RESUMEN
CRB1 gene mutations can cause early- or late-onset retinitis pigmentosa, Leber congenital amaurosis, or maculopathy. Recapitulating human CRB1 phenotypes in animal models has proven challenging, necessitating the development of alternatives. We generated human induced pluripotent stem cell (iPSC)-derived retinal organoids of patients with retinitis pigmentosa caused by biallelic CRB1 mutations and evaluated them against autologous gene-corrected hiPSCs and hiPSCs from healthy individuals. Patient organoids show decreased levels of CRB1 and NOTCH1 expression at the retinal outer limiting membrane. Proximity ligation assays show that human CRB1 and NOTCH1 can interact via their extracellular domains. CRB1 patient organoids feature increased levels of WDFY1+ vesicles, fewer RAB11A+ recycling endosomes, decreased VPS35 retromer complex components, and more degradative endolysosomal compartments relative to isogenic control organoids. Taken together, our data demonstrate that patient-derived retinal organoids enable modeling of retinal degeneration and highlight the importance of CRB1 in early endosome maturation receptor recycling in the retina.
Asunto(s)
Células Madre Pluripotentes Inducidas , Degeneración Retiniana , Retinitis Pigmentosa , Animales , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Retina/metabolismo , Degeneración Retiniana/genética , Retinitis Pigmentosa/genética , Mutación , Organoides/metabolismo , Proteínas del Ojo/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismoRESUMEN
Subunit switches in the BAF chromatin remodeler are essential during development. ARID1B and its paralog ARID1A encode for mutually exclusive BAF subunits. De novo ARID1B haploinsufficient mutations cause neurodevelopmental disorders, including Coffin-Siris syndrome, which is characterized by neurological and craniofacial features. Here, we leveraged ARID1B+/- Coffin-Siris patient-derived iPSCs and modeled cranial neural crest cell (CNCC) formation. We discovered that ARID1B is active only during the first stage of this process, coinciding with neuroectoderm specification, where it is part of a lineage-specific BAF configuration (ARID1B-BAF). ARID1B-BAF regulates exit from pluripotency and lineage commitment by attenuating thousands of enhancers and genes of the NANOG and SOX2 networks. In iPSCs, these enhancers are maintained active by ARID1A-containing BAF. At the onset of differentiation, cells transition from ARID1A- to ARID1B-BAF, eliciting attenuation of the NANOG/SOX2 networks and triggering pluripotency exit. Coffin-Siris patient cells fail to perform the ARID1A/ARID1B switch, and maintain ARID1A-BAF at the pluripotency enhancers throughout all stages of CNCC formation. This leads to persistent NANOG/SOX2 activity which impairs CNCC formation. Despite showing the typical neural crest signature (TFAP2A/SOX9-positive), ARID1B-haploinsufficient CNCCs are also aberrantly NANOG-positive. These findings suggest a connection between ARID1B mutations, neuroectoderm specification and a pathogenic mechanism for Coffin-Siris syndrome.
Asunto(s)
Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteína Homeótica Nanog/metabolismo , Cresta Neural/metabolismo , Factores de Transcripción/metabolismo , Western Blotting , Proteínas de Unión al ADN/genética , Citometría de Flujo , Células HEK293 , Humanos , Mutación/genética , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción/genéticaRESUMEN
Combined Oxidative Phosphorylation Deficiency 8 (COXPD8) is an autosomal recessive disorder causing lethal childhood-onset hypertrophic cardiomyopathy. Homozygous or compound heterozygous mutations in the nuclear-encoded mitochondrial alanyl-tRNA synthetase 2 (AARS2) gene underly the pathology. We generated induced pluripotent stem cells (hiPSCs) from two patients carrying the heterozygous compound c.1774 C>T, c.2188 G>A and c.2872 C>T AARS2 mutations, as well as a related healthy control carrying the c.2872 C>T AARS2 mutation. All hiPSC-lines expressed pluripotency markers, maintained a normal karyotype, and differentiated towards the three germ layer derivatives in vitro. These lines can be used to model COXPD8 or mitochondrial dysfunction.
