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1.
BMC Genomics ; 17(1): 817, 2016 10 21.
Artículo en Inglés | MEDLINE | ID: mdl-27769165

RESUMEN

BACKGROUND: Human-induced pluripotent stem cells (hiPSCs) are a potentially invaluable resource for regenerative medicine, including the in vitro manufacture of blood products. HiPSC-derived red blood cells are an attractive therapeutic option in hematology, yet exhibit unexplained proliferation and enucleation defects that presently preclude such applications. We hypothesised that substantial differential regulation of gene expression during erythroid development accounts for these important differences between hiPSC-derived cells and those from adult or cord-blood progenitors. We thus cultured erythroblasts from each source for transcriptomic analysis to investigate differential gene expression underlying these functional defects. RESULTS: Our high resolution transcriptional view of definitive erythropoiesis captures the regulation of genes relevant to cell-cycle control and confers statistical power to deploy novel bioinformatics methods. Whilst the dynamics of erythroid program elaboration from adult and cord blood progenitors were very similar, the emerging erythroid transcriptome in hiPSCs revealed radically different program elaboration compared to adult and cord blood cells. We explored the function of differentially expressed genes in hiPSC-specific clusters defined by our novel tunable clustering algorithms (SMART and Bi-CoPaM). HiPSCs show reduced expression of c-KIT and key erythroid transcription factors SOX6, MYB and BCL11A, strong HBZ-induction, and aberrant expression of genes involved in protein degradation, lysosomal clearance and cell-cycle regulation. CONCLUSIONS: Together, these data suggest that hiPSC-derived cells may be specified to a primitive erythroid fate, and implies that definitive specification may more accurately reflect adult development. We have therefore identified, for the first time, distinct gene expression dynamics during erythroblast differentiation from hiPSCs which may cause reduced proliferation and enucleation of hiPSC-derived erythroid cells. The data suggest several mechanistic defects which may partially explain the observed aberrant erythroid differentiation from hiPSCs.


Asunto(s)
Eritropoyesis/genética , Sangre Fetal/citología , Regulación del Desarrollo de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Transcriptoma , Diferenciación Celular/genética , Análisis por Conglomerados , Eritroblastos/citología , Eritroblastos/metabolismo , Perfilación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Humanos , Células Madre Pluripotentes Inducidas/citología
2.
Blood ; 117(13): e96-108, 2011 Mar 31.
Artículo en Inglés | MEDLINE | ID: mdl-21270440

RESUMEN

Understanding the pattern of gene expression during erythropoiesis is crucial for a synthesis of erythroid developmental biology. Here, we isolated 4 distinct populations at successive erythropoietin-dependent stages of erythropoiesis, including the terminal, pyknotic stage. The transcriptome was determined using Affymetrix arrays. First, we demonstrated the importance of using defined cell populations to identify lineage and temporally specific patterns of gene expression. Cells sorted by surface expression profile not only express significantly fewer genes than unsorted cells but also demonstrate significantly greater differences in the expression levels of particular genes between stages than unsorted cells. Second, using standard software, we identified more than 1000 transcripts not previously observed to be differentially expressed during erythroid maturation, 13 of which are highly significantly terminally regulated, including RFXAP and SMARCA4. Third, using matched filtering, we identified 12 transcripts not previously reported to be continuously up-regulated in maturing human primary erythroblasts. Finally, using transcription factor binding site analysis, we identified potential transcription factors that may regulate gene expression during terminal erythropoiesis. Our stringent lists of differentially regulated and continuously expressed transcripts containing many genes with undiscovered functions in erythroblasts are a resource for future functional studies of erythropoiesis. Our Human Erythroid Maturation database is available at https://cellline.molbiol.ox.ac.uk/eryth/index.html. [corrected].


