Characterisation of the motor neurons obtained from induced pluripotent stem cells (iPS) in Amyotrophic lateral sclerosis (ALS).

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder affecting upper and lower motoneurons (MN) and leading to death within 2-3 years from diagnosis. Our previous findings and the possibility to determine the transcription profile of MN derived from ALS patients as well as investigating their functional characteristics led us to set up a collaboration with Professor Jun Xu et al at Tongji University in China. Professor Jun Xu and Professor Zhengliang Gao have established very good stem cell research facility at Stem Cell Research Center at Tongji University. They have international recognised track records in stem cell research and have successfully produced motor neuron like cells from human fibroblast cells. This collaboration is a joint effort to efficiently progress in ALS research, merging technical expertise and a large number of biological samples. The application of microarray analysis to the induced pluripotent stem cells (iPS) derived motor neurones (MN) from patients and controls will uncover the genes and pathways dysregulated in Amyotrophic lateral sclerosis (ALS). This research programme will elucidate the selective vulnerability of MN allowing the identification of potential therapeutic targets. The aims of the proposed study are: 1. To produce iPS-derived MN from our collaborators at Tongji University in China and set up the technique in the host laboratory in Sheffield, UK. 2. To determine the expression profile of the iPS-derived MN 3. To compare the expression profile of MN derived from sporadic and familial ALS patients with control. 4. To determine the stress response of MN obtained from patients compared to controls as well as comparing the stress response of MN and fibroblasts from the same individual 5. To identify the cytokines and other factors released in the media by the iPS-derived ALS cells and their effect on control MN. 6. To compare the axonal transport characteristics of patient derived MN with control derived MN. 7. To test drugs able to antagonise or induce relevant pathways identified through the investigations outlined above.

Our research programme will start at Stem Cell Research Centre at Tongji University in order to learn the methodology for reprogramming fibroblasts into iPS cells through retrovirus-mediated transfection of the transcription factors Oct3/4, Sox2, c-Myc, and Klf4. iPS can then be directed towards the motoneuronal differentiation pathway by stimulation with sonic hedgehog and retinoic acid. We will then set up the technique at the University of Sheffield. One of the priorities of this project is to establish the gene expression profile of iPS-derived motor neuron (MN) and compare this with the transcription profile of the cells they are derived from, i.e. fibroblasts, and the cells they have been differentiated into MN. The gene expression profile of iPS-derived MN from ALS patients carrying different mutations responsible for the development of the disease, eg. SOD1, TDP43 and FUS, as well as from sporadic cases, and healthy controls will be carried out at the University of Sheffield. A minimum of 6 fibroblast-derived cell lines per genotype are derived. Data analysis will be carried out using the GeneSpring software (Agilent) applying multiple unpaired two-tailed T-tests and the Benjamini and Hochberg correction for false discovery rate. Pathway analysis will be facilitated by the use of the online database DAVID (http://david.abcc.ncifcrf.gov) and the MetaCore software. This study will establish the foundation for the subsequent part of the project by identifying the differentially expressed genes and pathways to investigate in more depth. Our strategy is to investigate the mechanisms involved in the development of ALS by focusing on the differences between the stress responses, axonal properties, cytokines/chemokines released in the media, test drugs.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder affecting upper and lower motoneurons (MN) and leading to death within 2-3 years from diagnosis. Between 90-95% of cases are sporadic in origin, while the remaining 5-10% of cases are familial. Of these, around 20% carry mutations in the gene encoding for the superoxide dismutase 1 enzyme (SOD1). Transgenic mice expressing mutant forms of human SOD1 are used as a model of familial ALS. Understanding how mutant SOD1 can alter the physiological functions of MN is one of the key questions addressed in Professor Shaw's laboratory in Sheffield. The current project will collect and analyse a minimum of 6 fibroblast-derived cell lines per genotype from gene expression profile of iPS-derived MN from ALS patients carrying different mutations responsible for the development of the disease, eg. SOD1, TDP43 and FUS, as well as from sporadic cases, and healthy controls. This study will establish the foundation for the subsequent part of the project by identifying the differentially expressed genes and pathways to investigate in more depth. The iPS derived MNs will be widely available for academic and research field in ALS.