RESUMEN

Phosphatase and tensin homolog (PTEN) deficiency is associated with development, progression, and metastasis of various cancers. However, changes in gene expression associated with PTEN deficiency have not been fully characterized. To explore genes with altered expression in PTEN­deficient cells, the present study generated a PTEN­knockout cell line (ΔPTEN) from a mouse prostate cancer­derived cell line using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR­associated protein 9 (CRISPR/Cas9) gene editing system. Following transfection of the CRISPR/Cas9 construct, DNA sequencing was performed to identify deletion of the Pten locus and PTEN inactivation was verified by western blotting. The ΔPTEN cell line exhibited enhanced RAC­alpha serine/threonine­protein kinase phosphorylation and cyclin D1 expression. In addition, an increase in cell proliferation and colony formation was observed in the ΔPTEN cell line. Gene expression profiling experiments were analyzed with microarray and microRNA (miRNA) arrays. In the microarray analysis, 111 genes exhibited ≥10­fold increased expression compared with the parent strain and mock cell line and 23 genes were downregulated. The only miRNA with increased expression of 10­fold or more was mmu­miR­210­3p. Genes with enhanced expression included genes involved in the development, progression, and metastasis of cancer such as Tet methylcytosine dioxygenase 1, twist family BHLH transcription factor 2, C­fos­induced growth factor and Wingless­Type MMTV Integration Site Family, Member 3, and genes involved in immunosuppression such as Arginase 1. The results of the present study suggest that PTEN deficiency mobilizes a variety of genes critical for cancer cell survival and host immune evasion.

Asunto(s)

Sistemas CRISPR-Cas/genética , Terapia Genética , Fosfohidrolasa PTEN/genética , Neoplasias de la Próstata/genética , Animales , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Inmunidad Celular/inmunología , Masculino , Ratones , MicroARNs/genética , Análisis por Micromatrices , Próstata/inmunología , Próstata/patología , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-akt/genética

RESUMEN

Duchenne muscular dystrophy (DMD) is a progressive and fatal muscle degenerating disease caused by a dystrophin deficiency. Effective suppression of the primary pathology observed in DMD is critical for treatment. Patient-derived human induced pluripotent stem cells (hiPSCs) are a promising tool for drug discovery. Here, we report an in vitro evaluation system for a DMD therapy using hiPSCs that recapitulate the primary pathology and can be used for DMD drug screening. Skeletal myotubes generated from hiPSCs are intact, which allows them to be used to model the initial pathology of DMD in vitro. Induced control and DMD myotubes were morphologically and physiologically comparable. However, electric stimulation of these myotubes for in vitro contraction caused pronounced calcium ion (Ca(2+)) influx only in DMD myocytes. Restoration of dystrophin by the exon-skipping technique suppressed this Ca(2+) overflow and reduced the secretion of creatine kinase (CK) in DMD myotubes. These results suggest that the early pathogenesis of DMD can be effectively modelled in skeletal myotubes induced from patient-derived iPSCs, thereby enabling the development and evaluation of novel drugs.

Asunto(s)

Células Madre Pluripotentes Inducidas/patología , Modelos Biológicos , Distrofia Muscular de Duchenne/etiología , Distrofia Muscular de Duchenne/patología , Adulto , Calcio/metabolismo , Diferenciación Celular/efectos de los fármacos , Distrofina/metabolismo , Estimulación Eléctrica , Exones/genética , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Lactante , Masculino , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Proteína MioD/metabolismo , Oligonucleótidos Antisentido/farmacología , Tetraciclina/farmacología , Transfección

