Engineering human neural networks

Currently our knowledge of the human brain is restricted by ignorance of the most basic cellular functions of its fundamental unit, the neurone/astrocyte combination. Animal models of brain function are limited by their physical which differ in some vital respects to ours. In addition, the animal brains cannot be built from scratch in the manner that is necessary to understand any complex system or machine. The primary aim of the project is no less than to build a model of the most basic functions of the human brain to cast light on how living human neurones process and retain information and how they expand their memory of information-how they 'learn'. To begin we using a growth arrested human tumour cell line (NT2/D1 cells), which is then developed into an intimately connected mixture of neurones and astrocytes in similar proportions to those in our own brains using the 'differentiation' process which mimics human embryo development over the first few weeks of life and results in a miniature human neuronal tissue if grown in three dimensions. Once formed these cultures behave like brain slices of animals, as the astrocytes pulse at the same frequencies and the neurones can communicate in a network as the human brain does. We can fully manipulate the process of differentiation and even insert extra genes in the cells to visualize functions using multiphoton laser technology and calcium imaging. The key features of the system are that unlike an animal slice model, the cells are untraumatized and not slowly dying. The cultures can live for up to 6 months and so the most basic neuronal/astrocytic functions can be followed second by second as they happen in real time. The culture system has thus two considerable advantages over existing animal models, in that it is 'custom built' as required in order to understand the most basic steps of complex neuronal processes and it can be non-invasively monitored to study more advanced neuronal/astrocytic behaviour. To build a functioning model of the most basic functions of the human brain requires both cellular models, initially the NT2/D1 cells and then human stem cells, as well as the engineering and assembly of 3-D matrixes to construct biological neuronal networks (bNNs) which can should be able to store information as memory, as well as process data through a network of cells and then make a form of response. As even small areas of human brain tissue contain the ability to process and store and communicate with proximal and distal tissues, so we must build in a multi-compartmental approach where several basic biological networks are connected facilitating more advanced processing. Ultimately, the NT2.D1 cells (and eventually human stem cells) will be grown along 3-D neuronal networks which will have provable networking capability, which will be monitored using multi-photon laser technology as well as calcium imaging. Once the basic bNN networks have been formed, these will be challenged electronically to try to induce the cells to form new links (synapses) with each other to retain information, which is the basis of memory. This process, known as synaptic plasticity, is a vital step connection of multiple bNNs to determine if a more advanced processing such as visual memory development can take place. This is a very elementary form of 'training' where the linked bNNs can communicate, process and 'remember' information. If successful, this will be the most advanced model of basic human neuronal processing in existence and will pave the way for the adaptation to many other explorations of human neuronal activity, both compared with existing animal data and will facilitate the construction of bNNs which can be made to simulate different human conditions.

The creation of an in-vitro model of the human brain (iBrain) will involve a 'bottom-up' approach by engineering and assembly of 'elements' (synapses), 'units' (neural networks) and 'domains' (structured compartments). The proposed project, as stage I of iBrain, focuses on the formation of a biological neural network (bNN), capable of learning, initially from NT2.D1 cultures and then human stem cells. Specifically we will differentiate human stem and NT2.D1 cells into functional neurons, selecting specific scaffolds and optimising culture conditions to direct their growth into 3-D architectures, so facilitating synaptic connection formation. Such screening and optimisation studies will involve multiple parallel perfused microbioreactors. The NT2.D1s will be transfected with channelrhodopsin 2 capability, facilitating axon stimulation through digital light processing. The bNN's will be formed using guided cell self assembly and tissue engineering and will involve a blue-light stimulated input layer, (473 nm), followed by an output layer (monitored with Ca2+ imaging). A middle layer will be created using collagen-based constructs. Functional measurement through electrophysiological and optogenetic approaches will monitor action potentials in individual neurons, and synaptic currents will be detected with grid electrodes to observe spiking at the output layer, and 2-photon microscopy will record Ca2+ transients as a measure of net activity. 'Training' of bNNs in pattern recognition by inducing targeted synaptic plasticity will be by repetitive stimulation, so showing ability to distinguish between stimuli. Once self-organized, the bNN's will respond to inputs (supervised learning). Functional bNNs will be monitored in neural plasticity during aging and degeneration. Ultimately, we will create 'functional 'domains', i.e., a large cluster of inter-connected multi-compartment clusters of bNNs, which is effectively artificial human brain tissue.

The iBrain will revolutionize basic research into human neuronal function, as well as having an incalculable impact on CNS trauma and neurodegenerative disease, which cause immense suffering worldwide. This model will facilitate future stimulation of repair and regeneration of the human brain, which is unattainable with current models. The iBrain will be a world 'first', making the UK a global leader in human CNS model development and study. Current timescales of decades in the amelioration of the compromised human CNS will be revised downwards to less than 10 years if this enterprise is successful. The successful iBrain concept will reduce public dread of neurodegenerative disease, its impact on individuals and their families, as well as catastrophic burden of millions of elderly people suffering from conditions such as dementia. Among the different applications fostered by the iBrain's flexibility and adaptability include commercial interests in the field of anti-neurodegenerative drug discovery will wish to identify specific drug targets in compromised processes of learning and memory using the iBrain. The combination of functionality and structural human relevance of the iBrain will be ideally suited to the evaluation of pharmacologically active agents as well as the assessment of the neurological effects of environmental pollutants in terms of impairment of iBrain structure and function. In addition, the European Union is engaged in the promotion of models of human tissues in specific initiatives which are not suited to large-scale animal research, such as REACH, , the assessment of the impact of environmental chemicals. The human relevance of the iBrain in assessing chemicals which have revealed neurological perturbation would be seen in terms of impact on structure and functional capability of the model. The existence of a functional model of the human brain may contribute to the reassessment of the overall targeting of research council funding in neurological research, with particular reference to academic and commercial joint ventures which involve the use of the iBrain system in developing different neurological models. Research Skill Development and Training: this pioneering iBrain approach will provide a unique and unmatched opportunity to combine cellular molecular biological manipulation skills, tissue engineering and stem cell technology which provide the iBrain's structure, with the electroneurophysiological skills which can measure its function. This skills overlap will also include handling the most advanced equipment aimed at cell manipulation at the nucleotide level as well as in terms of ionic, electronic and neurotransmitter monitoring. This is an ideal project for the training of future research stars. Publicity and public engagement: just Professor Coleman's recent preliminary work with the NT2/D1 cells alone reached worldwide audiences with live interviews with BBC and Sky News, Radio 5 Live and others. There is clearly a huge world-wide appetite for advances in the development of models of basic human brain function and its application to neurodegenerative disease. This publicity occurred before the iBrain concept has been fully realised. Both Oxford and Aston Universities are highly skilled in publicising major research events and there is no doubt that the fruits of the iBrain project will reach world audiences in appropriate detail and level. We are currently open to collaboration with any interested parties who wish to extend the scope of the iBrain concept and we are all well experienced in sustaining collaborations, both national and international in the pursuit of a long-term goal. Our joint experience has shown us that there will be absolutely no difficulty in engaging the world's scientists and media in our future achievements with the iBrain.