Asunto(s)
Células Precursoras Eritroides/metabolismo , Células Precursoras Eritroides/fisiología , Eritropoyesis/genética , Perfilación de la Expresión Génica , Análisis por Micromatrices , Diferenciación Celular/genética , Células Cultivadas , Análisis por Conglomerados , Eritroblastos/metabolismo , Eritroblastos/fisiología , Células Precursoras Eritroides/química , Eritropoyesis/fisiología , Citometría de Flujo , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica , Humanos , Análisis por Micromatrices/métodos , Reacción en Cadena de la Polimerasa
3.
Blood ; 114(1): 20-5, 2009 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-19342478

RESUMEN

Hereditary hemochromatosis is an iron overload disorder that can lead to the impairment of multiple organs and is caused by mutations in one or more different genes. Type 1 hemochromatosis is the most common form of the disease and results from mutations in the HFE gene. Juvenile hemochromatosis (JH) is the most severe form, usually caused by mutations in hemojuvelin (HJV) or hepcidin (HAMP). The autosomal dominant form of the disease, type 4, is due to mutations in the SLC40A1 gene, which encodes for ferroportin (FPN). Hereditary hemochromatosis is commonly found in populations of European origin. By contrast, hemochromatosis in Asia is rare and less well understood and can be masked by the presence of iron deficiency and secondary iron overload from thalassemia. Here, we provide a comprehensive report of hemochromatosis in a group of patients of Asian origin. We have identified novel mutations in HJV, HAMP, and SLC40A1 in countries not normally associated with hereditary hemochromatosis (Pakistan, Bangladesh, Sri Lanka, and Thailand). Our family studies show a high degree of consanguinity, highlighting the increased risk of iron overload in many countries of the developing world and in countries in which there are large immigrant populations from these regions.


Asunto(s)
Sobrecarga de Hierro/genética , Adolescente , Adulto , Secuencia de Aminoácidos , Péptidos Catiónicos Antimicrobianos/genética , Asia , Pueblo Asiatico/genética , Proteínas de Transporte de Catión/genética , Niño , Consanguinidad , Femenino , Genotipo , Hemocromatosis/genética , Proteína de la Hemocromatosis , Hepcidinas , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Masculino , Proteínas de la Membrana/genética , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutación , Linaje , Fenotipo , Homología de Secuencia de Aminoácido , Adulto Joven
4.
Blood Cells Mol Dis ; 43(2): 180-93, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19487139

RESUMEN

Hereditary hemochromatosis is most frequently associated with mutations in HFE, which encodes a class Ib histocompatibility protein. HFE binds to the transferrin receptor-1 (TfR1) in competition with iron-loaded transferrin (Fe-Tf). HFE is released from TfR1 by increasing concentrations of Fe-Tf, and free HFE may then regulate iron homeostasis by binding other ligands. To search for new HFE ligands we expressed recombinant forms of HFE in the human cell line 293T. HFE protein was purified, biotinylated and made into fluorescently labelled tetramers. HFE tetramers bound to TfR1 in competition with Tf, but in addition we detected a binding activity on some cell types that was not blocked by Fe-Tf or by mutations in HFE that prevent binding to TfR1. We identified this second HFE ligand as the cation independent mannose-6-phosphate receptor (CI-MPR, also known as the insulin-like growth factor-2 receptor, IGF2R). HFE:CI-MPR binding was mediated through phosphorylated mannose residues on HFE. Recombinant murine Hfe also bound to CI-MPR. HFE bound to TfR1 was prevented from binding CI-MPR until released by increasing concentrations of Fe-Tf, a feature consistent with an iron sensing mechanism. However, it remains to be determined whether endogenous HFE in vivo also acquires the mannose-6 phosphate modification and binds to CI-MPR.


Asunto(s)
Antígenos CD/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Manosafosfatos/metabolismo , Proteínas de la Membrana/metabolismo , Receptor IGF Tipo 2/metabolismo , Receptores de Transferrina/metabolismo , Transferrina/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Proteína de la Hemocromatosis , Humanos , Ligandos , Ratones , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo
5.
Blood Cells Mol Dis ; 43(2): 194-8, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19477142

RESUMEN

On admission to hospital Caucasian 61 year old male with jaundice was found to have unexplained increased serum iron indices. He had bilateral peripheral arthritis. On further investigation he had grade II hepatocellular iron staining and a hepatic index of 5.4 leading to a diagnosis of hereditary hemochromatosis. He lacked the common C282Y HFE mutation. We sequenced the complete HFE gene and found that he was heterozygous for a novel single nucleotide deletion (c.del478) in exon 3 of HFE. He lacks any other mutation in HFE or HJV, TFR2, HAMP and SLC40A1. The HFE mutation causes a frameshift (p.P160fs) that introduces a premature termination codon leading to mRNA degradation by nonsense-mediated decay. Haploinsufficiency of HFE may be one possible explanation for hemochromatosis in this patient.


