RESUMO
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.
Assuntos
Proteína Axina/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Via de Sinalização Wnt , Animais , Proteína Axina/genética , Sistemas CRISPR-Cas , Marcação de Genes/métodos , Proteínas de Fluorescência Verde/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Timo/citologia , Timo/metabolismoAssuntos
Células-Tronco Pluripotentes Induzidas/fisiologia , Timo/fisiologia , Animais , Diferenciação Celular , Células Cultivadas , Fatores de Transcrição Forkhead/genética , Humanos , Camundongos , Camundongos Knockout , Camundongos Nus , Morfogênese , Técnicas de Cultura de Órgãos , Transplante de Órgãos , Timo/transplante , Transplante HeterólogoRESUMO
The functions of non-coding regulatory elements (NCREs), which constitute a major fraction of the human genome, have not been systematically studied. Here we report a method involving libraries of paired single-guide RNAs targeting both ends of an NCRE as a screening system for the Cas9-mediated deletion of thousands of NCREs genome-wide to study their functions in distinct biological contexts. By using K562 and 293T cell lines and human embryonic stem cells, we show that NCREs can have redundant functions, and that many ultra-conserved elements have silencer activity and play essential roles in cell growth and in cellular responses to drugs (notably, the ultra-conserved element PAX6_Tarzan may be critical for heart development, as removing it from human embryonic stem cells led to defects in cardiomyocyte differentiation). The high-throughput screen, which is compatible with single-cell sequencing, may allow for the identification of druggable NCREs.
Assuntos
Sistemas CRISPR-Cas , RNA Guia de Sistemas CRISPR-Cas , Humanos , RNA Guia de Sistemas CRISPR-Cas/genética , Células K562 , Sistemas CRISPR-Cas/genética , Células HEK293 , Genoma Humano/genética , Diferenciação Celular/genética , Miócitos Cardíacos/metabolismo , RNA não Traduzido/genética , Células-Tronco Embrionárias Humanas/metabolismo , Fator de Transcrição PAX6/genética , Fator de Transcrição PAX6/metabolismo , Biblioteca GênicaRESUMO
Genetic screens carried out in lower organisms such as yeast, Drosophila melanogaster and Caenorhabditis elegans have revealed many signaling pathways. For example, components of the RAS signaling cascade were identified using a mutant eye phenotype in D. melanogaster as a readout. Screening is usually based on enhancing or suppressing a phenotype by way of a known mutation in a particular signaling pathway. Such in vivo screens have been difficult to carry out in mammals, however, owing to their relatively long generation times and the limited number of animals that can be screened. Here we describe an in vivo mammalian genetic screen used to identify components of pathways contributing to oncogenic transformation. We applied retroviral insertional mutagenesis in Myc transgenic (E mu Myc) mice lacking expression of Pim1 and Pim2 to search for genes that can substitute for Pim1 and Pim2 in lymphomagenesis. We determined the chromosomal positions of 477 retroviral insertion sites (RISs) derived from 38 tumors from E mu Myc Pim1(-/-) Pim2(-/-) mice and 27 tumors from E mu Myc control mice using the Ensembl and Celera annotated mouse genome databases. There were 52 sites occupied by proviruses in more than one tumor. These common insertion sites (CISs) are likely to contain genes contributing to tumorigenesis. Comparison of the RISs in tumors of Pim-null mice with the RISs in tumors of E mu Myc control mice indicated that 10 of the 52 CISs belong to the Pim complementation group. In addition, we found that Pim3 is selectively activated in Pim-null tumor cells, which supports the validity of our approach.
Assuntos
Transformação Celular Neoplásica , Linfoma/genética , Retroviridae/genética , Transdução de Sinais , Animais , Transformação Celular Neoplásica/genética , Genes myc , Linfoma/fisiopatologia , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-pim-1 , Provírus/genética , Integração ViralRESUMO
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.
Assuntos
Células-Tronco Pluripotentes Induzidas , Degeneração Retiniana , Retinose Pigmentar , Animais , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Retina/metabolismo , Degeneração Retiniana/genética , Retinose Pigmentar/genética , Mutação , Organoides/metabolismo , Proteínas do Olho/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismoRESUMO
For most, but not all, types of severe combined immunodeficiency (SCID) the underlying molecular defects are known, in principle allowing the cure of affected children via gene therapy. Typically such approaches have used autologous hematopoietic stem cells modified to express a therapeutic gene via γ-retroviral vectors. Insertional mutagenesis has emerged as a significant risk for successful application of this type of gene therapy. Therefore, lentiviral vectors with a self-inactivating design have been developed. Recent advances in stem cell technology using induced pluripotent stem cells (iPSCs) allow an entire different approach to gene therapy for SCID and other genetic disorders, namely by correction of the affected gene in patient-specific iPSCs followed by hematopoietic differentiation. Here, we review these recent advances in the field from an efficacy and safety point of view.