Asunto(s)
Cardiomiopatía Hipertrófica , Células Madre Pluripotentes Inducidas , Enfermedades Mitocondriales , Niño , Heterocigoto , Homocigoto , Humanos , MutaciónRESUMEN
Research on acute and chronic lung diseases would greatly benefit from reproducible availability of alveolar epithelial cells (AEC). Primary alveolar epithelial cells can be derived from human lung tissue but the quality of these cells is highly donor dependent. Here, we demonstrated that culture of EpCAM+ cells derived from human induced pluripotent stem cells (hiPSC) at the physiological air-liquid interface (ALI) resulted in type 2 AEC-like cells (iAEC2) with alveolar characteristics. iAEC2 cells expressed native AEC2 markers (surfactant proteins and LPCAT-1) and contained lamellar bodies. ALI-iAEC2 were used to study alveolar repair over a period of 2 weeks following mechanical wounding of the cultures and the responses were compared with those obtained using primary AEC2 (pAEC2) isolated from resected lung tissue. Addition of the Wnt/ß-catenin activator CHIR99021 reduced wound closure in the iAEC2 cultures but not pAEC2 cultures. This was accompanied by decreased surfactant protein expression and accumulation of podoplanin-positive cells at the wound edge. These results demonstrated the feasibility of studying alveolar repair using hiPSC-AEC2 cultured at the ALI and indicated that this model can be used in the future to study modulation of alveolar repair by (pharmaceutical) compounds.
Asunto(s)
Células Epiteliales Alveolares/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Modelos Biológicos , Células Epiteliales Alveolares/citología , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Línea Celular , Células Cultivadas , Humanos , Técnicas In Vitro , Células Madre Pluripotentes Inducidas/citología , Alveolos Pulmonares/lesiones , Alveolos Pulmonares/fisiología , Alveolos Pulmonares/fisiopatología , Regeneración/fisiología , Cicatrización de Heridas/fisiologíaRESUMEN
RAG2 severe combined immune deficiency (RAG2-SCID) is a lethal disorder caused by the absence of functional T and B cells due to a differentiation block. Here, we generated induced pluripotent stem cells (iPSCs) from a RAG2-SCID patient to study the nature of the T cell developmental blockade. We observed a strongly reduced capacity to differentiate at every investigated stage of T cell development, from early CD7-CD5- to CD4+CD8+. The impaired differentiation was accompanied by an increase in CD7-CD56+CD33+ natural killer (NK) cell-like cells. T cell receptor D rearrangements were completely absent in RAG2SCID cells, whereas the rare T cell receptor B rearrangements were likely the result of illegitimate rearrangements. Repair of RAG2 restored the capacity to induce T cell receptor rearrangements, normalized T cell development, and corrected the NK cell-like phenotype. In conclusion, we succeeded in generating an iPSC-based RAG2-SCID model, which enabled the identification of previously unrecognized disorder-related T cell developmental roadblocks.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Modelos Biológicos , Inmunodeficiencia Combinada Grave/inmunología , Inmunodeficiencia Combinada Grave/patología , Linfocitos T/inmunología , Animales , Antígenos CD/metabolismo , Diferenciación Celular , Linaje de la Célula , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Hematopoyesis , Humanos , Células Asesinas Naturales/inmunología , Ratones SCIDRESUMEN
Natural killer (NK) cells are innate immune cells, characterized by their cytotoxic capacity, and chemokine and cytokine secretion upon activation. Human NK cells are identified by CD56 expression. Circulating NK cells can be further subdivided into the CD56bright (~10%) and CD56dim NK cell subsets (~90%). NK cell-like cells can also be derived from human induced pluripotent stem cells (iPSC). To study the chemokine and cytokine secretion profile of the distinct heterogenous NK cell subsets, intracellular flow cytometry staining can be performed. However, this assay is challenging when the starting material is limited. Alternatively, NK cell subsets can be enriched, sorted, stimulated, and functionally profiled by measuring secreted effector molecules in the supernatant by Luminex. Here, we provide a rapid and straightforward protocol for the isolation and stimulation of primary NK cells or iPSC-derived NK cell-like cells, and subsequent detection of secreted cytokines and chemokines, which is also applicable for a low number of cells.
