RESUMO
Extracellular vesicles (EVs) released from healthy endothelial cells (ECs) have shown potential for promoting angiogenesis, but their therapeutic efficacy remains poorly understood. We have previously shown that transplantation of a human embryonic stem cell-derived endothelial cell product (hESC-ECP), promotes new vessel formation in acute ischemic disease in mice, likely via paracrine mechanism(s). Here, we demonstrated that EVs from hESC-ECPs (hESC-eEVs) significantly increased EC tube formation and wound closure in vitro at ultralow doses, whereas higher doses were ineffective. More important, EVs isolated from the mesodermal stage of the differentiation (hESC-mEVs) had no effect. Small RNA sequencing revealed that hESC-eEVs have a unique transcriptomic profile and are enriched in known proangiogenic microRNAs (miRNAs, miRs). Moreover, an in silico analysis identified three novel hESC-eEV-miRNAs with potential proangiogenic function. Differential expression analysis suggested that two of those, miR-4496 and miR-4691-5p, are highly enriched in hESC-eEVs. Overexpression of miR-4496 or miR-4691-5p resulted in increased EC tube formation and wound closure in vitro, validating the novel proangiogenic function of these miRNAs. In summary, we demonstrated that hESC-eEVs are potent inducers of EC angiogenic response at ultralow doses and contain a unique EV-associated miRNA repertoire, including miR-4496 and miR-4691-5p, with novel proangiogenic function.
Assuntos
Vesículas Extracelulares , MicroRNAs , Humanos , Animais , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Células Endoteliais/metabolismo , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Diferenciação Celular/genética , Células-Tronco/metabolismoRESUMO
Pluripotent stem cell-derived differentiated endothelial cells offer high potential in regenerative medicine in the cardiovascular system. With the aim of translating the use of a human stem cell-derived endothelial cell product (hESC-ECP) for treatment of critical limb ischemia (CLI) in man, we report a good manufacturing practice (GMP)-compatible protocol and detailed cell tracking and efficacy data in multiple preclinical models. The clinical-grade cell line RC11 was used to generate hESC-ECP, which was identified as mostly endothelial (60% CD31+/CD144+), with the remainder of the subset expressing various pericyte/mesenchymal stem cell markers. Cell tracking using MRI, PET, and qPCR in a murine model of limb ischemia demonstrated that hESC-ECP was detectable up to day 7 following injection. Efficacy in several murine models of limb ischemia (immunocompromised/immunocompetent mice and mice with either type I/II diabetes mellitus) demonstrated significantly increased blood perfusion and capillary density. Overall, we demonstrate a GMP-compatible hESC-ECP that improved ischemic limb perfusion and increased local angiogenesis without engraftment, paving the way for translation of this therapy.
Assuntos
Células Endoteliais/citologia , Membro Posterior/citologia , Isquemia/terapia , Neovascularização Fisiológica/fisiologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células Endoteliais/metabolismo , Membro Posterior/metabolismo , Humanos , Isquemia/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Pericitos/citologia , Pericitos/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Transplante de Células-Tronco/métodosRESUMO
Schizophrenia and bipolar disorder are leading causes of morbidity across all populations, with heritability estimates of approximately 80% indicating a substantial genetic component. Population genetics and genome-wide association studies suggest an overlap of genetic risk factors between these illnesses but it is unclear how this genetic component is divided between common gene polymorphisms, rare genomic copy number variants, and rare gene sequence mutations. We report evidence that the lipid transporter gene ABCA13 is a susceptibility factor for both schizophrenia and bipolar disorder. After the initial discovery of its disruption by a chromosome abnormality in a person with schizophrenia, we resequenced ABCA13 exons in 100 cases with schizophrenia and 100 controls. Multiple rare coding variants were identified including one nonsense and nine missense mutations and compound heterozygosity/homozygosity in six cases. Variants were genotyped in additional schizophrenia, bipolar, depression (n > 1600), and control (n > 950) cohorts and the frequency of all rare variants combined was greater than controls in schizophrenia (OR = 1.93, p = 0.0057) and bipolar disorder (OR = 2.71, p = 0.00007). The population attributable risk of these mutations was 2.2% for schizophrenia and 4.0% for bipolar disorder. In a study of 21 families of mutation carriers, we genotyped affected and unaffected relatives and found significant linkage (LOD = 4.3) of rare variants with a phenotype including schizophrenia, bipolar disorder, and major depression. These data identify a candidate gene, highlight the genetic overlap between schizophrenia, bipolar disorder, and depression, and suggest that rare coding variants may contribute significantly to risk of these disorders.