(18)2021 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-34575977

RESUMO

Amidst the global shortfalls in blood supply, storage limitations of donor blood and the availability of potential blood substitutes for transfusion applications, society has pivoted towards in vitro generation of red blood cells (RBCs) as a means to solve these issues. Many conventional research studies over the past few decades have found success in differentiating hematopoietic stem and progenitor cells (HSPCs) from cord blood, adult bone marrow and peripheral blood sources. More recently, techniques that involve immortalization of erythroblast sources have also gained traction in tackling this problem. However, the RBCs generated from human induced pluripotent stem cells (hiPSCs) still remain as the most favorable solution due to many of its added advantages. In this review, we focus on the breakthroughs for high-density cultures of hiPSC-derived RBCs, and highlight the major challenges and prospective solutions throughout the whole process of erythropoiesis for hiPSC-derived RBCs. Furthermore, we elaborate on the recent advances and techniques used to achieve cost-effective, high-density cultures of GMP-compliant RBCs, and on their relevant novel applications after downstream processing and purification.


Assuntos
Substitutos Sanguíneos/uso terapêutico , Eritrócitos/citologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Diferenciação Celular/genética , Transfusão de Eritrócitos , Eritropoese/genética , Sangue Fetal/citologia , Humanos
7.
Int J Mol Sci ; 22(17)2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34502385

RESUMO

Erythropoietin (Epo) is the critical hormone for erythropoiesis. In adults, Epo is mainly produced by a subset of interstitial fibroblasts in the kidney, with minor amounts being produced in the liver and the brain. In this study, we used the immortalized renal interstitial fibroblast cell line FAIK F3-5 to investigate the ability of the bioactive sphingolipid sphingosine 1-phosphate (S1P) to stimulate Epo production and to reveal the mechanism involved. Stimulation of cells with exogenous S1P under normoxic conditions (21% O2) led to a dose-dependent increase in Epo mRNA and protein levels and subsequent release of Epo into the medium. S1P also enhanced the stabilization of HIF-2α, a key transcription factor for Epo expression. S1P-stimulated Epo mRNA and protein expression was abolished by HIF-2α mRNA knockdown or by the HIF-2 inhibitor compound 2. Furthermore, the approved S1P receptor modulator FTY720, and its active form FTY720-phosphate, both exerted a similar effect on Epo expression as S1P. The effect of S1P on Epo was antagonized by the selective S1P1 and S1P3 antagonists NIBR-0213 and TY-52156, but not by the S1P2 antagonist JTE-013. Moreover, inhibitors of the classical MAPK/ERK, the p38-MAPK, and inhibitors of protein kinase (PK) C and D all blocked the effect of S1P on Epo expression. Finally, the S1P and FTY720 effects were recapitulated in the Epo-producing human neuroblastoma cell line Kelly, suggesting that S1P receptor-dependent Epo synthesis is of general relevance and not species-specific. In summary, these data suggest that, in renal interstitial fibroblasts, which are the primary source of plasma Epo, S1P1 and 3 receptor activation upregulates Epo under normoxic conditions. This may have a therapeutic impact on disease situations such as chronic kidney disease, where Epo production is impaired, causing anemia, but it may also have therapeutic value as Epo can mediate additional tissue-protective effects in various organs.


Assuntos
Eritropoetina/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Linhagem Celular , Células Cultivadas , Eritropoese , Eritropoetina/fisiologia , Fibroblastos/metabolismo , Cloridrato de Fingolimode/metabolismo , Humanos , Hipóxia/metabolismo , Hipóxia/fisiopatologia , Rim/metabolismo , Lisofosfolipídeos/metabolismo , Camundongos , Ligação Proteica , Receptores de Lisoesfingolipídeo/metabolismo , Insuficiência Renal Crônica/metabolismo , Transdução de Sinais , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato/fisiologia
8.
Blood Adv ; 5(18): 3726-3735, 2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34516644

