RESUMEN
Induced pluripotent stem (iPS) cells may be of use in regenerative medicine. However, the low efficiency of reprogramming is a major impediment to the generation of patient-specific iPS cell lines. Here we report the first selection system for the isolation of human iPS cells. We developed the EOS (Early Transposon promoter and Oct-4 (Pou5f1) and Sox2 enhancers) lentiviral vector to specifically express in mouse and human embryonic stem cells but not in primary fibroblasts. The bicistronic EOS vector marked emerging mouse and human iPS cell colonies with EGFP, and we used puromycin selection to aid the isolation of iPS cell lines that expressed endogenous pluripotency markers. These lines differentiated into cell types from all three germ layers. Reporter expression was extinguished upon differentiation and therefore monitored the residual pluripotent cells that form teratomas. Finally, we used EOS selection to establish Rett syndrome-specific mouse and human iPS cell lines with known mutations in MECP2.
Asunto(s)
Desdiferenciación Celular/genética , Separación Celular/métodos , Genes Reporteros/genética , Vectores Genéticos/genética , Lentivirus/genética , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Elementos Transponibles de ADN/genética , Embrión de Mamíferos/citología , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Elementos de Facilitación Genéticos/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteína 2 de Unión a Metil-CpG/genética , Ratones , Ratones Endogámicos NOD , Ratones Mutantes , Ratones SCID , Regiones Promotoras Genéticas/genética , Síndrome de Rett/genética , Síndrome de Rett/patología , Teratoma/patologíaRESUMEN
We have identified a large multigenerational Austrian family displaying a novel form of X-linked recessive myopathy. Affected individuals develop an adult-onset scapulo-axio-peroneal myopathy with bent-spine syndrome characterized by specific atrophy of postural muscles along with pseudoathleticism or hypertrophy and cardiac involvement. Known X-linked myopathies were excluded by simple-tandem-repeat polymorphism (STRP) and single-nucleotide polymorphism (SNP) analysis, direct gene sequencing, and immunohistochemical analysis. STRP analysis revealed significant linkage at Xq25-q27.1. Haplotype analysis based on SNP microarray data from selected family members confirmed this linkage region on the distal arm of the X chromosome, thereby narrowing down the critical interval to 12 Mb. Sequencing of functional candidate genes led to the identification of a missense mutation within the four and a half LIM domain 1 gene (FHL1), which putatively disrupts the fourth LIM domain of the protein. Mutation screening of FHL1 in a myopathy family from the UK exhibiting an almost identical phenotype revealed a 3 bp insertion mutation within the second LIM domain. FHL1 on Xq26.3 is highly expressed in skeletal and cardiac muscles. Western-blot analysis of muscle biopsies showed a marked decrease in protein expression of FHL1 in patients, in concordance with the genetic data. In summary, we have to our knowledge characterized a new disorder, X-linked myopathy with postural muscle atrophy (XMPMA), and identified FHL1 as the causative gene. This is the first FHL protein to be identified in conjunction with a human genetic disorder and further supports the role of FHL proteins in the development and maintenance of muscle tissue. Mutation screening of FHL1 should be considered for patients with uncharacterized myopathies and cardiomyopathies.
Asunto(s)
Genes Ligados a X , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Musculares/genética , Atrofia Muscular/genética , Enfermedades Musculares/genética , Adulto , Austria , Análisis Mutacional de ADN , Femenino , Genes Recesivos , Humanos , Proteínas con Dominio LIM , Masculino , Atrofia Muscular/fisiopatología , Enfermedades Musculares/fisiopatología , Mutación Missense , Linaje , Estructura Terciaria de Proteína , Reino UnidoRESUMEN
As the population of childhood heart disease survivors grows, a better understanding of the genetic underpinnings of heart disease is needed to improve diagnostics, therapeutics and outcomes. The Trans-Atlantic Research Network, GenomeHeart and The SickKids Heart Centre Biobank hosted an international symposium on childhood heart disease titled 'Personalized Medicine in the Genomics Era'. Experts in cardiology, developmental biology, genomics, pharmacology, bioinformatics, stem cell biology, ethics and biobanking shared their knowledge and expertise. The 2-day symposium hosted participants from North America, Europe and Asia including scientists, physicians, nurses, trainees and representatives from industry partners, federal and provincial funding agencies, and patient and community groups. The symposium focused on international research partnerships and application of current state-of-the-art in genomics and stem cell medicine towards personalized healthcare for childhood onset heart disease.
Asunto(s)
Genómica/métodos , Cardiopatías Congénitas/genética , Cooperación Internacional , Medicina de Precisión/métodos , Congresos como Asunto , Genómica/tendencias , Cardiopatías Congénitas/terapia , Humanos , Farmacogenética , Medicina de Precisión/tendencias , Trasplante de Células MadreRESUMEN
Generation of induced pluripotent stem (iPS) cells from patients has exciting applications for studying molecular mechanisms of diseases, screening drugs and ultimately for use in cell therapies. However, the low efficiency and heterogeneous nature of reprogramming is a major impediment to the generation of personalized iPS cell lines. We reported in Nature Methods (6, 370-376, 2009) the first selection system to enrich for reprogrammed human iPS cells. Using a lentiviral vector that specifically expresses the enhanced green fluorescence protein and puromycin resistance genes in pluripotent stem cells, it is now possible to mark and enrich for human iPS cell colonies expressing endogenous pluripotency markers. In this study, we describe a detailed protocol for the production of the pluripotent state-specific lentiviral vector and the selection system for the induction of healthy and disease-specific human iPS cells. Overall, preparation of the selection system takes 2 weeks, and the generation of human iPS cells takes approximately 2 months.