Asunto(s)
Ácidos Nucleicos Libres de Células/análisis , Síndrome de Klinefelter/diagnóstico , Adulto , Ácidos Nucleicos Libres de Células/sangre , Ácidos Nucleicos Libres de Células/genética , Reacciones Falso Positivas , Femenino , Humanos , Síndrome de Klinefelter/genética , Embarazo , Estudios RetrospectivosRESUMEN
Chorion, amnion and villi are reservoirs of mesenchymal stromal cells (StC) and the hypothesis that StC from fetal tissues retain higher plasticity compared to adult StC has been suggested. Aimed at investigating this aspect, a series of in vitro experiments were performed with StC isolated from first trimester human chorionic villi (CVStC). CVStC were cultured in: (i) standard mesenchymal medium (MM) and (ii) AmniomaxII® (AM), specifically designed to grow amnion-derived cells in prenatal diagnostic procedures. Cells were then exposed to distinct differentiation treatments and distinguished according to morphology, immunophenotype and molecular markers. Human StC obtained from adult bone marrow (BMStC) were used as control. CVStC cultured either in MM or AM presented stromal morphology and immunophenotype, were negative for pluripotency factors (Nanog, Oct-4 and Sox-2), lacked detectable telomerase activity and retained high genomic stability. In AM, however, CVStC exhibited a faster proliferation rate compared to BMStC or CVStC kept in MM. During differentiation, CVStC were less efficient than BMStC in acquiring adipocytes and osteocytes features; the cardiomyogenic conversion occurred at low efficiency in both cell types. Remarkably, in the presence of pro-angiogenic factors, CVStC reprogrammed toward an endothelial-like phenotype at significantly higher efficiency than BMStC. This effect was particularly evident in CVStC expanded in AM. Mechanistically, the reduced CVStC expression of anti-angiogenic microRNA could support this process. The present study demonstrates that, despite of fetal origin, CVStC exhibit restricted plasticity, distinct from that of BMStC and predominantly directed toward the endothelial lineage.
Asunto(s)
Diferenciación Celular , Linaje de la Célula , Medios de Cultivo , Endotelio/crecimiento & desarrollo , Células Madre Mesenquimatosas/citología , Médula Ósea/crecimiento & desarrollo , Proliferación Celular , Vellosidades Coriónicas/crecimiento & desarrollo , Endotelio/citología , Inestabilidad Genómica , Proteínas de Homeodominio/metabolismo , Humanos , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factores de Transcripción SOXB1/metabolismo , Telomerasa/metabolismoRESUMEN
Dilated cardiomyopathy (DCM) is a common heart disorder caused by genetic and non-genetic etiologies, characterized by left ventricular dilatation and contractile dysfunction. Here, we created a human induced pluripotent stem cell line from peripheral blood mononuclear cells using non-integrating vectors from a patient carrying a heterozygous LMNA variant (c.481G > A, p.Glu161Lys, NM_170707.4). The obtained EURACi015-A line, showed the typical morphology of pluripotent cells, normal karyotype and exhibited pluripotency markers and a trilineage differentiation potential. This cell line can be successfully differentiated into cardiomyocytes and endothelial cells. This line represents a human in vitro model to study the genetic basis of DCM.
Asunto(s)
Cardiomiopatía Dilatada , Células Madre Pluripotentes Inducidas , Humanos , Cardiomiopatía Dilatada/genética , Células Madre Pluripotentes Inducidas/metabolismo , Lamina Tipo A/genética , Células Endoteliales/metabolismo , Leucocitos Mononucleares/metabolismo , MutaciónRESUMEN
The SNCA gene encodes the presynaptic α-synuclein (aSyn) protein, and its mutations are associated with autosomal dominant Parkinson's disease (PD). We describe the generation of an induced pluripotent stem cell (iPSC) line of a patient carrying a pathogenic Ala53Thr missense mutation in the SNCA gene. Human dermal fibroblasts were reprogrammed using a non-integrating episomal method. The generated iPSC line (EURACi014-A; iPS-1.1) shows expression of pluripotency markers, the potential to differentiate into all three germ layers, and a stable karyotype. Hence, this line represents a valuable resource for the study and modeling of the processes directly controlled by aSyn.