RESUMO

Myeloid dysplastic syndrome (MDS) reflects a preleukemic bone marrow (BM) disorder with limited treatment options and poor disease survival. As only a minority of MDS patients are eligible for curative hematopoietic stem cell transplantation, there is an urgent need to develop alternative treatment options. Chronic activation of Wnt/ß-catenin has been implicated to underlie MDS formation and recently assigned to drive MDS transformation to acute myeloid leukemia. Wnt/ß-catenin signaling therefore may harbor a pharmaceutical target to treat MDS and/or prevent leukemia formation. However, targeting the Wnt/ß-catenin pathway will also affect healthy hematopoiesis in MDS patients. The control of Wnt/ß-catenin in healthy hematopoiesis is poorly understood. Whereas Wnt/ß-catenin is dispensable for steady-state erythropoiesis, its activity is essential for stress erythropoiesis in response to BM injury and anemia. Manipulation of Wnt/ß-catenin signaling in MDS may therefore deregulate stress erythropoiesis and even increase anemia severity. Here, we provide a comprehensive overview of the most recent and established insights in the field to acquire more insight into the control of Wnt/ß-catenin signaling in healthy and inefficient erythropoiesis as seen in MDS.


Assuntos
Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Eritropoese , Humanos , Via de Sinalização Wnt
10.
FASEB J ; 35(10): e21915, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34496088

RESUMO

During development, erythroid cells are generated by two waves of hematopoiesis. In zebrafish, primitive erythropoiesis takes place in the intermediate cell mass region, and definitive erythropoiesis arises from the aorta-gonad mesonephros. TALE-homeoproteins Meis1 and Pbx1 function upstream of GATA1 to specify the erythroid lineage. Embryos lacking Meis1 or Pbx1 have weak gata1 expression and fail to produce primitive erythrocytes. Nevertheless, the underlying mechanism of how Meis1 and Pbx1 mediate gata1 transcription in erythrocytes remains unclear. Here we show that Hif1α acts downstream of Meis1 to mediate gata1 expression in zebrafish embryos. Inhibition of Meis1 expression resulted in suppression of hif1a expression and abrogated primitive erythropoiesis, while injection with in vitro-synthesized hif1α mRNA rescued gata1 transcription in Meis1 morphants and recovered their erythropoiesis. Ablation of Hif1α expression either by morpholino knockdown or Crispr-Cas9 knockout suppressed gata1 transcription and abrogated primitive erythropoiesis. Results of chromatin immunoprecipitation assays showed that Hif1α associates with hypoxia-response elements located in the 3'-flanking region of gata1 during development, suggesting that Hif1α regulates gata1 expression in vivo. Together, our results indicate that Meis1, Hif1α, and GATA1 indeed comprise a hierarchical regulatory network in which Hif1α acts downstream of Meis1 to activate gata1 transcription through direct interactions with its cis-acting elements in primitive erythrocytes.


Assuntos
Células Eritroides/metabolismo , Eritropoese , Fator de Transcrição GATA1/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteína Meis1/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Imunoprecipitação da Cromatina , Eritrócitos/citologia , Eritrócitos/metabolismo , Células Eritroides/citologia , Eritropoese/genética , Fator de Transcrição GATA1/genética , Regulação da Expressão Gênica no Desenvolvimento , Subunidade alfa do Fator 1 Induzível por Hipóxia/deficiência , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Proteína Meis1/deficiência , Proteína Meis1/genética , Fator de Transcrição 1 de Leucemia de Células Pré-B/deficiência , Fator de Transcrição 1 de Leucemia de Células Pré-B/genética , Transcrição Genética , Peixe-Zebra/sangue , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética
11.
Sci Rep ; 11(1): 18557, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34535703

RESUMO

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.


Assuntos
Moléculas de Adesão Celular/genética , Hemoglobina Fetal/análise , Fatores de Transcrição Kruppel-Like/genética , Sistema do Grupo Sanguíneo Lutheran/genética , gama-Globinas/análise , Células Cultivadas , Células Eritroides/citologia , Células Eritroides/metabolismo , Eritropoese , Hemoglobina Fetal/genética , Humanos , Mutação , Polimorfismo de Nucleotídeo Único , gama-Globinas/genética
12.
Blood Adv ; 5(15): 3002-3015, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34351390

