Study of Human Epilepsy
PROFESSOR MIKE COLEMAN
ALONG WITH E J HILL & D A NAGEL
PROJECT TITLE: Development of a human in vitro model of epilepsy using induced pluripotent stem cells
PROJECT TIMESCALE: 10 October 2016 – 9 October 2019
PHD STUDENT: Alastair Grainger
PROGRESS SO FAR -September to February 2017
The first task of any cell-based project is to make routine what is a very complex process of growing the cells and then instructing them to fulfil their potential as stem cells. That is, to become a specialized group capable of mimicking a real tissue.
In this study, we wish to go further than simply encourage the cells to become neurones and astrocytes, the basic units of the human brain. The project is aimed at producing highly specialized cortical neurones and astrocytes, which can perform the functions of the cerebral cortex of the human brain, which hosts our thoughts and our personalities. The differentiation process is very slow, as you would imagine, as the development of an aspect of the most complex system known is likely to take some time.
Figure 3 shows the cells at an early stage, when they are ‘neuronal precursors’ and when they form a characteristic ‘cortical rosette’, which is seen in the development of cortical neurone systems in the human brain.
Figure 4 shows how the process of changing a ‘blank canvass’ stem cell runs towards the formation of mature neurones and astrocytes. This ‘differentiation’ process orchestrates the formation of networks whereby they can communicate with each other and exchange information, as happens in our brains.
Figure 5 shows the start of the process of seeding the neuronal precursors onto MEA plates for future analysis. Will leave these for roughly 30 days and assess for electrophysiological activity. Any model of epilepsy must be based on a realistic cellular platform, which behaves exactly as our own brain tissue does, before we can then look at the effects of drugs which can change brain tissue network capacity towards epileptiform behaviour.
Alastair has already demonstrated that our preliminary work was sound in principle and we can produce human neurones and astrocytes which are developing normally and will be capable of stimulation by certain realistic cellular environments to show seizure-type electrical activity. Our preliminary work with anticonvulsant drugs must now be reproduced and confirmed so that we can start showing the scientific community a relevant, robust and effective method of studying the ability of chemicals to cause seizures and whether they can be stopped. Once such a model is published and can be developed, over the next decade, many thousands of animals will not need to undergo cruel, unnecessary and uninformative test processes to evaluate chemicals for the benefit of man. In addition, this project will provide training for a future scientist, who will be equipped with the latest biochemical, molecular biological and electrophysiological skills necessary to take this epilepsy model forward towards future regulatory validation.
JULY 2016 – The Beginning
DEVELOPMENT OF A HUMAN IN VITRO MODEL OF EPILEPSY USING INDUCED PLURIPOTENT STEM CELLS
Background-The Brain and Epilepsy
The brain is not only the seat of our personalities and consciousness, but it is also most complex as well as the most delicate organ in the human body. Whilst the brain is protected physically and biologically through the skull and the blood brain barrier, it remains extremely vulnerable to a vast number of chemicals in our food and atmosphere, as well as the drugs we take. The consequences for disruption to brain activity are much greater than for most other organs, with the exception of the heart. Violent changes in brain activity seriously imperil the individual and epilepsy is the best known manifestation of disordered brain activity. Whilst epilepsy can be caused through trauma or genetic reasons, chemicals such as drugs are capable of inducing seizures. Indeed, as part of all routine testing of new chemicals, which may be drugs, food additives or occupationally encountered agents, some means of evaluating the ability to cause seizures must be utilized.
Current Methods of Epilepsy Research
At the moment, the basic means of studying epilepsy involves large numbers of experimental animals, such as mice, rats and some larger animals such as cats and even primates. These animals are subjected to distressing seizures to explore the ability of chemicals to either cause or attenuate these effects, as part of regulatory and drug development processes. Many are under the impression that experimental primate use has declined to virtually nothing in recent years, however this is not the case. Indeed, genetically manipulated photosensitive baboons are used in advanced studies prior to human drug trials and in many models, the seizure-causing drugs are injected directly into animal brains to cause the experimental seizures.
