The brain is the most complex organ in the body. It controls the highest functions and regulates many processes which incorporate the complete physiological system. There is a significant risk that a new therapeutic agent could impact brain function and structure, which could result in serious pathologies and even death. As a result, CNS testing is one part of the ‘core battery’ of pharmacology studies.
Early stages of drug development rely primarily on using in vivo and in vitro animal models. Considerable costs are involved with animal work, as well as concerns regarding ethics and relevance. This has prompted researchers to turn to in silico or human options instead. Although the development of current medications and their safety profile would have been impossible without animal studies, these methods have obvious shortcomings.
Additionally, it can be hard to extrapolate between species because of metabolic and morphological differences. With this being the case, how can human drug screening models be generated without using invasive techniques? Induced pluripotent stem cells (iPSCs) could be the answer.
Takahashi and Yamanaka discovered iPSCs in 2006. They found that ectopic co-expression of four transcription factors that govern pluripotency (Oct4, c-Myc, Sox2 and Klf-4) with mouse fibroblasts successfully reverted fibroblasts into a pluripotent state (1). Protocols were developed over the following years using human fibroblasts to convert iPSCs into various adult cell types. Efficient and robust neural induction protocols were provided by Chambers et al. (2009) (2) and Shi et al. (2012) (3). These protocols allowed the conversion of neural stem cells into cortical neuronal cell types that are suitable for a variety of applications. The cortex is the executive, integrative center of the mammalian brain and therefore intimately associated with neurodegenerative and other CNS disease progressions and adverse reactions to drugs. Therefore, models of the cortex are desirable.
Do They Talk the Talk, and Do They Walk the Walk?
Ideally, iPSC-derived models should replicate the in vivo functionality and morphology of components as closely as possible. Cortical iPSC models should include cortical markers which can be identified using appropriate immunocytochemical methods. Although the cortex is a delicate six-layer arrangement of neurons and associated glial cells, many models omit glial cells from culture. This means that the importance of co-cultures containing both glia and neurons are often overlooked.
Despite being far fewer in number than their glial counterparts, neurons are the principal cell in the brain. Neurons facilitate synaptic transmission, respond to various stimuli and fire action potentials. Glia on the other hand, communicate directly with neurons, carry out their own transmission and notably, affect the metabolism of drugs. Therefore, generating a cortical co-culture from neural stem cells that contains both neurons and astrocytes is a much more representative model of the human system.
The presence of astrocytes matures and enhances neuronal electrical activity (Odawara et al., 2014) (4). Electrophysiological techniques can be used to characterize electrical activity of cultures. Although traditional invasive techniques, for example patch clamping, are a highly accurate measure of electrical activity and function, they are not suitable for high-throughput testing. A popular and effective high-throughput system for the assessment of spontaneous and synchronous activity and drug responses of neural cultures is multi-electrode arrays (MEA).
They are not invasive and enable the real-time analysis of activity in multiple locations in cultured neurons. MEAs consist of tightly spaced electrodes that form a grid, each electrode records putative action potentials and extracellular potentials from surrounding cultured neurons. They can also be used to monitor network responses to drugs and to see cell communication within a culture.
This is particularly useful for drugs that are known to decrease or increase neural activity. Newer systems mean that cells can be kept close to their optimum condition for survival because measurements can be taken from inside an incubator. This provides the most accurate results.
What are the Next Steps?
With several industries using iPSC-derived neural cultures as part of their drug screening process, their use is becoming more widespread (5). It remains to be seen whether these models can be refined to the extent that they are relevant to basic level human brain function. It is very likely that co-culturing astrocytes alongside neurons will help to achieve this, rather than just culturing neurons.
The nervous system is intricate and cannot be completely modeled in two-dimensions with the use of astrocytes and neurons alone. Oligodendrocytes and microglia also play important roles within the CNS and adding these cells increases the complexity and influences drug metabolism and responses to agents. Work is underway to create these co-cultures in three-dimensions. This work aims to create layers of the cortex as seen in vivo and also provide a relevant and robust platform that looks and behaves like a human cortex.
- Takahashi, K. & Yamanaka, S., 2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell , Volume 126, pp. 663-676.
- Chambers, SM. et al., 2009. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signalling. Nature Biotechnology , Volume 27(3), pp. 275-280.
- Shi, Y., Kirwan, P. & Livesey, F., 2012b. Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks. Nature , 7(10), pp. 1836-1846.
- Odawara, A. et al., 2014. Long-term electrophysiological activity and pharmacological response of a human induced pluripotent stem cell-derived neuron and astrocyte co-culture. Biochemical and Biophysical Research Communications , Volume 443, pp. 1176-1181.
- Authier, S. et al., 2016. Safety pharmacology investigations on the nervous system: an industry survey. Journal of Pharmacological and Toxicological Methods , Volume 81, pp. 37-46.
About AXOL Biosciences
Axol specializes in human cell culture.
Axol produces high quality human cell products and critical reagents such as media and growth supplements. We have a passion for great science, delivering epic support and innovating future products to help our customers advance faster in their research.
Our expertise includes reprogramming cells to iPSCs and then differentiating to various cell types. We supply differentiated cells derived from healthy donors and patients of specific disease backgrounds. As a service, we also take cells provided by customers (primary or iPSC) and then do the reprogramming (when necessary) and differentiation. Clearly, by offloading the burden of generating cells, your time is freed up to focus on the research. Axol holds the necessary licenses that are required to do iPSC work.
The package wouldn't be complete without optimized media, coating solutions and other reagents. Our in-house R&D team works hard to improve on existing media and reagents as well as innovate new products for human cell culture. We also supply a growing range of human primary cells; making Axol your first port of call for your human cell culture needs.
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