Asunto(s)
Células Madre Pluripotentes Inducidas/fisiología , Timo/fisiología , Animales , Diferenciación Celular , Células Cultivadas , Factores de Transcripción Forkhead/genética , Humanos , Ratones , Ratones Noqueados , Ratones Desnudos , Morfogénesis , Técnicas de Cultivo de Órganos , Trasplante de Órganos , Timo/trasplante , Trasplante HeterólogoRESUMEN
Human retinal organoids from induced pluripotent stem cells (hiPSCs) can be used to confirm the localization of proteins in retinal cell types and to test transduction and expression patterns of gene therapy vectors. Here, we compared the onset of CRB protein expression in human fetal retina with human iPSC-derived retinal organoids. We show that CRB2 protein precedes the expression of CRB1 in the developing human retina. Our data suggest the presence of CRB1 and CRB2 in human photoreceptors and Müller glial cells. Thus the fetal CRB complex formation is replicated in hiPSC-derived retina. CRB1 patient iPSC retinal organoids showed disruptions at the outer limiting membrane as found in Crb1 mutant mice. Furthermore, AAV serotype 5 (AAV5) is potent in infecting human Müller glial cells and photoreceptors in hiPSC-derived retinas and retinal explants. Our data suggest that human photoreceptors can be efficiently transduced by AAVs in the presence of photoreceptor segments.
Asunto(s)
Proteínas Portadoras/metabolismo , Células Ependimogliales/metabolismo , Proteínas del Ojo/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Organoides/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Adulto , Proteínas Portadoras/genética , Células Cultivadas , Dependovirus/genética , Células Ependimogliales/citología , Células Ependimogliales/ultraestructura , Proteínas del Ojo/genética , Femenino , Feto , Humanos , Inmunohistoquímica , Células Madre Pluripotentes Inducidas/citología , Proteínas de la Membrana/genética , Microscopía Inmunoelectrónica , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteínas del Tejido Nervioso/genética , Organoides/citología , Células Fotorreceptoras de Vertebrados/ultraestructura , Embarazo , Retina/citología , Retina/embriologíaRESUMEN
The transcription factor Sox2 controls the fate of pluripotent stem cells and neural stem cells. This gatekeeper function requires well-regulated Sox2 levels. We postulated that Sox2 regulation is partially controlled by the Sox2 overlapping long non-coding RNA (lncRNA) gene Sox2ot. Here we show that the RNA levels of Sox2ot and Sox2 are inversely correlated during neural differentiation of mouse embryonic stem cells (ESCs). Through allele-specific enhanced transcription of Sox2ot in mouse Sox2eGFP knockin ESCs we demonstrate that increased Sox2ot transcriptional activity reduces Sox2 RNA levels in an allele-specific manner. Enhanced Sox2ot transcription, yielding lower Sox2 RNA levels, correlates with a decreased chromatin interaction of the upstream regulatory sequence of Sox2 and the ESC-specific Sox2 super enhancer. Our study indicates that, in addition to previously reported in trans mechanisms, Sox2ot can regulate Sox2 by an allele-specific mechanism, in particular during development.
Asunto(s)
Células Madre Embrionarias de Ratones/citología , Neurogénesis , ARN Largo no Codificante/genética , Factores de Transcripción SOXB1/genética , Alelos , Animales , Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica , Técnicas de Sustitución del Gen , Ratones , Transcripción GenéticaRESUMEN
In vitro retinal organoid modeling from human pluripotent stem cells is becoming more common place in many ophthalmic laboratories worldwide. These organoids mimic human retinogenesis through formation of organized layered retinal structures that display markers for typical retinal cell types. Pivotally these humanized retinal models provide a stepping stone to the clinic as therapeutic tools and are expected to provide a promising alternative to current animal models. Thus pluripotent stem cell based healthy as well as diseased human retinal organoids are attractive for use in drug potency assays and gene augmentation therapeutics. Here we outline an established protocol for generation of these retinal organoids and how they can be used in conjunction with adeno-associated virus vectors for transgene expression assays.