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/fisiologia , Predisposição Genética para Doença , Polimorfismo Genético , Sequência de Aminoácidos , Transtorno Bipolar/genética , Estudos de Casos e Controles , Códon sem Sentido , Citogenética , Análise Mutacional de DNA , Depressão/genética , Éxons , Feminino , Ligação Genética , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Esquizofrenia/genética , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: Pluripotent stem cells are attractive progenitor cells for the generation of erythroid cells in vitro as have expansive proliferative potential. However, although embryonic (ESC) and induced pluripotent (iPSC) stem cells can be induced to undergo erythroid differentiation, the majority of cells fail to enucleate and the molecular basis of this defect is unknown. One protein that has been associated with the initial phase of erythroid cell enucleation is the intermediate filament vimentin, with loss of vimentin potentially required for the process to proceed. METHODS: In this study, we used our established erythroid culture system along with western blot, PCR and interegation of comparative proteomic data sets to analyse the temporal expression profile of vimentin in erythroid cells differentiated from adult peripheral blood stem cells, iPSC and ESC throughout erythropoiesis. Confocal microscopy was also used to examine the intracellular localisation of vimentin. RESULTS: We show that expression of vimentin is turned off early during normal adult erythroid cell differentiation, with vimentin protein lost by the polychromatic erythroblast stage, just prior to enucleation. In contrast, in erythroid cells differentiated from iPSC and ESC, expression of vimentin persists, with high levels of both mRNA and protein even in orthochromatic erythroblasts. In the vimentin-positive iPSC orthochromatic erythroblasts, F-actin was localized around the cell periphery; however, in those rare cells captured undergoing enucleation, vimentin was absent and F-actin was re-localized to the enucleosome as found in normal adult orthrochromatic erythroblasts. CONCLUSION: As both embryonic and adult erythroid cells loose vimentin and enucleate, retention of vimentin by iPSC and ESC erythroid cells indicates an intrinsic defect. By analogy with avian erythrocytes which naturally retain vimentin and remain nucleated, retention in iPSC- and ESC-derived erythroid cells may impede enucleation. Our data also provide the first evidence that dysregulation of processes in these cells occurs from the early stages of differentiation, facilitating targeting of future studies.
Assuntos
Eritropoese/fisiologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteômica/métodos , Vimentina/metabolismo , Diferenciação Celular , Células Cultivadas , Células Eritroides , Humanos , Células-Tronco Pluripotentes Induzidas/citologiaRESUMO
Squamous epithelium in mammals has evolved an atypical stress response involving down-regulation of the classic HSP70 protein and induction of sets of proteins including one named SEP53. This atypical stress response might be due to the unusual environmental pressures placed on squamous tissue. In fact, SEP53 plays a role as an anti-apoptotic factor in response to DNA damage induced by deoxycholic acid stresses implicated in oesophageal reflux disease. SEP53 also has a genetic signature characteristic of an adaptively and rapidly evolving gene, and this observation has been used to imply a role for SEP53 in immunity. Physiological models of squamous tissue are required to further define the regulation and function of SEP53. We examined whether porcine squamous epithelium would be a good model to study SEP53, since this animal suffers from a bile-reflux disease in squamous oesophageal tissue. We have (1) cloned and sequenced the porcine SEP53 locus from porcine bacterial artificial chromosome genomic DNA, (2) confirmed the strikingly divergent nature of the C-terminal portion of the SEP53 gene amongst mammals, (3) discovered that a function of the conserved N-terminal domain of the gene is to maintain cytoplasmic localisation, and (4) examined SEP53 expression in normal and diseased porcine pars oesophagea. SEP53 expression in porcine tissue was relatively confined to gastric squamous epithelium, consistent with its expression in normal human squamous epithelium. Immunohistochemical staining for SEP53 protein in normal and damaged pars oesophagea demonstrated significant stabilisation of SEP53 protein in the injured tissue. These results suggest that porcine squamous epithelium would be a robust physiological model to examine the evolution and function of the SEP53 stress pathway in modulating stress-induced responses in squamous tissue.