Asunto(s)
Células Madre Pluripotentes Inducidas , Enfermedad de Parkinson , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mutación/genética , Mutación Missense , Enfermedad de Parkinson/patología , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
Mutations in the Parkin (PRKN) gene are the most frequent known cause of autosomal recessive early-onset Parkinson's disease (PD). Heterozygous mutations might predispose to disease with a highly reduced penetrance. We generated iPSC lines from two individuals carrying a heterozygous deletion of exon 7 in the PRKN gene and two controls from the same family. PBMCs were reprogrammed using non-integrating episomal plasmids. The iPSC lines exhibit expression of pluripotency markers, the potential to differentiate into the three germ layers, and a stable karyotype. These lines will serve to study mechanisms of reduced penetrance in heterozygous PRKN mutation carriers.
Asunto(s)
Células Madre Pluripotentes Inducidas , Enfermedad de Parkinson , Exones/genética , Heterocigoto , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mutación , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Arrhythmogenic Cardiomyopathy (ACM) is a genetically based cardiomyopathy associated with ventricular arrhythmias and fibro-fatty substitution of cardiac tissue. It is characterized by incomplete penetrance. We generated human iPSCs by episomal reprogramming of blood cells from three members of the same family: the proband, affected by ACM and carrying the heterozygous plakophillin2 p.N346Lfs*12 mutation, one asymptomatic carrier of the same mutation and one apparently healthy control. hiPSCs were characterized according to standard protocols including karyotyping, pluripotency marker expression and differentiation towards the three germ layers. These hiPSC lines can be used to study the mechanisms of ACM incomplete penetrance in vitro.
Asunto(s)
Cardiomiopatías , Células Madre Pluripotentes Inducidas , Diferenciación Celular , Heterocigoto , Humanos , MutaciónRESUMEN
Becker Muscular dystrophy (BMD) is an X-linked syndrome characterized by progressive muscle weakness. BMD is generally less severe than Duchenne Muscular Dystrophy. BMD is caused by mutations in the dystrophin gene that normally give rise to the production of a truncated but partially functional dystrophin protein. We generated an induced pluripotent cell line from dermal fibroblasts of a BMD patient carrying a splice mutation in the dystrophin gene (c.1705-8 T>C). The iPSC cell-line displayed the characteristic pluripotent-like morphology, expressed pluripotency markers, differentiated into cells of the three germ layers and had a normal karyotype.
Asunto(s)
Células Madre Pluripotentes Inducidas , Distrofia Muscular de Duchenne , Distrofina/genética , Exones , Humanos , Distrofia Muscular de Duchenne/genética , MutaciónRESUMEN
Duchenne's muscular dystrophy (DMD) is a neuromuscular disorder affecting skeletal and cardiac muscle function, caused by mutations in the dystrophin (DMD) gene. Dermal fibroblasts, isolated from a DMD patient with a reported deletion of exons 51 to 53 in the DMD gene, were reprogramed into induced pluripotent stem cells (iPSCs) by electroporation with episomal vectors containing the reprograming factors: OCT4, SOX2, LIN28, KLF4, and L-MYC. The obtained iPSC line showed iPSC morphology, expression of pluripotency markers, possessed trilineage differentiation potential and was karyotypically normal.
