RESUMEN
Thymic epithelial cells (TECs) play a critical role in T cell maturation and tolerance induction. The generation of TECs from in vitro differentiation of human pluripotent stem cells (PSCs) provides a platform on which to study the mechanisms of this interaction and has implications for immune reconstitution. To facilitate analysis of PSC-derived TECs, we generated hESC reporter lines in which sequences encoding GFP were targeted to FOXN1, a gene required for TEC development. Using this FOXN1 (GFP/w) line as a readout, we developed a reproducible protocol for generating FOXN1-GFP(+) thymic endoderm cells. Transcriptional profiling and flow cytometry identified integrin-ß4 (ITGB4, CD104) and HLA-DR as markers that could be used in combination with EpCAM to selectively purify FOXN1(+) TEC progenitors from differentiating cultures of unmanipulated PSCs. Human FOXN1(+) TEC progenitors generated from PSCs facilitate the study of thymus biology and are a valuable resource for future applications in regenerative medicine.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Moléculas de Adhesión Celular/metabolismo , Células Epiteliales/metabolismo , Factores de Transcripción Forkhead/metabolismo , Antígenos HLA-DR/metabolismo , Integrina beta4/metabolismo , Células Madre Pluripotentes/citología , Timo/citología , Diferenciación Celular , Células Cultivadas , Molécula de Adhesión Celular Epitelial , Células Epiteliales/citología , Humanos , Células Madre Pluripotentes/metabolismoRESUMEN
NKX2-5 is expressed in the heart throughout life. We targeted eGFP sequences to the NKX2-5 locus of human embryonic stem cells (hESCs); NKX2-5(eGFP/w) hESCs facilitate quantification of cardiac differentiation, purification of hESC-derived committed cardiac progenitor cells (hESC-CPCs) and cardiomyocytes (hESC-CMs) and the standardization of differentiation protocols. We used NKX2-5 eGFP(+) cells to identify VCAM1 and SIRPA as cell-surface markers expressed in cardiac lineages.
Asunto(s)
Separación Celular/métodos , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/metabolismo , Mioblastos Cardíacos/citología , Miocitos Cardíacos/citología , Factores de Transcripción/metabolismo , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Biomarcadores/análisis , Diferenciación Celular , Perfilación de la Expresión Génica , Proteína Homeótica Nkx-2.5 , Proteínas de Homeodominio/genética , Humanos , Mioblastos Cardíacos/metabolismo , Miocitos Cardíacos/metabolismo , Reacción en Cadena de la Polimerasa , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Factores de Transcripción/genética , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
We have used homologous recombination in human embryonic stem cells (hESCs) to insert sequences encoding green fluorescent protein (GFP) into the NKX2.1 locus, a gene required for normal development of the basal forebrain. Generation of NKX2.1-GFP(+) cells was dependent on the concentration, timing, and duration of retinoic acid treatment during differentiation. NKX2.1-GFP(+) progenitors expressed genes characteristic of the basal forebrain, including SHH, DLX1, LHX6, and OLIG2. Time course analysis revealed that NKX2.1-GFP(+) cells could upregulate FOXG1 expression, implying the existence of a novel pathway for the generation of telencephalic neural derivatives. Further maturation of NKX2.1-GFP(+) cells gave rise to γ-aminobutyric acid-, tyrosine hydroxylase-, and somatostatin-expressing neurons as well as to platelet-derived growth factor receptor α-positive oligodendrocyte precursors. These studies highlight the diversity of cell types that can be generated from human NKX2.1(+) progenitors and demonstrate the utility of NKX2.1(GFP/w) hESCs for investigating human forebrain development and neuronal differentiation.
Asunto(s)
Linaje de la Célula/genética , Rastreo Celular/métodos , Células Madre Embrionarias/metabolismo , Proteínas Nucleares/genética , Prosencéfalo/embriología , Factores de Transcripción/genética , Animales , Animales Recién Nacidos , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Células Madre Embrionarias/citología , Citometría de Flujo/métodos , Genes Reporteros , Humanos , Ratones , Ratones Transgénicos , Terapia Molecular Dirigida/métodos , Neurogénesis/genética , Neurogénesis/fisiología , Proteínas Nucleares/metabolismo , Prosencéfalo/citología , Prosencéfalo/fisiología , Factor Nuclear Tiroideo 1 , Factores de Transcripción/metabolismoRESUMEN
This unit describes a series of technical procedures to form clonal human embryonic stem cell (hESC) lines that are genetically modified by homologous recombination. To develop a reporter knock-in hESC line, a vector is configured to contain a reporter gene adjacent to a positive selection cassette. These core elements are flanked by homologous sequences that, following electroporation into hESCs, promote the integration of the vector into the appropriate genomic locus. The positive selection cassette facilitates the enrichment and isolation of genetically modified hESC colonies that are then screened by PCR to identify correctly targeted lines. The selection cassette, flanked by loxP sites, is subsequently excised from the positively targeted hESCs via the transient expression of Cre recombinase. This is necessary because the continued presence of the cassette may interfere with the regulation of the reporter or neighboring genes. Finally, these genetically modified hESCs are clonally isolated using single-cell deposition flow cytometry. Reporter knock-in hESC lines are valuable tools that allow easy and rapid identification and isolation of specific hESC derivatives.
