Rational strategies for neuronal production and maturation from human cells.

Recent exciting finding have shown that normal cells from the skin (fibroblasts) can be converted directly to nerve cells by the addition of only 3 or 4 new specific proteins including so-called "proneural factors". This has opened up a wealth of possibilities for using these neurons to study nerve defects in conditions such as Parkinsons outside the body, and as a platform to test drugs that may enhance neural cell function. Moreover, findings in this area may ultimately underpin developments of methods to produce nerve cells to transplant back into patients with diverse neurological deficits.

However, for these nerve cells to be used to model disease in a petri dish, or indeed as a supply to replace nerve cells lost in neurological disease, we need to be able to generate lots of mature nerve cells from fibroblasts that come straight from patients (as opposed to fibroblasts that have been chosen specifically for their ability to grow well in the lab). In addition, we need a way to direct the fibroblasts to form the correct type of nerve cell (e.g. cortical neurons for Stroke, dopaminergic nerve cells for Parkinsons disease, motor neurons for spinal cord injury or motor neuron disease). We are using our knowledge of the regulation of the proteins, and specifically a hyperactive protein that we think will drive much better maturation of these nerves as it does in the developing embryo, to create more effective protocols for making mature nerve cells from patient samples. We will see whether nerve cells generated in this way from patient fibroblasts look and behave the same as nerves produced from different patient-derived cells that have been produced in another way, via a stage that converts them to a "naive" form usually found in the early embryo (so called induced pluripotent cells).

If we can use our hyperactive protein to enhance generation of mature nerves of specific types, we will then look at the genes turned on in these nerves to further explore why our neuronal differentiation factor is hyperactive.

Generation of human neurons in vitro is prerequisite for development of disease-in-a-dish models of neurodegenerative disorders, and ultimately for producing neurons for transplantation. However, for these applications to be realized, neurons generated must be mature, of defined subtype and with normal electrophysiological function. Recently, neurons have been generated from human fibroblasts and ES cells in vitro (iN cells) using defined transcription factors, with and without additional small molecules. However, the neurons generated have frequently been disappointingly immature.
The proneural transcription factor Ascl1, used extensively in these protocols, is a crucial regulator of multiple aspects of neurogenesis, including progenitor maintenance, neuronal differentiation, and neuronal subtype. We find that using a modified form of Ascl1 that cannot be phosphorylated on Ser-Pro sites greatly increases the morphological and functional maturity of iN cells produced from human fibroblasts. Our data indicates that phosphomutant Ascl1 will also enhance generation of mature neurons from ES cells.
We aim to manipulate phosphoregulation of Ascl1 to enhance maturation of human neurons generated in vitro, and to characterize the processes involved.
Experimentally, we will
1) Compare expression of WT and phosphomutant forms of Ascl1 to characterize effects on differentiation, maturation and neuronal subtype after iN cell generation from human fibroblasts and hES cells, in the presence and absence of signaling modulators.
2) Compare in vitro generated neurons produced by direct fibroblast transdifferentiation and via transcription factor expression in iPS cells from the same familial alzheimers patients to compare modelling methods.
3) Use genome-wide ChIPseq and RNA seq to identify key targets that respond differentially to Ascl1 phosphostatus and to use ChIP proteomics to identify potential differences in co-factor association controlled by phospho-status.

The primary aim of this proposal is to benefit patients with neurological deficits, the healthcare/biotech industry, the NHS and the tax-payer by enhancing neuronal differentiation and maturation in vitro along with controlling neuronal subtype choice to significantly enhance the utility of patient-specific disease modeling and generation of neurons for cell replacement therapies. In detail:

Who will benefit?
Patients, health-care providers and biomedical science and allied industries will benefit.

This proposal is centred on developing improved disease models for neurological disorders and neurodegenerative conditions that are widely prevalent and are rising fast in our aging community. In addition this project will potentially enhance methods undertaken to generate neurons in vitro for cell replacement therapies. Our work in basic biology has uncovered a way to promote neuronal differentiation and maturation from human cells in vitro more effectively than current protocols, and may simultaneously influence neuronal subtype choice, benefiting all those groups with an interest in improving and enhancing these processes.

How will they benefit?
This work, grounded in the basic developmental biology of proneural protein regulation, indicates a way to enhance neuronal differentiation of human ES cells and human fibroblasts in vitro. Moreover, it points towards a regulate-able way to manipulate the type of neuron generated in vitro. Both these advances will lead to better "disease-in-a-dish" models for neurological deficits. These are likely to be suitable both for detailed phenotypic analysis of neurons and also to test novel therapeutics for efficacy before use in animal models and human trials so benefitting patients, health-care providers and biomedical science and allied industries. Moreover, the scientific community will benefit from an enhanced understanding of the basic biology of neuronal differentiation, maturation and subtype choice, as well as development of better methods for neuron production. These benefits will be realised within the life of this grant or shortly after.

Generation of more mature human neurons of specific subtypes in vitro is a prerequisite step for producing neurons to repair or replace the underlying neurological deficits of a number of devastating and prevalent conditions, for instance nigral dopaminergic neurons in Parkinsons disease, cortical neurons for stroke and motor neurons for spinal cord injury. For these reasons, patients with neurological deficits will benefit from better patient-specific disease modeling and potentially enhanced cell based therapies, while health-care providers and allied industries will benefit by developing these therapies. While the advance at producing neurons should be very rapid, within the life-time of this grant, it would take at least 5 years to take these advances further into patients, and rodent xenograft trials would certainly be the next step (already planed with Prof Roger Barker).

Findings about post-translational regulation of proneural proteins is likely to be applicable to proneural proteins active in other tissues such as Ngn3 that controls differentiation of pancreatic beta cells. Implications of this work may therefore extend beyond neurosciences. We aim to maximise this impact by running parallel programs in the lab on complimentary aspects of proneural control in the nervous system and pancreas (an area where we have an MRC Research grant already funded).