Asunto(s)
Distrofina/genética , Células Madre Pluripotentes Inducidas/citología , Distrofia Muscular de Duchenne/patología , Diferenciación Celular , Línea Celular , Reprogramación Celular , Dermis/citología , Exones , Fibroblastos/citología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Cariotipo , Factor 4 Similar a Kruppel , Masculino , Distrofia Muscular de Duchenne/genética , Eliminación de Secuencia , Factores de Transcripción/genéticaRESUMEN
Mutations in the PRKN gene, encoding parkin, are the most frequent known cause of recessive Parkinson's disease (PD). We report the generation of an induced pluripotent stem cell (iPSC) line of a patient carrying a homozygous deletion of exon 3 in the PRKN gene. Skin fibroblasts were reprogrammed using non-integrating episomal plasmids. The generated cell line (EURACi005-A; iPS-2011) exhibits expression of pluripotency markers, the potential to differentiate into all three germ layers, and a stable karyotype. This iPSC line provides a valuable resource for further research on the pathomechanism and drug testing for PRKN-linked PD.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Línea Celular/patología , Exones/genética , Células Madre Pluripotentes Inducidas/patología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Eliminación de Secuencia/genética , Ubiquitina-Proteína Ligasas/genética , Secuencia de Bases , Femenino , Homocigoto , Humanos , Reproducibilidad de los ResultadosRESUMEN
Arrhythmogenic Cardiomyopathy (ACM) is an inherited cardiac disease characterized by arrhythmias and fibro-fatty replacement in the ventricular myocardium. Causative mutations are mainly reported in desmosomal genes, especially in plakophilin2 (PKP2). Here, using a virus-free reprogramming approach, we generated induced pluripotent stem cells (iPSCs) from skin fibroblasts of one ACM patient carrying the frameshift heterozygous PKP2 mutation c.2569_3018del50. The iPSC line (EURACi004-A) showed the typical morphology of pluripotent cells, possessed normal karyotype and exhibited pluripotency markers and trilineage differentiation potential, including cardiomyogenic capability. Thus, this line can represent a human in vitro model to study the molecular basis of ACM.
Asunto(s)
Fibroblastos/citología , Células Madre Pluripotentes Inducidas/citología , Placofilinas/genética , Piel/citología , Arritmias Cardíacas/metabolismo , Cardiomiopatías/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Fibroblastos/metabolismo , Heterocigoto , Humanos , Mutación/genéticaRESUMEN
Becker muscular dystrophy (BMD) is a dystrophinopathy caused by mutations in the dystrophin gene on chromosome Xp21. BMD mutations result in truncated semi-functional dystrophin isoforms. Consequently, less severe clinical symptoms become apparent later in life compared to Duchenne muscular dystrophy. Dermal fibroblasts from a BMD patient were electroporated with episomal plasmids containing reprogramming factors to create the induced pluripotent stem cell line: CCMi002BMD-A-9 that showed pluripotent markers, were karyotypically normal and capable of trilineage differentiation. MLPA analyses performed on DNA extracted from CCMi002BMD-A-9 showed an in-frame deletion of exons 45 to 55 (CCMi002BMD-A-9 Δ45-55).
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Distrofina/genética , Exones/genética , Células Madre Pluripotentes Inducidas/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patología , Eliminación de Secuencia/genética , Adulto , Humanos , MasculinoRESUMEN
Caveolinopathies are a heterogeneous family of genetic pathologies arising from alterations of the caveolin-3 gene (CAV3), encoding for the isoform specifically constituting muscle caveolae. Here, by reprogramming peripheral blood mononuclear cells, we report the generation of induced pluripotent stem cells (iPSCs) from three patients carrying the ΔYTT deletion, T78K and W101C missense mutations in caveolin-3. iPSCs displayed normal karyotypes and all the features of pluripotent stem cells in terms of morphology, specific marker expression and ability to differentiate in vitro into the three germ layers. These lines thus represent a human cellular model to study the molecular basis of caveolinopathies. Resource table.
Asunto(s)
Caveolina 3/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Caveolina 3/genética , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Reprogramación Celular/genética , Reprogramación Celular/fisiología , Citometría de Flujo , Humanos , Cariotipo , Mutación/genética , Mutación Missense/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Duchenne muscular dystrophy (DMD) is caused by abnormalities in the dystrophin gene and is clinically characterised by childhood muscle degeneration and cardiomyopathy. We produced an induced pluripotent stem cell line from a DMD patient's dermal fibroblasts by electroporation with episomal vectors containing: hL-MYC, hLIN28, hSOX2, hKLF4, hOCT3/4. The resultant DMD iPSC line (CCMi001DMD-A-3) displayed iPSC morphology, expressed pluripotency markers, possessed trilineage differentiation potential and was karyotypically normal. MLPA analyses performed on DNA extracted from CCMi001DMD-A-3 showed a deletion of exons 49 and 50 (CCMi001DMD-A-3, ∆49, ∆50).