Asunto(s)
Hemocromatosis/genética , Antígenos de Histocompatibilidad Clase I/genética , Proteínas de la Membrana/genética , Estabilidad del ARN , Codón sin Sentido/metabolismo , Exones/genética , Proteína de la Hemocromatosis , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple/genética , Eliminación de Secuencia , Transcripción Genética
6.
Stem Cell Res Ther ; 9(1): 351, 2018 12 19.
Artículo en Inglés | MEDLINE | ID: mdl-30567594

RESUMEN

BACKGROUND: Human bone marrow-derived mesenchymal stem/stromal cells (hBM MSCs) have multiple functions, critical for skeletal formation and function. Their functional heterogeneity, however, represents a major challenge for their isolation and in developing potency and release assays to predict their functionality prior to transplantation. Additionally, potency, biomarker profiles and defining mechanisms of action in a particular clinical setting are increasing requirements of Regulatory Agencies for release of hBM MSCs as Advanced Therapy Medicinal Products for cellular therapies. Since the healing of bone fractures depends on the coupling of new blood vessel formation with osteogenesis, we hypothesised that a correlation between the osteogenic and vascular supportive potential of individual hBM MSC-derived CFU-F (colony forming unit-fibroblastoid) clones might exist. METHODS: We tested this by assessing the lineage (i.e. adipogenic (A), osteogenic (O) and/or chondrogenic (C)) potential of individual hBM MSC-derived CFU-F clones and determining if their osteogenic (O) potential correlated with their vascular supportive profile in vitro using lineage differentiation assays, endothelial-hBM MSC vascular co-culture assays and transcriptomic (RNAseq) analyses. RESULTS: Our results demonstrate that the majority of CFU-F (95%) possessed tri-lineage, bi-lineage or uni-lineage osteogenic capacity, with 64% of the CFU-F exhibiting tri-lineage AOC potential. We found a correlation between the osteogenic and vascular tubule supportive activity of CFU-F clones, with the strength of this association being donor dependent. RNAseq of individual clones defined gene fingerprints relevant to this correlation. CONCLUSIONS: This study identified a donor-dependent correlation between osteogenic and vascular supportive potential of hBM MSCs and important gene signatures that support these functions that are relevant to their bone regenerative properties.


Asunto(s)
Células Madre Mesenquimatosas/metabolismo , Osteogénesis/genética , Adulto , Proliferación Celular , Células Cultivadas , Humanos , Adulto Joven
7.
PLoS One ; 10(3): e0119836, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25781011

RESUMEN

The role of infection in erythropoietic dysfunction is poorly understood. In children with P. falciparum malaria, the by-product of hemoglobin digestion in infected red cells (hemozoin) is associated with the severity of anemia which is independent of circulating levels of the inflammatory cytokine tumor necrosis alpha (TNF-α). To gain insight into the common and specific effects of TNF-α and hemozoin on erythropoiesis, we studied the gene expression profile of purified primary erythroid cultures exposed to either TNF-α (10 ng/ml) or to hemozoin (12.5 µg/ml heme units) for 24 hours. Perturbed gene function was assessed using co-annotation of associated gene ontologies and expression of selected genes representative of the profile observed was confirmed by real time PCR (rtPCR). The changes in gene expression induced by each agent were largely distinct; many of the genes significantly modulated by TNF-α were not affected by hemozoin. The genes modulated by TNF-α were significantly enriched for those encoding proteins involved in the control of type 1 interferon signalling and the immune response to viral infection. In contrast, genes induced by hemozoin were significantly enriched for functional roles in regulation of transcription and apoptosis. Further analyses by rtPCR revealed that hemozoin increases expression of transcription factors that form part of the integrated stress response which is accompanied by reduced expression of genes involved in DNA repair. This study confirms that hemozoin induces cellular stress on erythroblasts that is additional to and distinct from responses to inflammatory cytokines and identifies new genes that may be involved in the pathogenesis of severe malarial anemia. More generally the respective transcription profiles highlight the varied mechanisms through which erythropoiesis may be disrupted during infectious disease.


