Using microRNAs to enhance the generation of induced pluripotent stem cells.
ABSTRACT Somatic cells reprogrammed to acquire an ES-like state are termed iPS cells. In this unit, a protocol to use microRNAs as enhancers to increase the reprogramming efficiency is described. Mouse embryonic fibroblasts (MEFs) are isolated from E13.5 mouse embryos and seeded for reprogramming by defined factors. microRNA mimics are transfected into MEFs at two time points during this process to enhance the overall reprogramming efficiency. Two standard protocols for characterization of these miR-iPSCs, embryoid body formation and teratoma formation, are also provided. By using this method, the investigators can obtain a significantly higher number of bona-fide iPSC colonies and miR-iPSCs can be derived at a faster rate than with non-treated cells.
SourceAvailable from: Arlene V Drack[Show abstract] [Hide abstract]
ABSTRACT: eLife digest Retinitis pigmentosa is an inherited disorder in which the gradual degeneration of light-sensitive cells in the outer retina, known as photoreceptors, causes a progressive loss of sight. Retinitis pigmentosa can also occur as part of a wider syndrome: patients with Usher syndrome, for example, suffer from early-onset deafness and then develop retinitis pigmentosa later in life. Usher syndrome is caused by mutations in any of more than ten genes, but the most commonly affected is USH2A, which encodes a protein called usherin. Mutations in USH2A can also cause retinitis pigmentosa on its own. Clinical trials are underway to determine whether it is possible to treat various forms of inherited retinal degeneration using gene therapy. This involves inserting a functional copy of the gene associated with the disease into an inactivated virus, which is then injected into the eye. The virus carries the target gene to the light-sensitive photoreceptor cells where it can replace the faulty gene. This could be particularly useful for conditions such as Usher syndrome, in which the early-onset deafness makes it possible to diagnose retinitis pigmentosa before substantial numbers of photoreceptor cells have been lost. For gene therapy to become a widely used strategy for the treatment of retinal degenerative disease, identification and functional interrogation of the disease-causing gene/mutations will be critical. This is especially true for large highly polymorphic genes such as USH2A that often have mutations that are difficult to identify by standard sequencing techniques. Likewise, viruses that can carry large amounts of genetic material, or endogenous genome editing approaches, will need to be developed and validated in an efficient patient-specific model system. Tucker et al. might have found a way to address these problems. In their study, they used skin cells from a retinitis pigmentosa patient with mutations in USH2A to produce induced pluripotent stem cells. These are cells that can be made to develop into a wide variety of mature cell types, depending on the exact conditions in which they are cultured. Tucker et al. used these stem cells to generate photoreceptor precursor cells, which they transplanted into the retinas of immune-suppressed mice. The cells developed into normal-looking photoreceptor cells that expressed photoreceptor-specific proteins. These results have several implications. First, they support the idea that stem cell-derived retinal photoreceptor cells, generated from patients with unknown mutations, can be used to identify disease-causing genes and to interrogate disease pathophysiology. This will allow for a more rapid development of gene therapy strategies. Second, they demonstrate that USH2A mutations cause retinitis pigmentosa by affecting photoreceptors later in life rather than by altering their development. This suggests that it should, via early intervention, be possible to treat retinitis pigmentosa in adult patients with this form of the disease. Third, the technique could be used to generate animal models in which to study the effects of specific disease-causing mutations on cellular development and function. Finally, this study suggests that skin cells from adults with retinitis pigmentosa could be used to generate immunologically matched photoreceptor cells that can be transplanted back into the same patients to restore their sight. Many questions remain to be answered before this technique can be moved into clinical trials but, in the meantime, it will provide a new tool for research into this major cause of blindness. DOI: http://dx.doi.org/10.7554/eLife.00824.002eLife Sciences 08/2013; 2:e00824. DOI:10.7554/eLife.00824 · 8.52 Impact Factor
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ABSTRACT: Fibroblasts can be reprogrammed to induced pluripotent stem cells (iPSCs) by application of transcription factors octamer-binding protein 4 (Oct4), SRY-box containing gene 2 (Sox2), Kruppel-like factor 4 (Klf4), and c-Myelocytomatosis oncogene (c-Myc) (OSKM), but the underlying mechanisms remain unclear. Here, we report that exogenous Oct4 and Sox2 can bind at the promoter regions of mir-141/200c and mir-200a/b/429 cluster, respectively, and induce the transcription activation of miR-200 family during the OSKM-induced reprogramming. Functional suppression of miR-200s with specific inhibitors significantly represses the OSKM-caused mesenchymal-to-epithelial transition (MET, an early event in reprogramming of fibroblasts to iPSCs) and iPSC generation, whereas overexpression of miR-200s promotes the MET and iPSC generation. Mechanistic studies showed that miR-200s significantly repress the expression of zinc finger E-box binding homeobox 2 (ZEB2) through directly targeting its 3' UTR and direct inhibition of ZEB2 can mimic the effects of miR-200s on iPSC generation and MET process. Moreover, the effects of miR-200s during iPSC generation can be blocked by ZEB2 overexpression. Collectively, our findings not only reveal that members of the miR-200 family are unique mediators of the reprogramming factors Oct4/Sox2, but also demonstrate that the miR-200/ZEB2 pathway as one critical mechanism of Oct4/Sox2 to induce somatic cell reprogramming at the early stage.Proceedings of the National Academy of Sciences 02/2013; DOI:10.1073/pnas.1212769110 · 9.81 Impact Factor
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ABSTRACT: There are an estimated 7,000 "orphan diseases," but treatments are currently available for only about 5% of them. Recent progress in the advanced platforms of gene therapy, stem cell therapy, gene modification, and gene correction offers possibilities for new therapies and cures for rare diseases. Many rare diseases are genetic in origin, and gene therapy is being successfully applied to treat them. Human stem cell therapy, apart from bone marrow transplants, is still experimental. Genetic modification of stem cells can make stem cell-based products more effective. Autologous induced pluripotent stem (iPS) cells, when combined with new classes of artificial nucleases, have great potential in the ex vivo repair of specific mutated DNA sequences (zinc-finger proteins and transactivator-like effector nucleases). Patient-specific iPS cells can be corrected and transplanted back into the patient. Stem cells secrete paracrine factors that could become new therapeutic tools in the treatment of orphan diseases. Gene therapy and stem cell therapy with DNA repair are promising approaches to the treatment of rare, intractable diseases.Clinical Pharmacology & Therapeutics 06/2012; 92(2):182-92. DOI:10.1038/clpt.2012.82 · 6.85 Impact Factor