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1.
Stem Cell Res ; 44: 101744, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32220772

RESUMEN

Cystic Fibrosis (CF) is a genetic disease caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene which encodes for a chloride ion channel regulating the balance of salt and water across secretory epithelia. Here we generated an iPSC line from a CF patient homozygous for the p.Asn1303Lys mutation, a Class II folding defect mutation. This iPSC line provides a useful resource for disease modeling and to investigate the pharmacological response to CFTR modulators in iPSC derived epithelia.


Asunto(s)
Fibrosis Quística , Células Madre Pluripotentes Inducidas , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Homocigoto , Humanos , Mutación
2.
Stem Cell Reports ; 12(6): 1389-1403, 2019 06 11.
Artículo en Inglés | MEDLINE | ID: mdl-31080112

RESUMEN

Organotypic culture systems from disease-specific induced pluripotent stem cells (iPSCs) exhibit obvious advantages compared with immortalized cell lines and primary cell cultures, but implementation of iPSC-based high-throughput (HT) assays is still technically challenging. Here, we demonstrate the development and conduction of an organotypic HT Cl-/I- exchange assay using cystic fibrosis (CF) disease-specific iPSCs. The introduction of a halide-sensitive YFP variant enabled automated quantitative measurement of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) function in iPSC-derived intestinal epithelia. CFTR function was partially rescued by treatment with VX-770 and VX-809, and seamless gene correction of the p.Phe508del mutation resulted in full restoration of CFTR function. The identification of a series of validated primary hits that improve the function of p.Phe508del CFTR from a library of ∼42,500 chemical compounds demonstrates that the advantages of complex iPSC-derived culture systems for disease modeling can also be utilized for drug screening in a true HT format.


Asunto(s)
Aminofenoles/farmacología , Aminopiridinas/farmacología , Benzodioxoles/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Células Epiteliales , Ingeniería Genética , Células Madre Pluripotentes Inducidas , Quinolonas/farmacología , Secuencia de Aminoácidos , Línea Celular , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Evaluación Preclínica de Medicamentos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Eliminación de Secuencia
3.
BMC Res Notes ; 11(1): 54, 2018 Jan 22.
Artículo en Inglés | MEDLINE | ID: mdl-29357945

RESUMEN

OBJECTIVE: Pannexins are channel proteins important for the release of calcium and adenosine triphosphate, which are among other functions involved in early development. Here, the expression of pannexins was investigated in induced pluripotent stem cells derived from human cord blood endothelial cells (hCBiPS2), in hematopoietic stem cell-derived induced pluripotent stem cells (HSC_F1285_T-iPS2) and in human embryonic stem cells (HES-3). The expression of pannexin (Panx) 1-3 mRNAs was analyzed in all three undifferentiated stem cell lines. Stem cells then underwent undirected differentiation into embryoid bodies and were analyzed regarding expression of germ layer-specific genes. RESULTS: Panx1, Panx2, and Panx3 mRNAs were expressed in all undifferentiated stem cell lines investigated. In comparison, Panx1 showed the highest expression among all pannexins. The undirected differentiation resulted in a mixed germ layer genotype in all three stem cell lines. Whereas the expression of Panx1 was not affected by differentiation, the expression of Panx2 was slightly increased in differentiated hCBiPS2 cells, HSC_F1285_T-iPS2 as well as HES3 cells as compared to their undifferentiated counterparts. A slight increase of Panx3 expression was observed in differentiated hCBiPS2 cells only. In conclusion, pluripotent stem cells express all three pannexin genes.


Asunto(s)
Conexinas/genética , Expresión Génica , Proteínas del Tejido Nervioso/genética , Células Madre/metabolismo , Línea Celular , Células Progenitoras Endoteliales/citología , Células Progenitoras Endoteliales/metabolismo , Sangre Fetal/citología , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/citología
4.
Stem Cell Reports ; 10(1): 7-16, 2018 01 09.
Artículo en Inglés | MEDLINE | ID: mdl-29249666

RESUMEN

Mendelian susceptibility to mycobacterial disease (MSMD) is caused by inborn errors of interferon gamma (IFNγ) immunity and is characterized by severe infections by weakly virulent mycobacteria. Although IFNγ is the macrophage-activating factor, macrophages from these patients have never been studied. We demonstrate the generation of heterozygous and compound heterozygous (iMSMD-cohet) induced pluripotent stem cells (iPSCs) from a single chimeric patient, who suffered from complete autosomal recessive IFNγR1 deficiency and received bone-marrow transplantation. Loss of IFNγR1 expression had no influence on the macrophage differentiation potential of patient-specific iPSCs. In contrast, lack of IFNγR1 in iMSMD-cohet macrophages abolished IFNγ-dependent phosphorylation of STAT1 and induction of IFNγ-downstream targets such as IRF-1, SOCS-3, and IDO. As a consequence, iMSMD-cohet macrophages show impaired upregulation of HLA-DR and reduced intracellular killing of Bacillus Calmette-Guérin. We provide a disease-modeling platform that might be suited to investigate novel treatment options for MSMD and to gain insights into IFNγ signaling in macrophages.


Asunto(s)
Células Madre Pluripotentes Inducidas/inmunología , Interferón gamma/inmunología , Macrófagos/inmunología , Mycobacterium bovis/inmunología , Receptores de Interferón/deficiencia , Transducción de Señal/inmunología , Predisposición Genética a la Enfermedad , Humanos , Células Madre Pluripotentes Inducidas/microbiología , Células Madre Pluripotentes Inducidas/patología , Interferón gamma/genética , Macrófagos/microbiología , Macrófagos/patología , Receptores de Interferón/inmunología , Transducción de Señal/genética , Receptor de Interferón gamma
5.
Histochem Cell Biol ; 146(5): 529-537, 2016 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-27456332

RESUMEN

Gap junction proteins are essential for direct intercellular communication but also influence cellular differentiation and migration. The expression of various connexin gap junction proteins has been demonstrated in embryonic stem cells, with Cx43 being the most intensely studied. As Cx43 is the most prominent gap junction protein in the heart, cardiomyocyte-differentiated stem cells have been studied intensely. To date, however, little is known about the expression and the subcellular distribution of Cx43 in undifferentiated stem cells or about the structural arrangement of channels. We, therefore, here investigate expression of Cx43 in undifferentiated human cord-blood-derived induced pluripotent stem cells (hCBiPS2). For this purpose, we carried out quantitative real-time PCR and immunohistochemistry. For analysis of Cx43 ultrastructure and protein assembly, we performed freeze-fracture replica immunogold labeling (FRIL). Cx43 expression was detected at mRNA and protein level in hCBIPS2 cells. For the first time, ultrastructural data are presented on gap junction morphology in induced pluripotent stem (iPS) cells from cord blood: Our FRIL and electron microscopical analysis revealed the occurrence of gap junction plaques in undifferentiated iPS cells. In addition, these gap junctions were shown to contain the gap junction protein Cx43.


Asunto(s)
Conexina 43/ultraestructura , Sangre Fetal/citología , Sangre Fetal/metabolismo , Uniones Comunicantes/ultraestructura , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/ultraestructura , Células Cultivadas , Conexina 43/metabolismo , Uniones Comunicantes/metabolismo , Humanos
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