What drives this work is that whilst there are many anti-seizure drugs in use, most have serious side-effects which can be debilitating. In addition, up to 40% of those suffering from the condition cannot completely control the fits with existing drugs. Therefore, not only is there an ongoing search for new anti-seizure drugs, there is also a strong desire in the research community for more specialized models of epilepsy, such as those involving drug –resistant seizures. This has encouraged researchers to seek models which do not involve living animals, such as brain slice studies. Use of this model has increased dramatically in recent years, as it can provide more data than live experiments, but of course it still results in deaths of many animals.
It is clear that the lack of efficacy of existing anticonvulsant drugs is linked with the poorly predictive and inadequate animal models that caused these agents to be selected for clinical trial. Hence, there is a desire in the scientific community for more relevant test platforms for humans and whilst it is easy to demonize medical science for failing to seek models outside of whole animal usage, there are signs that these scientists will embrace a human experimental platform-if they can see its validity.
A New Human Epilepsy Model
Hence, our research mission is two-fold: firstly, we must develop a human-based cellular model which not only achieves everything the animal models do, but exceeds them in capacity and potential. Secondly, we must demonstrate through our future publications that this model will ‘do what it says on the side of the can’ to the satisfaction of our ‘target audience’, that is, neurophysiological scientists working in the area of epilepsy. How we will achieve this depends on our expertise, experience and belief in one of the most exciting arenas of medical research-stem cell technology.
In our current research we use a type of stem cell generated from skin cells, known as induced pluripotent stem cells. When these cells are expanded, they provide a plentiful supply of neural progenitor cells. By growing these progenitor cells in culture we can generate models of the human brain (neural networks) containing many of the different cell types found in the brain. This is a crucial step, as the brain is not one single organ but a collection of different neural organs and stem cell models allow us to re-create the cells that comprise these organs. Another key feature of our work is that we can monitor the electrical activity of the neural cells without damaging them-this ‘non-invasive’ process, which involves using multi-electrode arrays, means that we can gather information on the ability of a chemical to cause seizures in real time and observe while the effects are modulated or abolished by other drugs and treatments.
In order for any research project to be funded, the proposers (that is, ourselves) must provide some preliminary evidence to the Trust that they have some firm basis to work on, in terms of preliminary data. Our first step, was to grow the neuronal/ astrocyte networks in culture from the progenitor cells. We need both the neurones and the astrocytes, as both cell types are needed to perform the most basic brain operations-indeed, any model with just neurones is like an engine without the car! Once we have generated the neuronal/astrocyte networks, they have grown up together and are intimately wired up to each other so that they can carry out any function that our cortical brain cells will. The cells can be seen in Figure 1.
Figure 1. Human iPSC derived neural networks: these have been stained after 14 days in culture using a process known as immunofluorescence. (A, B) The green strain reveals neural cytoskeleton, the blue strain reveals neural axons and the red stain reveals the astrocytes. (C) Another green stain reveals the synaptic structure of the neural connections, both pre and post presynaptic structures. Scale bars = (A) 100μm, (B) 50μm and (C) 10μm.
Measuring Cellular Responses
As part of the process of building the cellular model, we must first demonstrate that we can ‘listen’ and ‘see’ what the cells are doing. In the same way we communicate with a computer through its keyboard and see the results on our monitor, we must be able to ‘watch’ the tiny normally invisible events that occur every second in the neuronal and astrocytic cells and visualize them in such a way we really can see an accurate representative of them. This can be achieved through two methods: one, is by adding a dye to the cells which fluoresces when it meets Calcium ions. Cellular network activity is always accompanied by pulses of Calcium ion release. Our microscope then can ‘see’ these Calcium movements. The second method is by using an apparatus known as a multi-electrode array or MEA (Figure 2).
Figure 2: an MEA (multi-electrode array) detects the electrical responses which are made by the cell networks and can translate them into impulses which can be analysed by our computer software. We can watch as the cell networks produce signals which are associated with appropriate cellular maturity-that they are ‘grown’ enough to work with and are of comparable capability to neurones and astrocytes in our own brains.
Finally, if we wish to study epilepsy, logically, we must see and measure ‘normal’ cellular behaviour and then ‘abnormal’ or epileptiform cell activity. Figure 3 shows the neuronal/astrocytic cultures of cells responding to an epileptiform stimulus, in this case low Magnesium concentrations.