RESUMO
Within the bone marrow microenvironment, endothelial cells (EC) exert important functions. Arterial EC support hematopoiesis while H-type capillaries induce bone formation. Here, we show that BM sinusoidal EC (BM-SEC) actively control erythropoiesis. Mice with stabilized ß-catenin in BM-SEC (Ctnnb1OE-SEC) generated by using a BM-SEC-restricted Cre mouse line (Stab2-iCreF3) develop fatal anemia. While activation of Wnt-signaling in BM-SEC causes an increase in erythroblast subsets (PII-PIV), mature erythroid cells (PV) are reduced indicating impairment of terminal erythroid differentiation/reticulocyte maturation. Transplantation of Ctnnb1OE-SEC hematopoietic stem cells into wildtype recipients confirms lethal anemia to be caused by cell-extrinsic, endothelial-mediated effects. Ctnnb1OE-SEC BM-SEC reveal aberrant sinusoidal differentiation with altered EC gene expression and perisinusoidal ECM deposition and angiocrine dysregulation with de novo endothelial expression of FGF23 and DKK2, elevated in anemia and involved in vascular stabilization, respectively. Our study demonstrates that BM-SEC play an important role in the bone marrow microenvironment in health and disease.
Assuntos
Anemia/genética , Medula Óssea/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Endotélio Vascular/metabolismo , Eritroblastos/metabolismo , Eritropoese/genética , beta Catenina/genética , Anemia/metabolismo , Anemia/mortalidade , Anemia/patologia , Animais , Medula Óssea/irrigação sanguínea , Capilares/citologia , Capilares/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Diferenciação Celular , Células Endoteliais/classificação , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Eritroblastos/classificação , Eritroblastos/citologia , Feminino , Fator de Crescimento de Fibroblastos 23/genética , Fator de Crescimento de Fibroblastos 23/metabolismo , Regulação da Expressão Gênica , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Integrases/genética , Integrases/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Osteogênese , Reticulócitos/citologia , Reticulócitos/metabolismo , Análise de Sobrevida , Via de Sinalização Wnt , beta Catenina/metabolismoRESUMO
Polycythaemia vera (PV) is a haematological disorder caused by an overproduction of erythroid cells. To date, the molecular mechanisms involved in the disease pathogenesis are still ambiguous. This study aims to identify aberrantly expressed proteins in erythroblasts of PV patients by utilizing mass spectrometry-based proteomic analysis. Haematopoietic stem cells (HSCs) were isolated from newly-diagnosed PV patients, PV patients who have received cytoreductive therapy, and healthy subjects. In vitro erythroblast expansion confirmed that the isolated HSCs recapitulated the disease phenotype as the number of erythroblasts from newly-diagnosed PV patients was significantly higher than those from the other groups. Proteomic comparison revealed 17 proteins that were differentially expressed in the erythroblasts from the newly-diagnosed PV patients compared to those from healthy subjects, but which were restored to normal levels in the patients who had received cytoreductive therapy. One of these proteins was S-methyl-5'-thioadenosine phosphorylase (MTAP), which had reduced expression in PV patients' erythroblasts. Furthermore, MTAP knockdown in normal erythroblasts was shown to enhance their proliferative capacity. Together, this study identifies differentially expressed proteins in erythroblasts of healthy subjects and those of PV patients, indicating that an alteration of protein expression in erythroblasts may be crucial to the pathology of PV.
Assuntos
Policitemia Vera/tratamento farmacológico , Policitemia Vera/metabolismo , Purina-Núcleosídeo Fosforilase , Adulto , Idoso , Proliferação de Células , Eritroblastos/metabolismo , Eritrócitos/citologia , Células Precursoras Eritroides/metabolismo , Feminino , Células-Tronco Hematopoéticas/citologia , Humanos , Técnicas In Vitro , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Proteoma , Proteômica/métodos , Fator de Células-Tronco/metabolismoRESUMO
The ß-thalassemia syndromes are the most prevalent genetic disorder globally, characterised by reduced or absent ß-globin chain synthesis. HbE/ß-thalassemia is a subtype of ß-thalassemia with extremely high frequency in Asia. Studying molecular defects behind ß-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/ß-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 ß-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.
Assuntos
Diferenciação Celular/fisiologia , Eritroblastos/citologia , Células Eritroides/citologia , Células-Tronco Hematopoéticas/citologia , Talassemia beta/sangue , Linhagem Celular , HumanosRESUMO
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal metabolic disorder caused by thymidine phosphorylase (TP) deficiency. Successful therapeutic interventions for this disease rely on a means for efficient and long-lasting circulation of the TP enzyme. In this study we exploit lentiviral transduction of hematopoietic stem cells and an erythroid cell line (BEL-A) to generate reticulocytes that contain active TP. Significant loss of overexpressed TP during erythroid differentiation can be reduced by addition of the ubiquitination inhibitor MG132. However, the ubiquitination sites are located in the substrate binding site in human TP, and their removal abolished enzyme activity. Examination of the TP structure and mechanism suggested that these sites are only exposed in the absence of substrate. We show that supplementation of culture media with thymidine during differentiation reduces enzyme degradation, doubling the amount of TP retained in reticulocytes. This study provides proof of principle that therapeutic reticulocytes expressing TP can be generated in vitro and that ubiquitin-mediated degradation can be subverted through masking ubiquitination sites to ensure retention of human TP in reticulocytes following erythroid differentiation.
