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Custom architecturally defined 3D stem cell derived functional human neural networks for transformative progress in neuroscience and medicine

Periodic Reporting for period 2 - MESO_BRAIN (Custom architecturally defined 3D stem cell derived functional human neural networks fortransformative progress in neuroscience and medicine)

Reporting period: 2017-09-01 to 2019-08-31

MESO-BRAIN aims to grow human brain cells in the laboratory and to image for the first time brain cells communicating with each other. This work will allow scientists to study how the human brain works and could one day help combat the damage caused by Alzheimer's and other brain traumas. To do this, new three-dimensional structures are needed to grow cells in a similar way to how they grow in our own brains. New techniques which allow the cells to be studied without damage will allow the basic functions of the brain to be better understood, ultimately leading to new therapies for brain related diseases and conditions.
The scientific team have generated many different textured surfaces and three-dimensional structures to assess the best materials for growing brain cells. New fluorescent compounds and genes which indicate what the cells are doing have been developed and shown to work in special imaging systems which use the low levels of light needed to study living cells. The cells used have been created from human cells by reprogramming samples of cells then controlling the type of cells they turn into. This means the results will be directly relevant to human brains, unlike studies often performed in animals.
The project envisioned the construction of tailored 3D neuronal circuits derived from human induced pluripotent stem cells (IPSCs), for future medical applications. The tools and resources developed along the 3-year duration of the project advanced the present state of the art in nanofabrication, cell culturing, imaging and data analysis, and opened interesting prospects for advances at a technological, economic and societal level.

1) Progress in nanofabrication - the creation of miniature 3D platform with integrated electrodes for the in vitro modelling of human cortex is unimaginable without the application of the unique technology ‘two-photon polymerisation (2PP)’. In the course of the project, we have developed and demonstrated the possibilities that this technology offers to create such a 3D platform.

The 3D scaffolds developed represent an attractive tool for in vitro modelling of many types of tissues due to their unique features, which can be produced and specified by the application of 2PP technology. 2PP produced 3D scaffolds are reproducible and can any achieve different grades of complexity. Therefore, this type of 3D platform represents an appealing and reliable tool for in vitro tissue modelling, which should quickly find the wide acceptance in basic research and pharmacological industry and, thereby, contribute to the technological and health sectors with an international and socio-economic impact.

2) Progress in cell culturing and tailored biological circuits
Developing functional brain cell types is a fundamental yet challenging basis for the goals of MESOBRAIN. Along the project, protocols for cell differentiation and maturation were optimized and utilized at Aston. Co-cultures of excitatory cortical neurons and astrocytes and co-cultures of excitatory, inhibitory neurons and astrocytes were grown. Single cell patch clamp recordings indicated that neurons could fire trains of action potentials in response to step depolarisations. Calcium imaging and MEA recordings revealed neurons exhibiting independent activity patterns and also participating in synchronized events. Pharmacological interventions revealed physiological functionality in synaptic transmission and synchronized responses to agonists which could be blocked by antagonists, revealing expression of a range of expected neurotransmitter receptors. All these studies indicate a functionality that suggests that the differentiated cells may indeed faithfully reproduce human neural activity. More importantly, the experiments were conducted with and without scaffolds, and therefore portray some of the most detailed analyses of the interaction of iPSCs with physical constraints in vitro. Additionally, the experiments of Aston provided a detailed characterization of the interaction between neurons and glia. This opens new avenues in medicine for understanding the role of glia in neurological disorders. In numerous neurodegenerative diseases, , it is known that altered neuron-glia interactions accelerates the degradation of the entire neuronal circuit. Thus, the protocols and expertise from Aston, in combination with patient derived iPSC cell lines, can be used to develop accurate in vitro models of the neuron-glia interactions in disease and advance towards the development of treatments.

3) Progress in imaging and industrial partnership.
Imaging neuronal activity in a 3D environment is a challenging endeavour, with strong industrial interest, and at the frontline in photonics and microscopy. Traditionally, imaging techniques are used for retrieving structural or functional information contained in a plane (i.e. in 2D). Although accessing the third dimension is possible, the current high-resolution imaging techniques are cumbersome and slow, as they are based on point scanning methodologies. In addition, current measurement devices cannot cover large volumes. Technological developments have therefore been urged by the need to explore noninvasively th
Image of Neuronal brain cells