Induction of pluripotent stem cells from adult human fibroblasts by defined factors.

Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan.
Cell (Impact Factor: 33.12). 12/2007; 131(5):861-72. DOI: 10.1016/j.cell.2007.11.019
Source: PubMed

ABSTRACT Successful reprogramming of differentiated human somatic cells into a pluripotent state would allow creation of patient- and disease-specific stem cells. We previously reported generation of induced pluripotent stem (iPS) cells, capable of germline transmission, from mouse somatic cells by transduction of four defined transcription factors. Here, we demonstrate the generation of iPS cells from adult human dermal fibroblasts with the same four factors: Oct3/4, Sox2, Klf4, and c-Myc. Human iPS cells were similar to human embryonic stem (ES) cells in morphology, proliferation, surface antigens, gene expression, epigenetic status of pluripotent cell-specific genes, and telomerase activity. Furthermore, these cells could differentiate into cell types of the three germ layers in vitro and in teratomas. These findings demonstrate that iPS cells can be generated from adult human fibroblasts.

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    ABSTRACT: Groundbreaking studies showed that differentiated somatic cells of mouse and human origin could be reverted to a stable pluripotent state by the ectopic expression of only four proteins. The resulting pluripotent cells, called induced pluripotent stem (iPS) cells, could be an alternative to embryonic stem cells, which are under continuous ethical debate. Hence, iPS cell-derived functional cells such as neurons may become the key for an effective treatment of currently incurable degenerative diseases. However, besides the requirement of efficacy testing of the therapy also its long-term safety needs to be carefully evaluated in settings mirroring the clinical situation in an optimal way. In this context, we chose the long-lived common marmoset monkey (Callithrix jacchus) as a non-human primate species to generate iPS cells. The marmoset monkey is frequently used in biomedical research and is gaining more and more preclinical relevance due to the increasing number of disease models. Here, we describe, to our knowledge, the first-time generation of marmoset monkey iPS cells from postnatal skin fibroblasts by non-viral means. We used the transposon-based, fully reversible piggyback system. We cloned the marmoset monkey reprogramming factors and established robust and reproducible reprogramming protocols with a six-factor-in-one-construct approach. We generated six individual iPS cell lines and characterized them in comparison with marmoset monkey embryonic stem cells. The generated iPS cells are morphologically indistinguishable from marmoset ES cells. The iPS cells are fully reprogrammed as demonstrated by differentiation assays, pluripotency marker expression and transcriptome analysis. They are stable for numerous passages (more than 80) and exhibit euploidy. In summary, we have established efficient non-viral reprogramming protocols for the derivation of stable marmoset monkey iPS cells, which can be used to develop and test cell replacement therapies in preclinical settings.
    PLoS ONE 01/2015; 10(3):e0118424. DOI:10.1371/journal.pone.0118424 · 3.53 Impact Factor
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    ABSTRACT: Duchenne Muscular Dystrophy (DMD) is caused by mutations in the dystrophin gene (DMD), and characterized by progressive weakness in skeletal and cardiac muscles. Currently, dilated cardiomyopathy due to cardiac muscle loss, becomes one of the major lethal causes of late-stage DMD patients. To study the molecular mechanisms underlying dilated cardiomyopathy in DMD heart, we generated cardiomyocytes (CMs) from DMD and healthy control induced pluripotent stem (iPS) cells. DMD iPS cell-derived CMs (iPSC-CMs) displayed dystrophin deficiency, as well as the elevated levels of resting calcium, mitochondrial damage and cell apoptosis. Additionally, we found an activated mitochondria-mediated signaling network underlying the enhanced apoptosis in DMD iPSC-CMs. Furthermore, we treated DMD iPSC-CMs with a membrane sealant Poloxamer 188, which significantly decreased the resting cytosolic calcium level, repressed CASP3 activation and consequently suppressed apoptosis in DMD iPSC-CMs. Altogether, using DMD patient-derived iPSC-CMs, we established an in vitro model to manifest the major phenotypes of dilated cardiomyopathy in DMD patients, and uncovered a potential novel disease mechanism. Our study benefits mechanistic study of human muscular dystrophy, as well as the future preclinical testing of novel therapeutic compounds for dilated cardiomyopathy in DMD patients. © 2015. Published by The Company of Biologists Ltd.
    Disease Models and Mechanisms 03/2015; DOI:10.1242/dmm.019505 · 4.96 Impact Factor

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