Asunto(s)
Exones/genética , Células Madre Pluripotentes Inducidas/citología , Distrofia Muscular de Duchenne/enzimología , Adulto , Células Cultivadas , Reprogramación Celular/genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , MasculinoRESUMEN
Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation. In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.
Asunto(s)
Células Madre Pluripotentes Inducidas/fisiología , Plásmidos/genética , Reprogramación Celular/genética , Centrifugación/métodos , Criopreservación/métodos , Humanos , Células Madre Pluripotentes Inducidas/citología , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/fisiologíaRESUMEN
BACKGROUND: An increasing number of foetuses are recognized as having double Y because of the widespread use of prenatal screening using chorionic villus sampling and amniocentesis. 47, XYY karyotype occurs in about one out of 1,000 newborn males, but it is not often detected unless it is diagnosed during prenatal testing. Despite the fact that unbiased follow-up studies demonstrate largely normal post-natal development of young men with 47, XYY, there is a scarcity of controlled studies about the neurological, cognitive and behavioural phenotype which remains the main reason for anxiety and anticipatory negative attitudes of parents. Furthermore, prejudices still exist among professionals and the general population concerning the relationship between this sex chromosome aneuploidy and aggressive and antisocial behaviours. METHODS: We report on the clinical follow-up of children diagnosed prenatally with a 47,XYY karyotype, whose parents received multidisciplinary counselling and support at time of diagnosis. The specific focus of our study is on auxology, facial features, developmental milestones, behaviour, detection of aggressiveness as well as the evaluation of parental attitudes toward prenatal counselling. Clinical evaluations including auxological measurements and dysmorphological descriptions were as conducted on 13 boys aged 9 month -7 years. The Child Behavior Check List test specific for age and a 15 item questionnaire were administered to both parents. An update of ongoing problems was carried out by means of a telephone interview two years later. RESULTS: Our results show that, from birth, weight, height and head circumference were above average values while some facial features such mild hypertelorism are overrepresented when compared to parents' facial features. Language delay was detected in 8 out of 11 children older than 20 months. Parental attitudes were found to be favourable toward prenatal diagnoses of sexual chromosome aneuploidies. CONCLUSIONS: Our data, although limited, is similar to other observational studies, and serves to alert clinicians about opportunities to delineate new and appropriate educational interventions that target the specific learning challenges of XYY boys. Our experience better defines the early manifestation of XYY and should aid those involved in prenatal counselling and paediatric surveillance.
Asunto(s)
Asesoramiento Genético , Padres/psicología , Trastornos de los Cromosomas Sexuales/diagnóstico , Cariotipo XYY/diagnóstico , Agresión , Femenino , Humanos , Recién Nacido , Entrevistas como Asunto , Italia , Masculino , Fenotipo , Vigilancia de la Población , Embarazo , Diagnóstico Prenatal , Trastornos de los Cromosomas Sexuales/genética , Encuestas y Cuestionarios , Cariotipo XYY/genéticaRESUMEN
AIMS: Bone marrow mesenchymal stromal cell (BMStC) transplantation into the infarcted heart improves left ventricular function and cardiac remodelling. However, it has been suggested that tissue-specific cells may be better for cardiac repair than cells from other sources. The objective of the present work has been the comparison of in vitro and in vivo properties of adult human cardiac stromal cells (CStC) to those of syngeneic BMStC. METHODS AND RESULTS: Although CStC and BMStC exhibited a similar immunophenotype, their gene, microRNA, and protein expression profiles were remarkably different. Biologically, CStC, compared with BMStC, were less competent in acquiring the adipogenic and osteogenic phenotype but more efficiently expressed cardiovascular markers. When injected into the heart, in rat a model of chronic myocardial infarction, CStC persisted longer within the tissue, migrated into the scar, and differentiated into adult cardiomyocytes better than BMStC. CONCLUSION: Our findings demonstrate that although CStC and BMStC share a common stromal phenotype, CStC present cardiovascular-associated features and may represent an important cell source for more efficient cardiac repair.