Assuntos
Terapia Genética/métodos , Células-Tronco Pluripotentes Induzidas/transplante , Modelos Biológicos , Pediatria/métodos , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/terapia , Transplante de Células-Tronco/métodos , Criança , Vetores Genéticos/genética , Humanos , Mutagênese Insercional , Pediatria/tendênciasRESUMO
The development of T lymphocytes in the thymus and their stem cell precursors in the bone marrow is controlled by Wnt signaling in strictly regulated, cell-type specific dosages. In this study, we investigated levels of canonical Wnt signaling during hematopoiesis and T cell development within the Axin2-mTurquoise2 reporter. We demonstrate active Wnt signaling in hematopoietic stem cells (HSCs) and early thymocytes, but also in more mature thymic subsets and peripheral T lymphocytes. Thymic epithelial cells displayed particularly high Wnt signaling, suggesting an interesting crosstalk between thymocytes and thymic epithelial cells (TECs). Additionally, reporter mice allowed us to investigate the loss of Axin2 function, demonstrating decreased HSC repopulation upon transplantation and the partial arrest of early thymocyte development in Axin2Tg/Tg full mutant mice. Mechanistically, loss of Axin2 leads to supraphysiological Wnt levels that disrupt HSC differentiation and thymocyte development.
Assuntos
Proteína Axina , Hematopoese , Linfopoese , Animais , Proteína Axina/genética , Proteína Axina/metabolismo , Diferenciação Celular , Hematopoese/genética , Células-Tronco Hematopoéticas , Linfopoese/genética , Camundongos , Via de Sinalização WntRESUMO
The technology to generate autologous pluripotent stem cells (iPS cells) from almost any somatic cell type has brought various cell replacement therapies within clinical research. Besides the challenge to optimize iPS protocols to appropriate safety and GMP levels, procedures need to be developed to differentiate iPS cells into specific fully differentiated and functional cell types for implantation purposes. In this article, we describe a protocol to differentiate mouse iPS cells into oligodendrocytes with the aim to investigate the feasibility of IPS stem cell-based therapy for demyelinating disorders, such as multiple sclerosis. Our protocol results in the generation of oligodendrocyte precursor cells (OPCs) that can develop into mature, myelinating oligodendrocytes in-vitro (co-culture with DRG neurons) as well as in-vivo (after implantation in the demyelinated corpus callosum of cuprizone-treated mice). We report the importance of complete purification of the iPS-derived OPC suspension to prevent the contamination with teratoma-forming iPS cells.
Assuntos
Transplante de Tecido Encefálico/métodos , Células-Tronco Pluripotentes Induzidas/fisiologia , Células-Tronco Pluripotentes Induzidas/transplante , Oligodendroglia/fisiologia , Transplante de Células-Tronco/métodos , Animais , Técnicas de Cultura de Células/métodos , Células Cultivadas , Técnicas de Cocultura , Fibroblastos/citologia , Fibroblastos/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Camundongos Endogâmicos C57BL , Oligodendroglia/citologia , Ratos , Ratos Wistar , Transfecção/métodosRESUMO
Targeted ectopic expression of genes in the adult brain is an invaluable approach for studying many biological processes. This can be accomplished by generating transgenic mice or by virally mediated gene transfer, but these methods are costly and labor intensive. We devised a rapid strategy that allows localized in vivo transfection of plasmid DNA within the adult neurogenic niches without detectable brain damage. Injection of plasmid DNA into the ventricular system or directly into the hippocampus of adult mice, followed by application of electrical current via external electrodes, resulted in transfection of neural stem or progenitor cells and mature neurons. We showed that this strategy can be used for both fate mapping and gain- or loss-of-function experiments. Using this approach, we identified an essential role for cadherins in maintaining the integrity of the lateral ventricle wall. Thus, in vivo electroporation provides a new approach to study the adult brain.
Assuntos
Ventrículos Cerebrais/fisiologia , DNA/administração & dosagem , DNA/genética , Eletroporação/métodos , Neurônios/fisiologia , Transfecção/métodos , Animais , CamundongosRESUMO
Two decades ago, the existence of long non-coding RNAs (lncRNAs) was discovered. In the following genomics era more transcribed non-coding genomic regions were identified. These were initially regarded as transcriptional noise and did not receive a lot of attention. Emerging data on several of these long non-coding transcripts have refuted this hypothesis by demonstrating that non-coding RNAs (ncRNAs) are important for regulating transcription and cell signaling. A special subset of the lncRNAs affecting gene transcription appears to orchestrate major developmental programs. Here, we discuss the mechanisms by which lncRNAs regulate transcription, and review the evidence that links this class of lncRNAs to a role in development.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , RNA não Traduzido/fisiologia , Animais , Inativação Gênica , Genoma , Genômica , Humanos , Modelos BiológicosRESUMO
Fibroblasts from two patients carrying a heterozygous mutation in the translation initiation codon (c.2 T > G) of the kelch-like protein 24 (KLHL24) gene were used to generate human induced pluripotent stem cells (hiPSCs), using non-integrating Sendai virus to deliver reprogramming factors. CRISPR-Cas9 editing was used for genetic correction of the mutation in the patient-hiPSCs. The top-predicted off-target sites were not altered. Patient and isogenic hiPSCs showed typical morphology, expressed pluripotency-associated markers, had the capacity for in vitro differentiation into the three germ layers and displayed a normal karyotype. These isogenic pairs will enable in vitro modelling of KLHL24-associated heart and skin conditions.
