RESUMEN
The generation of targeted mutants is a crucial step toward studying the biomedical effect of genes of interest. The generation of such mutants in human induced pluripotent stem cells (iPSCs) is of an utmost importance as these cells carry the potential to be differentiated into any cell lineage. Using the CRISPR/Cas9 nuclease system for induction of targeted double-strand breaks, gene editing of target loci in iPSCs can be achieved with high efficiency. This chapter covers protocols for the preparation of reagents to target loci of interest, the transfection, and for the genotyping of single cell-derived iPSC clones. Furthermore, we provide a protocol for the convenient generation of plasmids enabling multiplex gene targeting.
Asunto(s)
Sistemas CRISPR-Cas/genética , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Edición Génica/métodos , Genoma Humano/genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Plásmidos/genéticaRESUMEN
Fibro-adipogenic progenitors (FAPs) are resident mesenchymal progenitors in adult skeletal muscle that support muscle repair, but also give rise to fibrous and adipose infiltration in response to disease and chronic injury. FAPs are identified using cell surface markers that do not distinguish between quiescent FAPs and FAPs actively engaged in the regenerative process. We have shown previously that FAPs are derived from cells that express the transcription factor Osr1 during development. Here we show that adult FAPs express Osr1 at low levels and frequency, however upon acute injury FAPs reactivate Osr1 expression in the injured tissue. Osr1+ FAPs are enriched in proliferating and apoptotic cells demonstrating that Osr1 identifies activated FAPs. In vivo genetic lineage tracing shows that Osr1+ activated FAPs return to the resident FAP pool after regeneration as well as contribute to adipocytes after glycerol-induced fatty degeneration. In conclusion, reporter LacZ or eGFP-CreERt2 expression from the endogenous Osr1 locus serves as marker for FACS isolation and tamoxifen-induced manipulation of activated FAPs.
Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Músculo Esquelético/metabolismo , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/fisiología , Proteínas de Unión al Calcio , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Quistes , Citometría de Flujo , Edición Génica , Regulación de la Expresión Génica , Glucosidasas/genética , Glucosidasas/metabolismo , Factor Nuclear 4 del Hepatocito/genética , Factor Nuclear 4 del Hepatocito/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Hepatopatías , Músculo Esquelético/citología , Factores de TranscripciónRESUMEN
Fibro-adipogenic progenitors (FAPs) are an interstitial cell population in adult skeletal muscle that support muscle regeneration. During development, interstitial muscle connective tissue (MCT) cells support proper muscle patterning, however the underlying molecular mechanisms are not well understood and it remains unclear whether adult FAPs and embryonic MCT cells share a common lineage. We show here that mouse embryonic limb MCT cells expressing the transcription factor Osr1, differentiate into fibrogenic and adipogenic cells in vivo and in vitro defining an embryonic FAP-like population. Genetic lineage tracing shows that developmental Osr1+ cells give rise to a subset of adult FAPs. Loss of Osr1 function leads to a reduction of myogenic progenitor proliferation and survival resulting in limb muscle patterning defects. Transcriptome and functional analyses reveal that Osr1+ cells provide a critical pro-myogenic niche via the production of MCT specific extracellular matrix components and secreted signaling factors.
Asunto(s)
Embrión de Mamíferos/citología , Extremidades/embriología , Desarrollo de Músculos , Mioblastos/citología , Factores de Transcripción/metabolismo , Envejecimiento/metabolismo , Animales , Tipificación del Cuerpo , Tejido Conectivo/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Regulación de la Expresión Génica , Ratones , Mioblastos/metabolismo , Transducción de Señal , Factor de Transcripción 4/metabolismoRESUMEN
Human induced pluripotent stem cells (hiPSCs) represent an ideal in vitro platform to study human genetics and biology. The recent advent of programmable nucleases makes also the human genome amenable to experimental genetics through either the correction of mutations in patient-derived iPSC lines or the de novo introduction of mutations into otherwise healthy iPSCs. The production of specific and sometimes complex genotypes in multiple cell lines requires efficient and streamlined gene editing technologies. In this article we provide protocols for gene editing in hiPSCs. We presently achieve high rates of gene editing at up to three loci using a modified iCRISPR system. This system includes a doxycycline inducible Cas9 and sgRNA/reporter plasmids for the enrichment of transfected cells by fluorescence-activated cell sorting (FACS). Here we cover the selection of target sites, vector construction, transfection, and isolation and genotyping of modified hiPSC clones.