Assuntos
Bile/metabolismo , Modelos Animais de Doenças , Epitélio/patologia , Esôfago/anatomia & histologia , Esôfago/patologia , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Epitélio/metabolismo , Esôfago/metabolismo , Humanos , Proteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , SuínosRESUMO
: This article describes a good manufacturing practice (GMP)-compatible, feeder-free and serum-free method to produce large numbers of erythroid cells from human pluripotent stem cells (hPSCs), either embryonic or induced. This multistep protocol combines cytokines and small molecules to mimic and surpass the early stages of development. It produces, without any selection or sorting step, a population of cells in which 91.8% ± 5.4% express CD34 at day 7, 98.6% ± 1.3% express CD43 at day 10, and 99.1% ± 0.95% of cells are CD235a positive by day 31 of the differentiation process. Moreover, this differentiation protocol supports extensive expansion, with a single hPSC producing up to 150 hematopoietic progenitor cells by day 10 and 50,000-200,000 erythroid cells by day 31. The erythroid cells produced exhibit a definitive fetal hematopoietic type, with 90%-95% fetal globin and variable proportion of embryonic and adult globin at the protein level. The presence of small molecules during the differentiation protocol has quantitative and qualitative effects; it increases the proportion of adult globin and decreases the proportion of embryonic globin. Given its level of definition, this system provides a powerful tool for investigation of the mechanisms governing early hematopoiesis and erythropoiesis, including globin switching and enucleation. The early stages of the differentiation protocol could also serve as a starting point for the production of endothelial cells and other hematopoietic cells, or to investigate the production of long-term reconstituting hematopoietic stem cells from hPSCs. SIGNIFICANCE: This differentiation protocol allows the production of a large amount of erythroid cells from pluripotent stem cells. Its efficiency is compatible with that of in vitro red blood cell production, and it can be a considerable asset for studying developmental erythropoiesis and red blood cell enucleation, thereby aiding both basic and translational research. In addition to red cells, the early stages of the protocol could also be used as a starting point for the large-scale production of other hematopoietic cell types, including the ultimate goal of generating long-term reconstituting hematopoietic stem cells.
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Eritrócitos/citologia , Células-Tronco Pluripotentes/citologia , Linhagem Celular , HumanosRESUMO
The mature sporadic T-cell malignancy, T-cell prolymphocytic leukemia (T-PLL) is remarkable for frequently harbouring somatic mutations of the Ataxia Telangiectasia (A-T) gene, ATM. Because some data suggest ATM is frequently rearranged in T-PLL, it was decided to investigate such rearrangements in detail by cloning breakpoints. Among 17 T-PLL tumour samples, three rearrangements were detected by Southern blotting. Two cases harboured a unique type of intragenic duplication in which breakpoints arose at the consensus sequence RGYW/WRCY. The third case harboured a large deletion terminating within the ATM gene. Also, 13 T-cell acute lymphoblastic leukemia (T-ALL) samples were examined and one sample harboured a deletion- insertion with the RGYW motif at the breakpoint in ATM. This is the first known deleterious mutation detected in ATM in T-ALL. Interestingly, the RGYW motif is the signal for a cell-cycle regulated DNA double strand break (DSB) that initiates somatic hypermutation of immunoglobulin and, probably, T-cell receptor genes. The structures of the ATM duplications suggest they may arise from an error in somatic hypermutation. We suggest that aberrant components of somatic hypermutation may contribute to the defective DSB repair characteristic of cancer.
Assuntos
Quebra Cromossômica/genética , Sequência Consenso/genética , Leucemia de Células T/genética , Mutação/genética , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Adolescente , Adulto , Idoso , Motivos de Aminoácidos , Sequência de Aminoácidos , Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia , Sequência de Bases , Proteínas de Ciclo Celular , Criança , Proteínas de Ligação a DNA , Éxons/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Recombinação Genética/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Supressoras de TumorRESUMO
Anticipation--earlier onset and more severe disease in the offspring generation--is a well documented feature of familial chronic lymphocytic leukaemia (CLL). In a number of Mendelian diseases, anticipation is caused by expansion of contiguous triplets of nucleotides. The severity of disease expression and penetrance is related to the extent of the triplet expansion. To investigate whether repeat nucleotide repeat expansion is a feature of CLL, the repeat expansion detection (RED) technique was applied to samples from 17 patients with familial disease and 32 patients with early-onset CLL disease. No potentially pathological CAG expansions were detected. We conclude that unstable CAG repeat expansion is not a feature of CLL and that other processes are likely to be involved in generating anticipation in familial forms of the disease.
Assuntos
Leucemia Linfocítica Crônica de Células B/genética , Expansão das Repetições de Trinucleotídeos/fisiologia , Adulto , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Antecipação Genética/genética , Southern Blotting , Análise Mutacional de DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Repetições de Trinucleotídeos/fisiologiaRESUMO
Acute myeloid leukaemia (AML) is the most common acute leukaemia in adults. Around 10-15% of individuals with recessively inherited Fanconi anaemia (FA) develop AML. FA is one of a group of recessive syndromes characterized by excessive spontaneous chromosomal breakage in which heterozygote carriers appear to display an increased risk of cancer and there is some indirect evidence that FA carriers may also be at increased risk of AML. This suggests that FA genes may play a role in the development of AML in the wider context. To examine this proposition, further, we have screened samples from 79 AML patients for mutations in the major FA gene, FANCA. No truncating FANCA mutations were detected. One missense mutation previously designated as pathogenic and five novel missense mutations causing non-conservative amino acid substitutions were detected. The data suggests that while FANCA mutations are rare, FANCA mutations may contribute to the development of the disease in a subset of AML.