Asunto(s)
Eritrocitos/citología , Eritropoyesis/fisiología , Enfermedades Hematológicas/etiología , Hemoproteínas/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Apoptosis/efectos de los fármacos , Células Cultivadas , Eritrocitos/metabolismo , Eritrocitos/parasitología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Hemoproteínas/metabolismo , Hemoproteínas/fisiología , Humanos , Inmunidad Celular , Interferones/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/fisiología
9.
Blood ; 106(3): 1092-7, 2005 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-15831700

RESUMEN

Ferroportin (FPN) mediates iron export from cells; FPN mutations are associated with the iron overloading disorder hemochromatosis. Previously, we found that the A77D, V162del, and G490D mutations inhibited FPN activity, but that other disease-associated FPN variants retained full iron export capability. The peptide hormone hepcidin inhibits FPN as part of a homeostatic negative feedback loop. We measured surface expression and function of wild-type FPN and fully active FPN mutants in the presence of hepcidin. We found that the Y64N and C326Y mutants of FPN are completely resistant to hepcidin inhibition and that N144D and N144H are partially resistant. Hemochromatosis-associated FPN mutations, therefore, either reduce iron export ability or produce an FPN variant that is insensitive to hepcidin. The former mutation type is associated with Kupffer-cell iron deposition and normal transferrin saturation in vivo, whereas patients with the latter category of FPN mutation have high transferrin saturation and tend to deposit iron throughout the liver parenchyma. FPN-linked hemochromatosis may have a variable pathogenesis depending on the causative FPN mutant.


Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Proteínas de Transporte de Catión/genética , Resistencia a Medicamentos/genética , Hemocromatosis/genética , Mutación Missense , Proteínas de Transporte de Catión/antagonistas & inhibidores , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Hemocromatosis/tratamiento farmacológico , Hemocromatosis/etiología , Hepcidinas , Humanos , Hierro/metabolismo , Radioisótopos de Hierro/metabolismo , Transferrina/metabolismo
10.
Blood ; 105(10): 4096-102, 2005 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-15692071

RESUMEN

Type IV hemochromatosis is associated with dominant mutations in the SLC40A1 gene encoding ferroportin (FPN). Known as the "ferroportin disease," this condition is typically characterized by high serum ferritin, reduced transferrin saturation, and macrophage iron loading. Previously FPN expression in vitro has been shown to cause iron deficiency in human cell lines and mediate iron export from Xenopus oocytes. We confirm these findings by showing that expression of human FPN in a human cell line results in an iron deficiency because of a 3-fold increased export of iron. We show that FPN mutations A77D, V162delta, and G490D that are associated with a typical pattern of disease in vivo cause a loss of iron export function in vitro but do not physically or functionally impede wild-type FPN. These mutants may, therefore, lead to disease by haploinsufficiency. By contrast the variants Y64N, N144D, N144H, Q248H, and C326Y, which can be associated with greater transferrin saturation and more prominent iron deposition in liver parenchyma in vivo, retained iron export function in vitro. Because FPN is a target for negative feedback in iron homeostasis, we postulate that the latter group of mutants may resist inhibition, resulting in a permanently "turned on" iron exporter.


Asunto(s)
Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Hemocromatosis/genética , Mutación/genética , Antígenos CD , Línea Celular , Ferritinas/metabolismo , Humanos , Espacio Intracelular/metabolismo , Hierro/metabolismo , Deficiencias de Hierro , Fenotipo , Unión Proteica , Receptores de Transferrina/metabolismo
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