RESUMEN
Induced pluripotent stem cells (iPSCs) were generated from blood outgrowth endothelial cells (BOECs) obtained from a healthy donor and from a patient diagnosed with Hermansky Pudlak Syndrome type 2 (HPS2), caused by compound heterozygous AP3B1 mutations (c.177delA and c.1839-1842delTAGA). BOECs were reprogrammed with a hOKSM self-silencing polycistronic lentiviral vector, where the generated iPSCs showed normal karyotype, expression of pluripotency associated markers and in vitro spontaneous differentiation towards the three germ layers. The generated iPSCs can be used to study HPS2 pathophysiology and the basic functions of AP3B1 protein in different cell types.
Asunto(s)
Síndrome de Hermanski-Pudlak , Células Madre Pluripotentes Inducidas , Complejo 3 de Proteína Adaptadora/genética , Subunidades beta de Complejo de Proteína Adaptadora/genética , Diferenciación Celular , Células Endoteliales , Heterocigoto , Humanos , MutaciónRESUMEN
Aberrant synaptic function is thought to underlie social deficits in neurodevelopmental disorders such as autism and schizophrenia. Although microRNAs have been shown to regulate synapse development and plasticity, their potential involvement in the control of social behaviour in mammals remains unexplored. Here, we show that deletion of the placental mammal-specific miR379-410 cluster in mice leads to hypersocial behaviour, which is accompanied by increased excitatory synaptic transmission, and exaggerated expression of ionotropic glutamate receptor complexes in the hippocampus. Bioinformatic analyses further allowed us to identify five "hub" microRNAs whose deletion accounts largely for the upregulation of excitatory synaptic genes observed, including Cnih2, Dlgap3, Prr7 and Src. Thus, the miR379-410 cluster acts a natural brake for sociability, and interfering with specific members of this cluster could represent a therapeutic strategy for the treatment of social deficits in neurodevelopmental disorders.
Asunto(s)
Conducta Animal , Euterios/genética , MicroARNs/genética , Familia de Multigenes , Conducta Social , Animales , Sitios de Unión , Euterios/metabolismo , Potenciales Postsinápticos Excitadores , Estudios de Asociación Genética , Marcadores Genéticos , Hipocampo/metabolismo , Ratones , Ratones Noqueados , Fenotipo , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas , Células Piramidales/metabolismo , Interferencia de ARN , Receptores de Glutamato/metabolismo , Transmisión SinápticaRESUMEN
Hematopoietic differentiation of human induced pluripotent stem cells (iPSCs) provide opportunities not only for fundamental research and disease modelling/drug testing but also for large-scale production of blood effector cells for future clinical application. Although there are multiple ways to differentiate human iPSCs towards hematopoietic lineages, there is a need to develop reproducible and robust protocols. Here we introduce an efficient way to produce three major blood cell types using a standardized differentiation protocol that starts with a single hematopoietic initiation step. This system is feeder-free, avoids EB-formation, starts with a hematopoietic initiation step based on a novel single cell-derived iPSC colony differentiation and produces multi-potential progenitors within 8-10â¯days. Followed by lineage-specific growth factor supplementation these cells can be matured into well characterized erythroid, megakaryocytic and myeloid cells with high-purity, without transcription factor overexpression or any kind of pre-purification step. This standardized differentiation system provides a simple platform to produce specific blood cells in a reproducible manner for hematopoietic development studies, disease modelling, drug testing and the potential for future therapeutic applications.
Asunto(s)
Técnicas de Cultivo de Célula , Diferenciación Celular , Células Eritroides , Células Madre Pluripotentes Inducidas , Megacariocitos , Células Mieloides , Células Eritroides/citología , Células Eritroides/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Megacariocitos/citología , Megacariocitos/metabolismo , Células Mieloides/citología , Células Mieloides/metabolismoRESUMEN
Peripheral blood mononuclear cells were isolated and cultured to a pure pro-EBL population and reprogrammed using episomal plasmids. The pluripotency of transgene-free induced pluripotent stem cell (iPSC) line was verified by the expression of pluripotency-associated markers and by in vitro spontaneous differentiation towards the 3 germ layers. The iPSC line showed normal karyotype. Peripheral blood is a non-invasive easy accessible cell source and combined with EBL outgrowth in vitro, a routine process obtaining sufficient amount of homogenous cells can be obtained within a week. Using episomal delivery, pro-EBLs can be reprogrammed in a transgene-free, cost effective system.
Asunto(s)
Eritroblastos/citología , Células Madre Pluripotentes Inducidas/citología , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Reprogramación Celular/genética , Reprogramación Celular/fisiología , Humanos , CariotipificaciónRESUMEN
The rare recessive developmental disorder Trichothiodystrophy (TTD) is characterized by brittle hair and nails. Patients also present a variable set of poorly explained additional clinical features, including ichthyosis, impaired intelligence, developmental delay and anemia. About half of TTD patients are photosensitive due to inherited defects in the DNA repair and transcription factor II H (TFIIH). The pathophysiological contributions of unrepaired DNA lesions and impaired transcription have not been dissected yet. Here, we functionally characterize the consequence of a homozygous missense mutation in the general transcription factor II E, subunit 2 (GTF2E2/TFIIEß) of two unrelated non-photosensitive TTD (NPS-TTD) families. We demonstrate that mutant TFIIEß strongly reduces the total amount of the entire TFIIE complex, with a remarkable temperature-sensitive transcription defect, which strikingly correlates with the phenotypic aggravation of key clinical symptoms after episodes of high fever. We performed induced pluripotent stem (iPS) cell reprogramming of patient fibroblasts followed by in vitro erythroid differentiation to translate the intriguing molecular defect to phenotypic expression in relevant tissue, to disclose the molecular basis for some specific TTD features. We observed a clear hematopoietic defect during late-stage differentiation associated with hemoglobin subunit imbalance. These new findings of a DNA repair-independent transcription defect and tissue-specific malfunctioning provide novel mechanistic insight into the etiology of TTD.
