Dr Nick Allen - PhD
Neural differentiation of human and mouse embryonic stem cells
Embryonic stem cells have tremendous potential to provide an unlimited source of therapeutic differentiated cells for tissue repair and regenerative medicine as well as providing specific cell types to biotechnology and pharmaceutical industry, for example for drug screening programmes.
Our labs interests are to investigate the mechanisms of neural differentiation of human and mouse embryonic stem cells, and to develop protocols to direct the differentiation of neural progenitors to acquire specific neural fates and phenotypes. ES cells are pluripotent, however we have shown that in chemically defined media differentiation is restricted towards the neural lineage. ES cell differentiation generates naïve neural progenitors and these are highly responsive to extrinsic developmental patterning cues such as growth factors and morphogens which can be used to direct their differentiation to acquire specific neural identities. Therefore specific cell types can be derived by a process of applied developmental biology.
Mouse (top) and human (bottom) ES cells differentiated with high efficiency in chemically defined medium to neural progenitors (left: nestin staining) and neurons (right: beta-tubulin III staining).
A major target for us is to derive suitable cells for therapeutic transplantation and in vitro drug screening for Huntington’s disease (HD). HD is a devastating neurodegenerative disease in which there is a selective vulnerability and loss of the medium spiney projection neurons in the striatum, caused by a toxic expansion of a polyglutamine stretch of amino acids in the N-terminus of the disease protein huntingtin. Therapeutic strategies include the development of drugs to counter the pathological effects of mutant huntingtin and prevent or slow the rate of neuronal loss, and secondly to repair the damaged brain by neural transplantation (See Prof Steve Dunnett). While future transplantation therapies will depend on the development of renewable stem cell resources, drug screening programmes will also benefit significantly from the development of human neuronal cell models of the disease. To meet both of these demands we are developing reporter human and mouse ES cell lines for forebrain neural lineage commitment and protocols to direct their differentiation into specified populations of striatal and other forebrain neural progenitors, and assessing the function of derived cells by neural transplantation.
Medium spiney neuron differentiation
To gain insights into mechanisms of MSN differentiation potentially applicable to ES derived neuronal cultures we are studying the development of a subpopulation of MSNs that express D3 dopamine receptors in vivo. Dopamine D3 receptor neurons project from the striatum caudally to the substantia nigra and form a major component of basal ganglia neural circuitry. To study these neurons and development of all of their axonal projections we used knock-in gene targeting to make a transgenic model in which the D3 receptor gene (Drd3) has been replaced by a tau-lacZ reporter gene. This reporter model is being used to identify mechanisms that enhance projection neuron differentiation and survival, and can also be used to identify cells for functional and transplantation studies.
Expression of the Drd3-tauLacZ reporter transgene. Targeting beta-galactosidase activity to the cytoskeleton by expressing it as a tau-beta-galactosidase fusion protein enables all axonal projections to be seen and reveals the entire neuroanatomy of D3 receptor expressing projection neurons. Neurons with cell bodies in the caudate putamen (Cpu) and nucleus accumbens (NAcc) project to the substantia nigra (SNR) and extend collaterals into the globus pallidus (GP), ventral pallidum (VP) and entopeduncular nucleus (EP). Staining is seen in all other brain regions known to express Drd3 including the Islands of Calleja (ICj) and CA1 of the hippocampus.
Regulation of neural stem cell development by dopamine
Analysis of Drd3-tauLacZ reporter mice shows that Drd3 expressing cells are present within neurogenic regions of the adult and fetal CNS. Addtionally, studies using dopamine receptor agonists and antagonists have demonstrated a role for dopamine in the regulation of adult neurogenesis. The precise functions of D1-like and D2-like receptors in dopamine regulated neurogenesis, and the contexts in which dopamine regulation is important are largely unknown. We are currently addressing the functions of the D2-like receptors (Drd2 and Drd3) using a combination of genetic and pharmacological approaches that manipulate dopamine receptor expression or activation.
Current Grant Support
- EU FP6
Prof Marc Peschanski
Dr Siddharthan Chandran (Cambridge)
Dr Richard Wade-Martins (Oxford)
Dr Alysia Battersby
Dalal Al Othman