Asunto(s)
Proteínas Bacterianas/genética , Sistemas CRISPR-Cas , Endonucleasas/genética , Edición Génica/métodos , Técnicas de Transferencia de Gen , ARN Guía de Kinetoplastida/genética , Proteínas Bacterianas/metabolismo , Proteína 9 Asociada a CRISPR , Línea Celular , Células Clonales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN/genética , ADN/metabolismo , Doxiciclina/farmacología , Electroporación/métodos , Endonucleasas/metabolismo , Citometría de Flujo , Marcación de Gen/métodos , Genes Reporteros , Genoma Humano , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Lípidos/química , Plásmidos/química , Plásmidos/metabolismo , ARN Guía de Kinetoplastida/metabolismoRESUMEN
The morphology of bones is genetically determined, but the molecular mechanisms that control shape, size and the overall gestalt of bones remain unclear. We previously showed that metacarpals in the synpolydactyly homolog (spdh) mouse, which carries a mutation in Hoxd13 similar to the human condition synpolydactyly (SPD), were transformed to carpal-like bones with cuboid shape that lack cortical bone and a perichondrium and are surrounded by a joint surface. Here we provide evidence that spdh metacarpal growth plates have a defect in cell polarization with a random instead of linear orientation. In parallel prospective perichondral cells failed to adopt the characteristic flattened cell shape. We observed a similar cell polarity defect in metacarpals of Wnt5a(-/-) mice. Wnt5a and the closely related Wnt5b were downregulated in spdh handplates, and HOXD13 induced expression of both genes in vitro. Concomitant we observed mislocalization of core planar cell polarity (PCP) components DVL2 and PRICKLE1 in spdh metacarpals indicating a defect in the WNT/PCP pathway. Conversely the WNT/ß-CATENIN pathway, a hallmark of joint cells lining carpal bones, was upregulated in the perichondral region. Finally, providing spdh limb explant cultures with cells expressing either HOXD13 or WNT5A led to a non-cell autonomous partial rescue of cell polarity the perichondral region and restored the expression of perichondral markers. This study provides a so far unrecognized link between HOX proteins and cell polarity in the perichondrium and the growth plate, a failure of which leads to transformation of metacarpals to carpal-like structures.
Asunto(s)
Cartílago/embriología , Placa de Crecimiento/embriología , Proteínas de Homeodominio/metabolismo , Huesos del Metacarpo/embriología , Factores de Transcripción/metabolismo , Proteínas Wnt/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Cartílago/metabolismo , Polaridad Celular , Células Cultivadas , Proteínas Dishevelled , Placa de Crecimiento/metabolismo , Proteínas de Homeodominio/genética , Humanos , Proteínas con Dominio LIM/metabolismo , Huesos del Metacarpo/metabolismo , Ratones , Ratones Noqueados , Morfogénesis/genética , Fosfoproteínas/metabolismo , Receptores de Fenciclidina/metabolismo , Sindactilia/genética , Factores de Transcripción/genética , Proteínas Wnt/genética , Proteína Wnt-5a , beta Catenina/metabolismoRESUMEN
The differentiation of HSCs into myeloid lineages requires the transcription factor PU.1. Whereas PU.1-dependent induction of myeloid-specific target genes has been intensively studied, negative regulation of stem cell or alternate lineage programs remains incompletely characterized. To test for such negative regulatory events, we searched for PU.1-controlled microRNAs (miRs) by expression profiling using a PU.1-inducible myeloid progenitor cell line model. We provide evidence that PU.1 directly controls expression of at least 4 of these miRs (miR-146a, miR-342, miR-338, and miR-155) through temporally dynamic occupation of binding sites within regulatory chromatin regions adjacent to their genomic coding loci. Ectopic expression of the most robustly induced PU.1 target miR, miR-146a, directed the selective differentiation of HSCs into functional peritoneal macrophages in mouse transplantation assays. In agreement with this observation, disruption of Dicer expression or specific antagonization of miR-146a function inhibited the formation of macrophages during early zebrafish (Danio rerio) development. In the present study, we describe a PU.1-orchestrated miR program that mediates key functions of PU.1 during myeloid differentiation.