RESUMO
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.
Assuntos
Cardiomiopatia Hipertrófica , Células-Tronco Pluripotentes Induzidas , Doenças Mitocondriais , Criança , Heterozigoto , Homozigoto , Humanos , MutaçãoRESUMO
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.
Assuntos
Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína Homeobox Nanog/metabolismo , Crista Neural/metabolismo , Fatores de Transcrição/metabolismo , Western Blotting , Proteínas de Ligação a DNA/genética , Citometria de Fluxo , Células HEK293 , Humanos , Mutação/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/genéticaRESUMO
We describe a protocol for efficient generation of human-induced pluripotent stem cells (hiPSCs) from urine-derived cells (UDCs) obtained from adult donors using self-replicative RNA containing the reprogramming factors OCT3/4, SOX2, KLF4, GLIS1, and c-MYC (ReproRNA-OKSGM). After electroporation, transfection efficiency is quantified by measuring OCT3/4-expressing UDCs using flow cytometry and should be ≥0.1%. hiPSC colonies emerge within 3 weeks after transfection and express multiple pluripotency markers. Moreover, the UDC-derived hiPSCs are able to differentiate into cells of all three germ layers and display normal karyotypes. ReproRNA-OKSGM is available commercially and only requires a single transfection step so that the protocol is readily accessible, as well as straightforward. In addition to a detailed step-by-step description for generating clonal hiPSCs from UDCs using ReproRNA-OKSGM, we provide guidance for basic pluripotency characterization of the hiPSC lines. © 2020 The Authors. Basic Protocol: Reprogramming of urine-derived cells using ReproRNA-OKSGM Support Protocol 1: Determination of the pluripotency status of hiPSCs by flow cytometry Support Protocol 2: Characterization of functional pluripotency of hiPSCs.
Assuntos
Técnicas de Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Urina/citologia , Células Cultivadas , Eletroporação , Humanos , Fator 4 Semelhante a Kruppel , RNA/metabolismoRESUMO
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.
RESUMO
Fibroblasts from a patient carrying a heterozygous 18bp deletion in exon 8 of the ACVRL1 gene (c.1120del18) were reprogrammed using episomal vectors. The in-frame deletion in ACVRL1 causes the loss of 6 amino acids of the protein, which is associated with Hereditary Hemorrhagic Telangiectasia (HHT) type 2 (Letteboer et al., 2005). CRISPR-Cas9 editing was used to genetically correct the mutation in the induced pluripotent stem cells (iPSCs). The top5-predicted off-target sites were not altered. Patient and isogenic iPSCs showed high pluripotent marker expression, in vitro differentiation capacity into all three germ layers and displayed a normal karyotype. The obtained isogenic pairs will enable proper in vitro disease modelling of HHT (Roman and Hinck, 2017).
Assuntos
Células-Tronco Pluripotentes Induzidas , Telangiectasia Hemorrágica Hereditária , Receptores de Activinas Tipo II/genética , Células Clonais , Heterozigoto , Humanos , Mutação/genética , Telangiectasia Hemorrágica Hereditária/genéticaRESUMO
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.
Assuntos
Células Epiteliais Alveolares/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Modelos Biológicos , Células Epiteliais Alveolares/citologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Humanos , Técnicas In Vitro , Células-Tronco Pluripotentes Induzidas/citologia , Alvéolos Pulmonares/lesões , Alvéolos Pulmonares/fisiologia , Alvéolos Pulmonares/fisiopatologia , Regeneração/fisiologia , Cicatrização/fisiologiaRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Imunodeficiência Combinada Severa/imunologia , Imunodeficiência Combinada Severa/patologia , Linfócitos T/imunologia , Animais , Antígenos CD/metabolismo , Diferenciação Celular , Linhagem da Célula , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Hematopoese , Humanos , Células Matadoras Naturais/imunologia , Camundongos SCIDRESUMO
The recent discovery that two partly overlapping sets of four genes induce nuclear reprogramming of mouse and even human cells has opened up new possibilities for cell replacement therapies. Although the combination of genes that induce pluripotency differs to some extent, Oct4 and Sox2 appear to be a prerequisite. The introduction of four genes, several of which been linked with cancer, using retroviral approaches is however unlikely to be suitable for future clinical applications. Towards developing a safer reprogramming protocol, we investigated whether cell types that express one of the most critical reprogramming genes endogenously are predisposed to reprogramming. We show here that three of the original four pluripotency transcription factors (Oct4, Klf4 and c-Myc or MYCER(TAM)) induced reprogramming of mouse neural stem (NS) cells exploiting endogenous SoxB1 protein levels in these cells. The reprogrammed neural stem cells differentiated into cells of each germ layer in vitro and in vivo, and contributed to mouse development in vivo. Thus a combinatorial approach taking advantage of endogenously expressed genes and inducible transgenes may contribute to the development of improved reprogramming protocols.