Dr Majid Hafezparast, from the University of Sussex, has used a project grant from the NC3Rs to generate iPS cells from mutant and wild type mice which can be used to grown neurons for motor neurone disease research.
Principal Investigator: Majid Hafezparast, Reader in Human/ Mammalian Molecular Genetics
Organisation: University of Sussex
Award: £216,696, in 2009, over 24 months
Title: Induced pluripotent stem cells to reduce animal usage in motor neurone disease
Read more about Dr Hafezparast's research
Motor neurone disease is a fatal neurodegenerative disease
Motor neurone disease (MND) encompasses a number of neurodegenerative disorders which affect the central nervous system, the most common of which is amyotrophic lateral sclerosis. Degeneration of the motor neurones leads to difficulty in movement, swallowing, speech and breathing. There is no cure for MND and only one drug, Riluzole, is licensed as a treatment to slow the progression of the disease. Approximately seven in 100,000 of the UK’s population have MND. Its aetiology is unknown, with only a small percentage of patients having a family history of the disease; most cases are sporadic and there are no known predisposing factors.
Mice with mutations that impair axonal transport are used to model motor neurone disease
Impaired axonal transport has been shown to play a key role in the pathogenesis of MND. Cytoplasmic dynein is a large protein involved in a range of cellular processes, including the movement of organelles, cell division and axonal retrograde transport. Mice with mutations in the cytoplasmic dynein gene lose sensory and motor neurones and are widely used as a model for studying dynein function and neurodegeneration.
Loa and Arl are mice with mutations in the neuronespecific functions of cytoplasmic dynein. Homozygous Loa mice cannot move or feed and die within 24 hours of birth. Heterozygotes, however, have a normal lifespan but show a progressive loss of locomotor function and perform significantly worse in tests of coordination and balance than wild type litter mates. Arl mice are homozygous lethal, but can be maintained as heterozygotes. These have a neuromuscular defect that results in impaired locomotor function and an abnormal gait, with early adulthood onset.
In a typical experiment, primary motor neurones are cultured from Loa and Arl embryos. Breeding of Loa and Arl mice is classified as moderate severity under the Animals (Scientific Procedures) Act 1986. Since the lines are maintained as heterozygotes, large numbers of animals are used to provide sufficient embryos of the correct genotype as a source of primary neurones. This is compounded by Arl mice being poor breeders and neurones from both lines only remaining viable in culture for up to three weeks.
Induced pluripotent stem cells can replace the use of primary motor neurones
With NC3Rs funding, Dr Majid Hafezparast, University of Sussex, has developed induced pluripotent stem cells (iPS) from Loa and Arl mouse embryonic fibroblasts, providing an alternative to using primary neuronal cell cultures. In defined culture conditions, using various signalling and growth factors, the iPS cells differentiate into sensory and motor neurones which have been shown to be equivalent to those obtained from mouse embryos. The use of iPS cells has allowed Dr Hafezparast to halve the use of mice in his laboratory to 2,000 per year. Use of the Loa and Arl derived iPS cells is being optimised and this should allow the number of mice used to be reduced to fewer than 700. The iPS cells also have other advantages. They can be further manipulated for use in molecular and cell biology studies, such as transfection with tagged genes, which is impractical in the primary neuronal cell cultures because of their low transfection efficiencies. This approach is now being used to study the role in motor neurone cell death of mutations in genes, such as superoxide dismutase 1, which have been implicated in neurodegenerative diseases.
New collaborations to extend the technology to other mouse mutants and patients
Two new collaborations have been made with UK groups, at the MRC Mammalian Genetics Unit and the Institute of Neurology, to produce iPS cells for other mouse models of neurodegenerative diseases. A collaboration has also been established with neurologists at the Brighton and Sussex Medical School to develop iPS cells from patients, which has the prospect of replacing mouse use for some studies of MND.
This case study was published in a review of our research portfolio in September 2011.