Asunto(s)
Factores de Transcripción TFII/genética , Síndromes de Tricotiodistrofia/genética , Diferenciación Celular/genética , Reprogramación Celular/genética , ADN Helicasas/genética , Reparación del ADN , Femenino , Humanos , Células Madre Pluripotentes Inducidas/patología , Masculino , Mutación , Mutación Missense , Especificidad de Órganos , Linaje , Factores de Transcripción TFII/metabolismo , Transcripción Genética , Síndromes de Tricotiodistrofia/metabolismo , Síndromes de Tricotiodistrofia/patologíaRESUMEN
Peripheral blood mononuclear cells were isolated from an individual harboring a heterozygous c.859CâT p.Q287* mutation in GFI1B, causing an autosomal dominant bleeding disorder, platelet type, 17 (BDPLT17). PBMCs were differentiated to erythroblasts and reprogrammed by lentiviral delivery of a self-silencing hOKSM polycistronic vector. Pluripotency of iPSC line was confirmed by expression of associated markers and by in vitro spontaneous differentiation towards the 3 germ layers. Normal karyotype confirmed the genomic integrity of iPSCs and the presence of disease causing mutation was shown by Sanger sequencing. The generated iPSCs can be used to study BDPLT17 pathophysiology and basic functions of GFI1B.
Asunto(s)
Plaquetas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Reprogramación Celular/genética , Reprogramación Celular/fisiología , Heterocigoto , Humanos , Cariotipo , MutaciónRESUMEN
Mobilized peripheral blood (MPB) CD34+ cells were differentiated to CD34+/CD41+ megakaryoblasts. Cells were sorted to obtain a pure megakaryoblast population which was reprogrammed with a hOKSM self-silencing polycistronic lentiviral vector. Resulting iPSC showed normal karyotype and expression of pluripotency associated markers and in vitro spontaneous differentiation towards the 3 germ layers confirmed pluripotency of iPSC lines. Besides normal iPSC applications, these lines can be used as a control line for other megakaryoid origin iPSC and could be applied for epigenetic based research.
Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Células Progenitoras de Megacariocitos/citología , Diferenciación Celular/fisiología , Células Cultivadas , Humanos , Inmunohistoquímica , CariotipificaciónRESUMEN
Mobilized peripheral blood (MPB) CD34+ cells were cultured to CD41+/CD34+ megakaryoblasts. Cells were sorted to obtain a pure megakaryoblast population that was reprogramed by a hOKSM self-silencing polycistronic vector using lentiviral delivery. The generated induced pluripotent stem cell (iPSC) lines were tested for silencing of the reprogramming construct by flow cytometry. Pluripotency of MML-6838-Cl2 iPSC line was confirmed by expression of associated markers and by in vivo spontaneous differentiation towards the 3 germ layers. The genomic integrity of iPSC line was shown by karyotyping. The MML-6838-Cl2 iPSC is, to our knowledge, the first to be generated from megakaryoblasts.
Asunto(s)
Reprogramación Celular , Células Madre Pluripotentes Inducidas/citología , Células Progenitoras de Megacariocitos/citología , Animales , Antígenos CD34/metabolismo , Línea Celular , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/trasplante , Cariotipo , Lentivirus/genética , Masculino , Células Progenitoras de Megacariocitos/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Glicoproteína IIb de Membrana Plaquetaria/metabolismo , Teratoma/metabolismo , Teratoma/patología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The E3 ubiquitin ligase Ube3a is an important regulator of activity-dependent synapse development and plasticity. Ube3a mutations cause Angelman syndrome and have been associated with autism spectrum disorders (ASD). However, the biological significance of alternative Ube3a transcripts generated in mammalian neurons remains unknown. We report here that Ube3a1 RNA, a transcript that encodes a truncated Ube3a protein lacking catalytic activity, prevents exuberant dendrite growth and promotes spine maturation in rat hippocampal neurons. Surprisingly, Ube3a1 RNA function was independent of its coding sequence but instead required a unique 3' untranslated region and an intact microRNA pathway. Ube3a1 RNA knockdown increased activity of the plasticity-regulating miR-134, suggesting that Ube3a1 RNA acts as a dendritic competing endogenous RNA. Accordingly, the dendrite-growth-promoting effect of Ube3a1 RNA knockdown in vivo is abolished in mice lacking miR-134. Taken together, our results define a noncoding function of an alternative Ube3a transcript in dendritic protein synthesis, with potential implications for Angelman syndrome and ASD.