Asunto(s)
Dependovirus/genética , Vectores Genéticos , Células Madre Pluripotentes Inducidas/metabolismo , Organoides/metabolismo , Retina/metabolismo , Transgenes/fisiología , Humanos , Células Madre Pluripotentes Inducidas/citología , Organoides/crecimiento & desarrollo , Retina/crecimiento & desarrolloRESUMEN
Systemic sclerosis is an autoimmune disease characterized by fibrosis of skin and multiple organs of which the pathogenesis is poorly understood. We studied differentially expressed coding and non-coding genes in relation to systemic sclerosis pathogenesis with a specific focus on antisense non-coding RNAs. Skin biopsy-derived RNAs from 14 early systemic sclerosis patients and six healthy individuals were sequenced with ion-torrent and analyzed using DEseq2. Overall, 4,901 genes with a fold change >1.5 and a false discovery rate <5% were detected in patients versus controls. Upregulated genes clustered in immunologic, cell adhesion, and keratin-related processes. Interestingly, 676 deregulated non-coding genes were detected, 257 of which were classified as antisense genes. Sense genes expressed opposite of these antisense genes were also deregulated in 42% of the observed sense-antisense gene pairs. The majority of the antisense genes had a similar effect sizes in an independent North American dataset with three genes (CTBP1-AS2, OTUD6B-AS1, and AGAP2-AS1) exceeding the study-wide Bonferroni-corrected P-value (PBonf < 0.0023, Pcombined = 1.1 × 10-9, 1.4 × 10-8, 1.7 × 10-6, respectively). In this study, we highlight that together with coding genes, (antisense) long non-coding RNAs are deregulated in skin tissue of systemic sclerosis patients suggesting a novel class of genes involved in pathogenesis of systemic sclerosis.
Asunto(s)
ARN Largo no Codificante/genética , Esclerodermia Sistémica/genética , Piel/metabolismo , Regulación hacia Arriba , Células Cultivadas , Humanos , ARN Largo no Codificante/biosíntesis , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patología , Piel/patología , Factores de Transcripción , Activación TranscripcionalRESUMEN
Precise genome editing involves homologous recombination between donor DNA and chromosomal sequences subjected to double-stranded DNA breaks made by programmable nucleases. Ideally, genome editing should be efficient, specific, and accurate. However, besides constituting potential translocation-initiating lesions, double-stranded DNA breaks (targeted or otherwise) are mostly repaired through unpredictable and mutagenic non-homologous recombination processes. Here, we report that the coordinated formation of paired single-stranded DNA breaks, or nicks, at donor plasmids and chromosomal target sites by RNA-guided nucleases based on CRISPR-Cas9 components, triggers seamless homology-directed gene targeting of large genetic payloads in human cells, including pluripotent stem cells. Importantly, in addition to significantly reducing the mutagenicity of the genome modification procedure, this in trans paired nicking strategy achieves multiplexed, single-step, gene targeting, and yields higher frequencies of accurately edited cells when compared to the standard double-stranded DNA break-dependent approach.CRISPR-Cas9-based gene editing involves double-strand breaks at target sequences, which are often repaired by mutagenic non-homologous end-joining. Here the authors use Cas9 nickases to generate coordinated single-strand breaks in donor and target DNA for precise homology-directed gene editing.
Asunto(s)
ADN/genética , Edición Génica , Sistemas CRISPR-Cas , Línea Celular , ADN/metabolismo , Roturas del ADN de Doble Cadena , Roturas del ADN de Cadena Simple , Reparación del ADN por Unión de Extremidades , Genoma Humano , Humanos , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismoRESUMEN
The canonical Wnt signalling pathway has been implicated in organogenesis and self-renewal of essentially all stem cell systems. In vivo reporter systems are crucial to assess the role of Wnt signalling in the biology and pathology of stem cell systems. We set out to develop a Turquoise (TQ) fluorescent protein based Wnt reporter. We used a CRISPR-Cas9 approach to insert a TQ fluorescent protein encoding gene into the general Wnt target gene Axin2, thereby establishing a Wnt reporter mouse similar to previously generated Wnt reporter mice but with the mTurquoise2 gene instead of E. coli-ß-galactosidase (LacZ). The use of mTurquoise2 is especially important in organ systems in which cells need to a be alive for further experimentation such as in vitro activation or transplantation studies. We here report successful generation of Axin2-TQ mice and show that cells from these mice faithfully respond to Wnt signals. High Wnt signals were detected in the intestinal crypts, a classical Wnt signalling site in vivo, and by flow cytometry in the thymus. These mice are an improved tool to further elucidate the role of Wnt signalling in vivo.