Asunto(s)
Técnicas de Cultivo de Célula , Células Madre Embrionarias/citología , Técnicas Genéticas , Recombinación Genética , Transgenes/genética , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Electroporación , Citometría de Flujo/métodos , Genes Reporteros , Vectores Genéticos , Humanos , Modelos Genéticos , Mutagénesis Insercional , Reacción en Cadena de la PolimerasaRESUMEN
A human embryonic stem cell (hESC) line that enabled globin-expressing cells to be easily recognized would facilitate optimization of erythroid differentiation in vitro and aid in the identification of hESC-derived erythroid cells in transplanted animals. We describe a genetically modified hESC line, ErythRED, in which expression of RFP, controlled by regulatory sequences from the human beta-globin locus control region, is restricted to maturing erythroid cells.
Asunto(s)
Células Madre Embrionarias/citología , Células Eritroides/citología , Animales , Diferenciación Celular , Línea Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/metabolismo , Células Eritroides/metabolismo , Regulación de la Expresión Génica , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Globinas beta/genética , Globinas beta/metabolismoRESUMEN
This unit describes a protocol for the large-scale expansion of karyotypically normal human embryonic stem cells (hESCs). hESCs can be maintained indefinitely as dense colonies that are mechanically cut into pieces, which are subsequently transferred to fresh organ culture dishes seeded with primary mouse embryonic fibroblasts (MEFs). hESCs can also be enzymatically passaged (bulk culture); however, over time, this style of culturing may lead to the acquisition of chromosomal abnormalities. Nevertheless, enzymatic passaging can be used for short periods (up to 25 passages) without the appearance of cells with abnormal karyotypes.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Células Madre Embrionarias/citología , Animales , Proliferación Celular , Células Cultivadas , Humanos , RatonesRESUMEN
This unit describes a protocol for the differentiation of human embryonic stem cells (hESCs). To generate spin embryoid bodies (EBs), known numbers of hESCs are deposited into low-attachment, round-bottomed 96-well plates in a serum-free medium supplemented with growth factors. The cells are then aggregated by centrifugation, initiating formation of EBs of uniform size. The spin EBs generated using this technique differentiate efficiently and synchronously along the lineages preferentially induced by the combinations of growth factors to which the cells are exposed. The 96-well format permits an assessment of the effects of different combinations of growth factors in the same experiment, facilitating the optimization of differentiation conditions for any given cell type. Up to 40 plates can be set up within a couple of hours by one experimenter, and aliquots of the differentiating EBs can be harvested at intervals and subjected to analyses using a variety of techniques.
Asunto(s)
Diferenciación Celular , Ensayo de Unidades Formadoras de Colonias/métodos , Embrión de Mamíferos/citología , Células Madre Embrionarias/citología , Células Madre Hematopoyéticas/citología , Animales , Técnicas de Cultivo de Célula/métodos , Proliferación Celular , Células Cultivadas , Humanos , Metilcelulosa , RatonesRESUMEN
The first step in the generation of genetically tagged human embryonic stem cell (HESC) reporter lines is the isolation of cells that contain a stably integrated copy of the reporter vector. These cells are identified by their continued growth in the presence of a specific selective agent, usually conferred by a cassette encoding antibiotic resistance. In order to mitigate potential interference between the regulatory elements driving expression of the antibiotic resistance gene and those controlling the reporter gene, it is advisable to remove the positive selection cassette once the desired clones have been identified. This report describes a protocol for the removal of loxP-flanked selection cassettes from genetically modified HESCs by transient transfection with a vector expressing Cre recombinase. An integrated procedure for the clonal isolation of these genetically modified lines using single-cell deposition flow cytometry is also detailed. When performed sequentially, these protocols take approximately 1 month.
Asunto(s)
Farmacorresistencia Microbiana/genética , Células Madre Embrionarias/metabolismo , Citometría de Flujo/métodos , Técnicas de Transferencia de Gen , Mutagénesis Insercional/métodos , Vectores Genéticos/genética , HumanosRESUMEN
The ability to genetically modify human embryonic stem cells (HESCs) will be critical for their widespread use as a tool for understanding fundamental aspects of human biology and pathology and for their development as a platform for pharmaceutical discovery. Here, we describe a method for the genetic modification of HESCs using electroporation, the preferred method for introduction of DNA into cells in which the desired outcome is gene targeting. This report provides methods for cell amplification, electroporation, colony selection and screening. The protocol we describe has been tested on four different HESC lines, and takes approximately 4 weeks from electroporation to PCR screening of G418-resistant clones.