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

Lead Research Organisation: University of Sheffield
Department Name: Neurosciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description Investigating mechanistic causes of C9ORF72-related amyotrophic lateral sclerosis (ALS)
Amount £372,522 (GBP)
Funding ID MR/M010864/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 12/2014 
End 11/2017
 
Description Collaboration in stem cell research 
Organisation Tongji University
Country China 
Sector Academic/University 
PI Contribution We have collaborated in stem cell research and supervised a joint PhD student. PhD student name: Yuri Ciervo We provide control and ALS patient fibroblasts for Tongji research team to make iPS lines for us to use in Sheffield. We differentiate the iPS lines into motor neurons for our proposed studies in Sheffield.
Collaborator Contribution Tongji research team make iPS lines for us and them for our proposed studies. We had a joint publication recently and expect more joint publications in the near future.
Impact Wang X, Dong C, Sun L, Zhu L, Sun C, Ma R, Ning K, Lu B, Zhang J, Xu J. (2016). Quantitative proteomic analysis of age-related subventricular zone proteins associated with neurodegenerative disease. Sci Rep. 6:37443.
Start Year 2015
 
Description In collaboration with Tongji University in Shanghai, China 
Organisation Tongji University
Department Medical School
Country China 
Sector Hospitals 
PI Contribution This work has been done in collaboration with Professor Jun Xu's research group at Tongji University Medical School in Shanghai, China. Primary fibroblast cells were collected at the University of Sheffield and sent to China to make iPS lines. iPS were differentiated into motor neurons at both sites and used for cell viability and functional studies. ALS patient specific cell models have been established and will be used for gene expression profiling and drug screen studies, as well as molecular mechanism and signalling pathway analysis.
Collaborator Contribution To make iPS lines by using fibroblast cells from Sheffield University. iPS were differentiated into motor neurons and used for cell viability and functional studies.
Impact A manuscript has been written for publication.
Start Year 2012