In WP1- Biotechnology – ESRs 1, 3 and 6 will be trained in microfluidics technologies and hPSC differentiation to together develop and implement brain-on-chip models of neurodegenerative disorders mainly focus on HD and PD. In these diseases the most affected cells are the striatal medium spiny neurons (MSNs) and the dopaminergic neurons (DAergic) which are interconnected to each other. Thus, the brain-on-chip model that will be developed in this project will connect hPSCs differentiated to either MSNs or DAergic neurons using patient derived induced hPSCs (hiPSCs). However, as cortical inputs are also relevant in this system and in HD, ESR1 will also implement the differentiation of hiPSCs into cortical neurons. ESR6 will be mainly dedicated to the development of the microfluidic system that will aid culture of the differentiated cells from ESR1 and 3. The microfluidic compartmentalisation of specific neuronal sub-types will also allow to improve selection of neurons to study the effects of optogenetic stimulus in one of the chambers. Differentiation of MSNs will be improved not only by ESR1 but also together with ESR2 which will also analyse the interaction of MSNs with microglial cells. Similarly, DAergic differentiation will be developed and improved by ESR3 but also by ESR4 and ESR5. ESR4 will add a new approach that is the direct reprograming of somatic cells into DAergic neurons.
Another approach to analyse the interaction and connectivity of cells is the use of 3D cerebral organoid. This approach will be developed by ESR5 and ESR7 and will allow examination of the role of cell-cell direct contact, including the role of glial cells. In this aspect, the system will be also useful for ESR2 to analyse the interaction of neurons and microglia. In addition, ESR7 will introduce the 3D bioprinter technology that will be improved together with ESR6. This technology will allow the consortium not only to analyse the interaction between cells but also with the scaffolds that are very relevant to WP3.
WP2 – Molecular biology is dedicated to the learning of molecular techniques for genetically modifying the cells and for cell characterization of differentiated cells by using single cell profiling technologies. So ESR4 will use genetic modification of somatic cells to direct their reprogramming into DAergic neurons, which in turn will be used in WP1 as explained before. ESR1 and ESR2 will use molecular techniques and single cell analyses to characterize the subpopulations of cells obtained after differentiation, as it has previously been described by the partner from KI. The subpopulations obtained, and the characterization of specific markers will be important to culture specific subpopulations into the brain-on-chip system and to analyse the role of specific neuronal afferents and efferents in the disease models in WP1 for ESR1 and ESR3. These studies will be complemented with the technology developed by Plasticell that will be employed by ESR8 to improve differentiation of hPSCs towards specific neuronal subpopulations. In WP2, ESR4 and ESR12 will genetically modify cells by direct reprogramming or for expression of the TVA/rabies system respectively, which will be used in WP3. Similarly, ESR9 and ESR10 will genetically modify mouse model to be used in WP3 to differentiate endogenous NSCs into neurons or oligodendrocytes.
Most of the ESRs and WPs contributes in knowledge or materials for WP3 – In vivo mouse manipulations. In this WP3, ESR9 and ESR10 will use the animals developed in WP2 to analyse the role of endogenous NSCs to regenerate neurons and glial cells. Endogenous cells will also be mobilized by ESR13 using direct reprograming strategies tested in vitro by ESR4 in WP2. ESR11, ESR12, and ESR14 will use information from WP1 to develop cells transplants strategies, using cells, scaffolds and administration techniques developed by ESR1, ESR5, ESR6 and ESR7. The collaboration between ESRs from WP1, WP2 and WP3 will be necessary to achieve the final goals developing advanced therapies to understand NDs and possible treatments.