Neural differentiation of patient specific iPS cells as a novel approach to study the pathophysiology of multiple sclerosis

Abstract

The recent introduction of technologies capable of reprogramming human somatic cells into induced pluripotent stem (iPS) cells offers a unique opportunity to study many aspects of neurodegenerative diseases in vitro that could ultimately lead to novel drug development and testing. Here, we report for the first time that human dermal fibroblasts from a patient with relapsing-remitting Multiple Sclerosis (MS) were reprogrammed to pluripotency by retroviral transduction using defined factors (OCT4, SOX2, KLF4, and c-MYC). The MSiPS cell lines resembled human embryonic stem (hES) cell-like colonies in morphology and gene expression and exhibited silencing of the retroviral transgenes after four passages. MSiPS cells formed embryoid bodies that expressed markers of all three germ layers by immunostaining and Reverse Transcriptase (RT)-PCR. The injection of undifferentiated iPS cell colonies into immunodeficient mice formed teratomas, thereby demonstrating pluripotency. The MSiPS cells were successfully differentiated into mature astrocytes, oligodendrocytes and neurons with normal karyotypes. Although MSiPS-derived neurons displayed some differences in their electrophysiological characteristics as compared to the control cell line, they exhibit properties of functional neurons, with robust resting membrane potentials, large fast tetrodotoxin-sensitive action potentials and voltage-gated sodium currents. This study provides for the first time proof of concept that disease cell lines derived from skin cells obtained from an MS patient can be generated and successfully differentiated into mature neural lineages. This represents an important step in a novel approach for the study of MS pathophysiology and potential drug discovery.

In vitro myelin formation using embryonic stem cells

“…Recently, myelin pathology of PMD has been replicated using patient-derived iPSCs (Numasawa-Kuroiwa et al, 2014). iPSCs from MS patients are also available (Douvaras et al, 2014;Song et al, 2012). It is possible to adapt these models to the microfluidic co-culture environment to study myelination.…”

Rho/ROCK pathway is essential to the expansion, differentiation, and morphological rearrangements of human neural stem/progenitor cells induced by lysophosphatidic acid

“…In particular, the main difference between the protocols for generating a monolayer of NS/PCs is in the derivation of the NS/PCs themselves. We use dissociated neurospheres to obtain a monolayered culture, whereas the NEP used by Hurst and colleagues were directly obtained from hESCs without going through a neurosphere stage ( 41,46 ). This variation in protocol might account for Supplemental Material can be found at: ( 74 ).…”

Lithium chloride improves the efficiency of induced pluripotent stem cell-derived neurospheres

“…Human iPSC (hiPSC) cell culture hiPSC lines were generated from a 35-year-old normal healthy female, as described previously ( Song et al, 2012 ). Briefl y, a 6-mm skin punch biopsy was obtained following an informed consent carried out under human research subject and stem cell protocols and approved by the institutional review boards of Monash University and Monash Medical Centre, Melbourne, Australia.…”

“…We characterized the neurosphere contents previously ( Song et al, 2012 ) and showed that neural progenitor markers were expressed ubiquitously. In the present study, therefore, we did not recharacterize neurosphere contents; however, we dissociated them and they were further cultured in neural differentiation medium.…”