Assuntos
Proteínas de Ligação a DNA , Anemia de Fanconi/genética , Leucemia Mieloide Aguda/genética , Proteínas/genética , Adolescente , Adulto , Idoso , Análise Mutacional de DNA , Primers do DNA/química , Éxons , Proteína do Grupo de Complementação A da Anemia de Fanconi , Genes Recessivos , Humanos , Pessoa de Meia-Idade , Mutação , Mutação de Sentido IncorretoRESUMO
Tgfbm1 (chromosome 5, P = 8 x 10(-5)) and Tgfbm3 (chromosome 12, P = 6 x 10(-11)) were identified as loci that modify developmental angiogenesis of Tgfb1 -/- mice. Congenic mice validated these loci and demonstrated epistatic interaction between them. The novel locus, Tgfbm3, encompasses approximately 22 genes, colocalizes with both tumor susceptibility and atherosclerosis susceptibility loci, and is enriched in genes regulating cell growth and morphogenesis. The use of gene knockout and/or transgenic mice that predispose to a complex trait, such as vascular development/angiogenesis, facilitates the identification of modifiers by simplifying genetic analysis. Identification of genes that modify response to lack of transforming growth factor beta1 (TGFbeta1) will enhance the understanding of TGFbeta1 action in vivo and may help predict which patients would respond well to anti-TGFbeta therapy. Identification of angiogenesis-modifying genes may provide new targets for angiogenesis therapies and analysis of polymorphisms therein may contribute to assessment of risk for diseases involving angiogenesis.
Assuntos
Epistasia Genética , Regulação da Expressão Gênica no Desenvolvimento , Neovascularização Fisiológica/genética , Locos de Características Quantitativas/genética , Fator de Crescimento Transformador beta/genética , Animais , Arteriosclerose/genética , Cromossomos/genética , Predisposição Genética para Doença/genética , Escore Lod , Camundongos , Camundongos Knockout , Neoplasias/genética , Especificidade da Espécie , Fator de Crescimento Transformador beta1 , Fator de Crescimento Transformador beta3RESUMO
Interindividual differences in susceptibility to hematologic malignancies may be mediated in part through polymorphic variability in the bioactivation and detoxification of carcinogens. The glutathione S-transferases (GSTs) have been implicated as susceptibility genes in this context for a number of cancers. The aim of this study was to examine whether polymorphic variation in GSTs confers susceptibility to chronic lymphocytic leukemia (CLL). GSTM1, GSTT1, and GSTP1 genotypes were determined in 138 patients and 280 healthy individuals. The frequency of both GSTM1 and GSTT1 null genotypes and the GSTP1-Ile allele was higher in cases than in controls. There was evidence of a trend in increasing risk with the number of putative "high-risk" alleles of the GST family carried (P =.04). The risk of CLL associated with possession of all 3 "high-risk" genotypes was increased 2.8-fold (OR = 2.8, 95% confidence interval: 1.1-6.9). Our findings suggest that heritable GST status may influence the risk of developing CLL.
Assuntos
Glutationa Transferase/genética , Isoenzimas/genética , Leucemia Linfocítica Crônica de Células B/genética , Polimorfismo Genético , Feminino , Frequência do Gene , Genótipo , Glutationa S-Transferase pi , Humanos , Leucemia Linfocítica Crônica de Células B/enzimologia , Masculino , Pessoa de Meia-IdadeRESUMO
It is now recognized that a subset of B-cell chronic lymphocytic leukemia (CLL) is familial. The genetic basis of familial CLL is poorly understood, but recently germ line mutations in the Ataxia Telangiectasia (ATM) gene have been proposed to confer susceptibility to CLL. The evidence for this notion is, however, not unequivocal. To examine this proposition further we have screened the ATM gene for mutations in CLLs from 61 individuals in 29 families. Truncating ATM mutations, including a known ATM mutation, were detected in 2 affected individuals, but the mutations did not cosegregate with CLL in the families. In addition, 3 novel ATM missense mutations were detected. Common ATM missense mutations were not overrepresented. The data support previous observations that ATM mutation is associated with B-CLL. However, ATM mutations do not account for familial clustering of the disease.