RESUMO

Erythropoiesis requires a combination of ubiquitous and tissue-specific transcription factors (TFs). Here, through DNA affinity purification followed by mass spectrometry, we have identified the widely expressed protein MAZ (Myc-associated zinc finger) as a TF that binds to the promoter of the erythroid-specific human α-globin gene. Genome-wide mapping in primary human erythroid cells revealed that MAZ also occupies active promoters as well as GATA1-bound enhancer elements of key erythroid genes. Consistent with an important role during erythropoiesis, knockdown of MAZ reduces α-globin expression in K562 cells and impairs differentiation in primary human erythroid cells. Genetic variants in the MAZ locus are associated with changes in clinically important human erythroid traits. Taken together, these findings reveal the zinc-finger TF MAZ to be a previously unrecognized regulator of the erythroid differentiation program.


Assuntos
Proteínas de Ligação a DNA , Eritropoese , Fatores de Transcrição , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Eritroides/metabolismo , Eritropoese/genética , Regulação da Expressão Gênica , Humanos , Células K562 , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
13.
Life Sci ; 284: 119900, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34453946

RESUMO

ZMIZ1 is a transcriptional coactivator that is related to members of the protein inhibitor of activated STAT (PIAS) family. ZMIZ1 regulates the activity of various transcription factors including the androgen receptor, p53, and Smad3. ZMIZ1 also interacts with Notch1 and selectively regulates Notch1 target genes relevant for T cell development and leukemogenesis in mammals. Human ZMIZ1 is additionally characterized as a latitude-dependent autoimmune disease (LDAD) risk gene, as it is responsive to vitamin D and has been associated with at least eleven blood cell traits. To address the function of ZMIZ1 in fish, we introduced CRISPR/Cas9 mutations in the zmiz1a gene in zebrafish. We observed that inactivation of zmiz1a in developing zebrafish larvae results in lethality at 15 days post fertilization (dpf) and delayed erythroid maturation. Differential gene expression analysis indicated that 15 dpf zmiz1a-null larvae had altered expression of autophagy genes, and erythrocytes that lacked Zmiz1a function exhibited an accumulation of mitochondrial DNA. Furthermore, we observed that autophagy gene expression was dysregulated at earlier stages of development, which suggests the involvement of Zmiz1a in the regulation of autophagy genes beyond the process of red blood cell differentiation. Finally, we showed that the loss of Zmiz1a decreased the capacity of the embryos to respond to vitamin D, indicating additional participation of Zmiz1a as a mediator of vitamin D activity.


Assuntos
Autofagia/genética , Eritropoese/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Mutação/genética , Vitamina D/farmacologia , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Autofagia/efeitos dos fármacos , Sequência de Bases , Diferenciação Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Embrião não Mamífero/patologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/patologia , Eritropoese/efeitos dos fármacos , Dosagem de Genes , Hemoglobinas/metabolismo , Inflamação/genética , Transcriptoma/genética , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/metabolismo
14.
Int J Mol Sci ; 22(16)2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34445665

RESUMO

Mast cell disease is an epigenetically and genetically determined disease entity with very diverse clinical manifestations in potentially every system and tissue due to inap pro priate release of variable subsets of mast cell mediators together with accumulation of either morphologically normal or altered mast cells. Easy bruising, excessive bleeding, and aberrancies of erythropoiesis can frequently be observed in patients with mast cell disease. A thorough history, including a family history, will guide the appropriate work-up, and laboratory evaluations may provide clues to diagnosis. In recent years, our understanding of the involvement of coagulation and anticoagulant pathways, the fibrinolytic system, and erythropoiesis in the pathophysiology of mast cell disease has increased considerably. This review summarizes current knowledge of the impact of the disturbed hemostatic and erythropoietic balance in patients with mast cell disease and describes options of treatment.