RESUMO
Blood transfusion is widely used in the clinic but the source of red blood cells (RBCs) is dependent on donors, procedures are susceptible to transfusion-transmitted infections and complications can arise from immunological incompatibility. Clinically-compatible and scalable protocols that allow the production of RBCs from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have been described but progress to translation has been hampered by poor maturation and fragility of the resultant cells. Genetic programming using transcription factors has been used to drive lineage determination and differentiation so we used this approach to assess whether exogenous expression of the Erythroid Krüppel-like factor 1 (EKLF/KLF1) could augment the differentiation and stability of iPSC-derived RBCs. To activate KLF1 at defined time points during later stages of the differentiation process and to avoid transgene silencing that is commonly observed in differentiating pluripotent stem cells, we targeted a tamoxifen-inducible KLF1-ERT2 expression cassette into the AAVS1 locus. Activation of KLF1 at day 10 of the differentiation process when hematopoietic progenitor cells were present, enhanced erythroid commitment and differentiation. Continued culture resulted the appearance of more enucleated cells when KLF1 was activated which is possibly due to their more robust morphology. Globin profiling indicated that these conditions produced embryonic-like erythroid cells. This study demonstrates the successful use of an inducible genetic programing strategy that could be applied to the production of many other cell lineages from human induced pluripotent stem cells with the integration of programming factors into the AAVS1 locus providing a safer and more reproducible route to the clinic. Stem Cells 2017;35:886-897.
Assuntos
Diferenciação Celular , Eritrócitos/citologia , Eritrócitos/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Fatores de Transcrição Kruppel-Like/metabolismo , Núcleo Celular/metabolismo , Proliferação de Células , Eritropoese/genética , Regulação da Expressão Gênica , Globinas/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células K562 , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismoRESUMO
A major barrier to the clinical use of erythrocytes generated in vitro from pluripotent stem cells or cord blood progenitors is failure of these erythrocytes to express adult hemoglobin. The key regulators of globin switching KLF1 and BCL11A are absent or at a lower level than in adult cells in K562 and erythroid cells differentiated in vitro from induced pluripotent stem cells and cord blood progenitors. Transfection or transduction of K562 and cord blood erythroid cells with either KLF1 or BCL11A-XL had little effect on ß-globin expression. In contrast, transduction with both transcription factors stimulated ß-globin expression. Similarly, increasing the level of BCL11A-XL in the induced pluripotent stem cell-derived erythroid cell line HiDEP-1, which has levels of endogenous KLF1 similar to adult cells but lacks BCL11A, resulted in levels of ß-globin equivalent to that of adult erythroid cells. Interestingly, this increase in ß-globin was coincident with a decrease in ε- and ζ-, but not γ-globin, implicating BCL11A in repression of embryonic globin expression. The data show that KLF1 and BCL11A-XL together are required, but sufficient to induce adult levels of ß-globin in induced pluripotent stem cell and cord blood-derived erythroid cells that intrinsically express embryonic or fetal globin.
Assuntos
Proteínas de Transporte/genética , Células Eritroides/metabolismo , Hemoglobina Fetal/genética , Expressão Gênica , Fatores de Transcrição Kruppel-Like/genética , Proteínas Nucleares/genética , Transdução Genética , Globinas beta/genética , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Células Eritroides/citologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células K562 , Fenótipo , Proteínas Repressoras , Transfecção , Globinas épsilon/genética , gama-Globinas/genéticaRESUMO
A number of MDC (metalloproteinase-like, disintegrin-like, cysteine-rich) proteins are exclusively or abundantly expressed on mammalian sperm from a range of species, with data from rodents supporting a role for some of these in oolemma binding. However, in the human, transcripts for three of the most likely candidates have been shown to contain deletions and in-frame termination codons, rendering them non-functional. In this paper we have addressed the expression, in humans, of an additional MDC protein, tMDC III (also known as ADAM 18), previously shown to be exclusively expressed on sperm from rodents and macaques. Using a PCR-based approach, we have determined the complete nucleotide sequence of human tMDC III cDNA which, unlike the three non-functional genes, contains an uninterrupted open reading frame encoding a full-length MDC protein. Furthermore, polyclonal antisera raised against human recombinant tMDC III demonstrated the presence of mature protein on human sperm. In common with orthologues from other species, human tMDC III contains a putative integrin-binding glu-cys-asp (ECD) motif, the relevance of which is discussed in the context of a possible role in oolemma binding.