RESUMEN

The establishment of human induced pluripotent stem cells (hiPSCs) has enabled the production of in vitro, patient-specific cell models of human disease. In vitro recreation of disease pathology from patient-derived hiPSCs depends on efficient differentiation protocols producing relevant adult cell types. However, myogenic differentiation of hiPSCs has faced obstacles, namely, low efficiency and/or poor reproducibility. Here, we report the rapid, efficient, and reproducible differentiation of hiPSCs into mature myocytes. We demonstrated that inducible expression of myogenic differentiation1 (MYOD1) in immature hiPSCs for at least 5 days drives cells along the myogenic lineage, with efficiencies reaching 70-90%. Myogenic differentiation driven by MYOD1 occurred even in immature, almost completely undifferentiated hiPSCs, without mesodermal transition. Myocytes induced in this manner reach maturity within 2 weeks of differentiation as assessed by marker gene expression and functional properties, including in vitro and in vivo cell fusion and twitching in response to electrical stimulation. Miyoshi Myopathy (MM) is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in DYSFERLIN. Using our induced differentiation technique, we successfully recreated the pathological condition of MM in vitro, demonstrating defective membrane repair in hiPSC-derived myotubes from an MM patient and phenotypic rescue by expression of full-length DYSFERLIN (DYSF). These findings not only facilitate the pathological investigation of MM, but could potentially be applied in modeling of other human muscular diseases by using patient-derived hiPSCs.

Asunto(s)

Diferenciación Celular/efectos de los fármacos , Miopatías Distales/genética , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas de la Membrana/genética , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/genética , Atrofia Muscular/genética , Proteína MioD/genética , Animales , Biomarcadores/metabolismo , Diferenciación Celular/genética , Células Cultivadas , Miopatías Distales/metabolismo , Miopatías Distales/patología , Doxiciclina/farmacología , Disferlina , Estimulación Eléctrica , Expresión Génica , Perfilación de la Expresión Génica , Vectores Genéticos , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Ratones , Ratones SCID , Modelos Biológicos , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/efectos de los fármacos , Proteínas Musculares/metabolismo , Atrofia Muscular/metabolismo , Atrofia Muscular/patología , Proteína MioD/metabolismo , Transfección

RESUMEN

Induced pluripotent stem (iPS) cells are generated from adult somatic cells by transduction of defined factors. Given their unlimited proliferation and differentiation potential, iPS cells represent promising sources for cell therapy and tools for research and drug discovery. However, systems for the directional differentiation of iPS cells toward paraxial mesodermal lineages have not been reported. In the present study, we established a protocol for the differentiation of mouse iPS cells into paraxial mesodermal lineages in serum-free culture. The protocol was dependent on Activin signaling in addition to BMP and Wnt signaling which were previously shown to be effective for mouse ES cell differentiation. Independently of the cell origin, the number of transgenes, or the type of vectors used to generate iPS cells, the use of serum-free monolayer culture stimulated with a combination of BMP4, Activin A, and LiCl enabled preferential promotion of mouse iPS cells to a PDGFR-α(+)/Flk-1(-) population, which represents a paraxial mesodermal lineage. The mouse iPS cell-derived paraxial mesodermal cells exhibited differentiation potential into osteogenic, chondrogenic, and myogenic cells both in vitro and in vivo and contributed to muscle regeneration. Moreover, purification of the PDGFR-α(+)/KDR(-) population after differentiation allowed enrichment of human iPS cell populations with paraxial mesodermal characteristics. The resultant PDGFR-α(+)/KDR(-) population derived from human iPS cells specifically exhibited osteogenic, chondrogenic, and myogenic differentiation potential in vitro, implying generation of paraxial mesodermal progenitors similar to mouse iPS cell-derived progenitors. These findings highlight the potential of protocols based on the serum-free, stepwise induction and purification of paraxial mesodermal cell lineages for use in stem cell therapies to treat diseased bone, cartilage, and muscle.

Asunto(s)

Mesodermo/citología , Modelos Biológicos , Células Madre Pluripotentes/citología , Células Madre/citología , Activinas/fisiología , Animales , Proteína Morfogenética Ósea 4/fisiología , Diferenciación Celular/fisiología , Linaje de la Célula , Medio de Cultivo Libre de Suero , Expresión Génica/fisiología , Técnicas In Vitro , Ratones , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/fisiología , Transducción de Señal , Transgenes , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/fisiología , Proteínas Wnt/metabolismo