Assuntos
Eritropoese/fisiologia , Hemostasia/fisiologia , Mastocitose/sangue , Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Eritropoese/efeitos dos fármacos , Fibrinólise/efeitos dos fármacos , Fibrinolíticos/farmacologia , Hemostasia/efeitos dos fármacos , Heparina/farmacologia , Humanos , Mastocitose/imunologia , Mastocitose/fisiopatologia
15.
Int J Mol Sci ; 22(15)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34360789

RESUMO

The erythroid Krüppel-like factor EKLF/KLF1 is a hematopoietic transcription factor binding to the CACCC DNA motif and participating in the regulation of erythroid differentiation. With combined use of microarray-based gene expression profiling and the promoter-based ChIP-chip assay of E14.5 fetal liver cells from wild type (WT) and EKLF-knockout (Eklf-/-) mouse embryos, we identified the pathways and direct target genes activated or repressed by EKLF. This genome-wide study together with the molecular/cellular analysis of the mouse erythroleukemic cells (MEL) indicate that among the downstream direct target genes of EKLF is Tal1/Scl. Tal1/Scl encodes another DNA-binding hematopoietic transcription factor TAL1/SCL, known to be an Eklf activator and essential for definitive erythroid differentiation. Further identification of the authentic Tal gene promoter in combination with the in vivo genomic footprinting approach and DNA reporter assay demonstrate that EKLF activates the Tal gene through binding to a specific CACCC motif located in its promoter. These data establish the existence of a previously unknow positive regulatory feedback loop between two DNA-binding hematopoietic transcription factors, which sustains mammalian erythropoiesis.


Assuntos
Eritropoese , Feto/embriologia , Hematopoese Extramedular , Fatores de Transcrição Kruppel-Like/metabolismo , Fígado/embriologia , Proteína 1 de Leucemia Linfocítica Aguda de Células T/metabolismo , Animais , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Knockout , Elementos de Resposta , Proteína 1 de Leucemia Linfocítica Aguda de Células T/genética
16.
Int J Mol Sci ; 22(15)2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34360557

RESUMO

Among the eight human glutathione peroxidase isoforms, glutathione peroxidase 4 (GPX4) is the only enzyme capable of reducing complex lipid peroxides to the corresponding alcohols. In mice, corruption of the Gpx4 gene leads to embryonic lethality and more detailed expression silencing studies have implicated the enzyme in several physiological processes (e.g., embryonal cerebrogenesis, neuronal function, male fertility). Experiments with conditional knockout mice, in which expression of the Gpx4 gene was silenced in erythroid precursors, indicated a role of Gpx4 in erythropoiesis. To test this hypothesis in a cellular in vitro model we transfected mouse erythroleukemia cells with a Gpx4 siRNA construct and followed the expression kinetics of erythropoietic gene products. Our data indicate that Gpx4 is expressed at high levels in mouse erythroleukemia cells and that expression silencing of the Gpx4 gene delays in vitro erythropoiesis. However, heterozygous expression of a catalytically inactive Gpx4 mutant (Gpx4+/Sec46Ala) did not induce a defective erythropoietic phenotype in different in vivo and ex vivo models. These data suggest that Gpx4 plays a role in erythroid differentiation of mouse erythroleukemia cells but that heterozygous expression of a catalytically inactive Gpx4 is not sufficient to compromise in vivo and ex vivo erythropoiesis.


Assuntos
Eritropoese , Leucemia Eritroblástica Aguda/patologia , Mitocôndrias/patologia , Estresse Oxidativo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/antagonistas & inibidores , RNA Interferente Pequeno/genética , Animais , Leucemia Eritroblástica Aguda/etiologia , Leucemia Eritroblástica Aguda/metabolismo , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/genética , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo
17.
Sci Rep ; 11(1): 15898, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354145

RESUMO

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.


Assuntos
Proteínas de Transporte/genética , Membrana Eritrocítica/fisiologia , Eritropoese/genética , Anemia/metabolismo , Animais , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Proteínas de Transporte/metabolismo , Membrana Eritrocítica/genética , Eritrócitos/metabolismo , Eritrócitos Anormais/metabolismo , Eritropoese/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Policitemia/genética , Baço
18.
Sci Rep ; 11(1): 17129, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34429458

