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  • br Conclusion br Materials and methods br


    Materials and methods
    Acknowledgments We thank M. Dubrovcakova and V. Frivalska for excellent technical assistance. We are grateful to J. Zmajkovic for technical help with fluorescence microscopy, J. Lakota, M.D. for providing us with material and M. Cihova for critical reading of the manuscript. This study was supported by the VEGA grants No. 2/0088/11 (L.K.) and 2/0146/10 (M.M.), and APVV grant No. APVV-0260-07 (L.K.).
    Introduction Multiple Sclerosis (MS) is the most common cause of non-traumatic neurological disability among young adults throughout the world. While the cause of MS is unknown, this disease has traditionally been classified as an autoimmune inflammatory disease of the white matter. In recent years much research has focused on the chronic neurodegenerative processes affecting the gray matter (Lassmann, 2010). MS patients have a genetic predisposition that is subsequently triggered by one or more environmental factors such as vitamin D deficiency or viral infection. Once the disease is initiated, it can result in self-limiting attacks of inflammatory infiltrations into the CNS ranging from several per year to one every 10years. In others, the course is slowly progressive over many years. Treatment with medications such as interferon β is only effective in some patients (Killestein and Polman, 2011). There are currently no reliable surrogate markers to predict disease severity, type or response to treatment. Despite the large resources now invested in MS research, there are clearly a number of significant questions regarding pathogenesis, disease subtypes and response to therapy that still need to be examined. Currently, analyses of MS samples have been limited to peripheral blood, DNA, cerebrospinal fluid, and either small biopsy samples or autopsy tissue from MS patients (Baranzini et al., 1999; Dutta and Trapp, 2011; Menon et al., 2011). Brain biopsies are rarely obtained from typical MS patients as this material is often not representative of chronic ‘end-stage’ MS. Not surprisingly, much research has relied on varieties of autoimmune-mediated demyelinating models for an insight into its pathophysiology. Despite their similarities with MS and benefits in understanding the disease, these models are different to the human disease which has led to ineffective and even detrimental MS treatments (Wekerle, 2008). The production of diseased-cell lines from individuals with MS could provide exciting new opportunities for MS researchers and clinicians to study relevant human cell types, including those from the CNS, and may potentially shed further insight into the pathogenesis of this severely debilitating disease. Recently the direct reprogramming of somatic EPZ-6438 through the induced expression of four transcription factors (OCT3/4, SOX2, KLF4 and c-MYC) to produce induced pluripotent stem (iPS) cells has proved to be a major advance in stem cell biology (Takahashi and Yamanaka, 2006; Nishikawa et al., 2008), attracting considerable worldwide attention in disease modeling, drug screening and regenerative medicine (Ebert et al., 2009). iPS cells derived from adult somatic cells such as fibroblasts are able to differentiate into cell types representing all three embryonic germ layers — essentially any cell in the body. This technology could enable the generation and detailed examination of oligodendrocytes, astrocytes and neurons in the laboratory to study MS. Moreover, such diseased cell lines would also be invaluable for testing new drug treatments on the patient\'s own cells.
    Discussion In this study we present evidence for the first time that differentiated oligodendrocytes, astrocytes and functional neurons can be obtained by direct reprogramming of skin fibroblasts from a patient with MS, thus opening new avenues for modeling this complex neurological disease. Consistent with previous studies (Takahashi et al., 2007; Park et al., 2008; Song et al., 2011), the MSiPS cell line we have established is similar to hES cells in many aspects including morphology, proliferation, feeder dependence, surface markers, gene expression, promoter activities, and teratoma formation. The minor differences in gene profiling and electrophysiology fall within the variability of embryonic cell lines from different sources and culture conditions (Richards et al., 2004). Variability in neural differentiation potential among pluripotent stem cell lines is well described (Osafune et al., 2008; Chamberlain et al., 2010; Kim et al., 2011). Similar differences are described in the efficiencies by which human ES and iPS cells undergo specification down the neural lineages, although both can be directed to form comparable neural progenitors. This suggests that once specified to a neural fate, the pluripotent cells are equally efficient at generating neural cells (Kim et al., 2011). In this context, it is noteworthy that a comprehensive and detailed study by Hu et al. (2010) demonstrated clearly that human iPS cells differentiate to neural lineages according to the same temporal program as hESCs and can produce functional neurons. However, the neural differentiation of human iPS cells is not only variable between the different iPS cell lines, but also unpredictable and much less efficient than hES cells (Hu et al., 2010). Consequently, any cell-based modeling using multiple iPS cell lines will need to take into consideration the issue of appropriate controls.