Asunto(s)
Proteína Axina/metabolismo , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Vía de Señalización Wnt , Animales , Proteína Axina/genética , Sistemas CRISPR-Cas , Marcación de Gen/métodos , Proteínas Fluorescentes Verdes/metabolismo , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Timo/citología , Timo/metabolismoRESUMEN
Hematopoietic stem cell transplantations have become a very successful therapeutic approach to treat otherwise life-threatening blood disorders. It is thought that stem cell transplantation may also become a feasible treatment option for many non-blood-related diseases. So far, however, the limited availability of human leukocyte antigen-matched donors has hindered development of some cell replacement therapies. The Nobel-prize rewarded finding that pluripotency can be induced in somatic cells via expression of a few transcription factors has led to a revolution in stem cell biology. The possibility to change the fate of somatic cells by expressing key transcription factors has been used not only to generate pluripotent stem cells, but also for directly converting somatic cells into fully differentiated cells of another lineage or more committed progenitor cells. These approaches offer the prospect of generating cell types with a specific genotype de novo, which would circumvent the problems associated with allogeneic cell transplantations. This technology has generated a plethora of new disease-specific research efforts, from studying disease pathogenesis to therapeutic interventions. Here we will discuss the opportunities in this booming field of cell biology and summarize how the scientists in the Netherlands have joined efforts in one area to exploit the new technology.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos , Reprogramación Celular , Animales , Diferenciación Celular , Tratamiento Basado en Trasplante de Células y Tejidos/efectos adversos , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Tratamiento Basado en Trasplante de Células y Tejidos/tendencias , Humanos , Países Bajos , Investigación con Células Madre , Trasplante de Células Madre/efectos adversos , Trasplante de Células Madre/métodos , Trasplante de Células Madre/tendencias , Células Madre/citología , Células Madre/metabolismoRESUMEN
Many life-threatening hematological diseases are now treated by bone marrow transplantations, i.e., infusion of hematopoietic stem cells (HSCs). HSC transplantations are a valid option for the treatment of a variety of metabolic disorders, and even for solid tumors and some refractory severe autoimmune diseases. Unfortunately, the frequency and outcome of HSC transplantations are limited by a shortage of suitable donors. Induced pluripotent stem cells (iPSCs)--somatic cells that have acquired pluripotent stem cell characteristics by the ectopic expression of pluripotency-inducing factors--have been proposed as an alternative source of HSCs. Possible applications include cells of autologous, of autologous and genetically modified, or of allogeneic origin. Here, we provide a perspective on the distinct opportunities of iPSCs and discuss the challenges that lie ahead.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/trasplante , Diferenciación Celular , Enfermedades Hematológicas/terapia , Neoplasias Hematológicas/terapia , Hematopoyesis , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Humanos , Seguridad , Trasplante Autólogo , Trasplante HomólogoRESUMEN
UNLABELLED: Stem cells are being used more frequently for research and experimental therapy, but as yet the clinical applications of stem cells are limited. Pluripotent stem cells, with embryonic stem cells as the most well know example, can differentiate into each cell type; in contrast, tissue specific stem cells can only form one or more cell types within one type of tissue. It has been possible for some time to reprogram different types of somatic cells into pluripotent stem cells. Such stem cells are termed induced pluripotent stem cells (iPS cells). iPS cells can also be created from cells of patients with genetic conditions. Research into mechanisms of pathology and new medicines can be carried out with these against a specific genetic background. Clinical application of such iPS cells is not to be expected in the short term. Facilities are being established in different Dutch academic centres to create iPS cells for scientific research. CONFLICT OF INTEREST: none declared.