RESUMO

Production of red blood cells relies on proper mitochondrial function, both for their increased energy demands during differentiation and for proper heme and iron homeostasis. Mutations in genes regulating mitochondrial function have been reported in patients with anemia, yet their pathophysiological role often remains unclear. PGC1ß is a critical coactivator of mitochondrial biogenesis, with increased expression during terminal erythroid differentiation. The role of PGC1ß has however mainly been studied in skeletal muscle, adipose and hepatic tissues, and its function in erythropoiesis remains largely unknown. Here we show that perturbed PGC1ß expression in human hematopoietic stem/progenitor cells from both bone marrow and cord blood results in impaired formation of early erythroid progenitors and delayed terminal erythroid differentiation in vitro, with accumulations of polychromatic erythroblasts, similar to MDS-related refractory anemia. Reduced levels of PGC1ß resulted in deregulated expression of iron, heme and globin related genes in polychromatic erythroblasts, and reduced hemoglobin content in the more mature bone marrow derived reticulocytes. Furthermore, PGC1ß knock-down resulted in disturbed cell cycle exit with accumulation of erythroblasts in S-phase and enhanced expression of G1-S regulating genes, with smaller reticulocytes as a result. Taken together, we demonstrate that PGC1ß is directly involved in production of hemoglobin and regulation of G1-S transition and is ultimately required for proper terminal erythroid differentiation.


Assuntos
Células Eritroides/metabolismo , Eritropoese , Proteínas de Ligação a RNA/metabolismo , Ciclo Celular , Células Cultivadas , Células Eritroides/citologia , Hemoglobinas/metabolismo , Humanos , Proteínas de Ligação a RNA/genética
19.
Stem Cell Res ; 55: 102476, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34339993

RESUMO

Inadequate production of erythropoietin (EPO) leads to anemia. Although erythropoiesis-stimulating agents can be used to treat anemia, these approaches are limited by high costs, adverse effects, and the need for frequent injections. Developing methods for the generation and transplantation of EPO-producing cells would allow for the design of personalized and complication-free therapeutic solutions. In mice, the first EPO source are neural crest cells (NCCs), which ultimately migrate to the fetal kidney to differentiate into EPO-producing fibroblasts. In humans however, it remains unknown whether NCCs can produce EPO in response to hypoxia. Here, we developed a new protocol to differentiate human induced pluripotent stem cells (hiPSCs) into NCCs and showed that cthese cells can produce functional EPO that can induce human CD34+ hematopoietic progenitor differentiation into erythroblasts in vitro. Moreover, we showed that hiPSC-derived NCCs can be embedded in clinical-grade atelocollagen scaffolds and subcutaneously transplanted into anemic mice to produce human EPO, accelerate hematocrit recovery, and induce erythropoiesis in the spleen. Our findings provide unprecedented evidence of the ability of human NCCs to produce functional EPO in response to hypoxia, and proof-of-concept for the potential clinical use of NCC-containing scaffolds as cell therapy for renal and non-renal anemia.


Assuntos
Anemia , Eritropoetina , Células-Tronco Pluripotentes Induzidas , Anemia/induzido quimicamente , Anemia/terapia , Animais , Eritropoese , Humanos , Camundongos , Crista Neural
20.
Front Immunol ; 12: 671648, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34386001

RESUMO

Despite recent advances in immunodeficient mouse models bearing human red blood cells (hRBCs), the elimination of circulating hRBCs by residual innate immune systems remains a significant challenge. In this study, we evaluated the role of mouse complement C3 in the elimination of circulating hRBCs by developing a novel NOG substrain harboring a truncated version of the murine C3 gene (NOG-C3ΔMG2-3). Genetic C3 deletion prolonged the survival of transfused hRBCs in the circulation. Chemical depletion and functional impairment of mouse macrophages, using clodronate liposomes (Clo-lip) or gadolinium chloride (GdCl3), respectively, further extended the survival of hRBCs in NOG-C3ΔMG2-3 mice. Low GdCl3 toxicity allowed the establishment of hRBC-bearing mice, in which hRBCs survived for more than 4 weeks with transfusion once a week. In addition, erythropoiesis of human hematopoietic stem cells (hHSCs) was possible in NOG-C3ΔMG2-3/human GM-CSF-IL-3 transgenic mice with Clo-lip treatment. These findings indicate that mouse models harboring hRBCs can be achieved using NOG-C3ΔMG2-3 mice, which could facilitate studies of human diseases associated with RBCs.


Assuntos
Complemento C3/deficiência , Eritrócitos , Modelos Animais , Animais , Transfusão de Eritrócitos/métodos , Eritrócitos/imunologia , Eritropoese/fisiologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/imunologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos
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