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Training on Advanced Stem Cell Technologies in Neurology

Periodic Reporting for period 1 - ASCTN-Training (Training on Advanced Stem Cell Technologies in Neurology)

Reporting period: 2018-10-01 to 2020-09-30

ASCTN-Training is addressing existing gaps within Human Stem Cell-based Neuronal disorders (NDs) Modelling (NDM) for research to develop new medicines for the treatment of neurological disorders (e.g. Parkinson´s (PD), Huntington´s (HD), Alzheimer’s (AD) and Demyelination’s (DM) diseases). These disorders have a high prevalence and are associated with short- and long-term impairments and disabilities with high emotional, financial and social burden to the patients, their families and their social network. NDs are mostly incurable and affect millions of people.
Building valid and robust in vitro models for brain circuitry and diseases will accelerate drug discovery and enhance efficacy of neurotoxicology testing. ASCTN-Training advanced technologies based on brain-on-chip and 3D minibrains will reproduce neural interactions and cell-cell contacts including not only neurons but also astrocytes, oligodendrocytes and microglia. In addition to disease modelling, in vivo strategies to better analyse stem-cell based treatment of NDs may be one of the biggest hopes for many human conditions.
The main objective of ASCTN-Training is to train a new generation of 14 highly inter-disciplinary early stage researchers (ESRs), capable of mastering techniques and methods across the borders of their own disciplines, and to recognize ideas applicable to their own work. Moreover, through experience of learning with and from those of other professions, ASCTN-Training equips ESRs with unique leadership qualities, with and respect for others’ cultures and to be well prepared for work on teams and in settings where multidisciplinary collaboration is key to success achieve the specific objectives:
1.- To model NDs using brain-on-chip and 3D technologies.
2.- To obtain specific neural progenitor (NPCs) subpopulations for in vitro and in vivo studies.
3.- To develop new scaffolds to improve integration, differentiation and connectivity of NPCs in vivo.
Besides the affection of the project by the pandemic, all ESRs have been working hard on their respective projects. They have learned the needed technologies already available at the host laboratories and have developed techniques and tools that will be useful to achieve the main project goals. At cellular level, all ESRs have obtained, expanded and cryostoraged the needed cell lines that include models for PD and HD. Using these cell lines, new differentiation protocols have been established and improved to obtain human pluripotent stem cells (hPSCs)-derived striatal or dopaminergic neurons, the most affected in HD and PD respectively; and microglial cells. The initial observations suggest a cell-autonomous effect of mutant HTT on microglia status and activity supporting the use of iPSC-derived microglia as a model to study neuroinflammation in HD.
Neuronal differentiation protocols have been adapted to growth on microcarriers, a necessary step for using the high-throughput CombiCult® screening platform. In addition, cell sorting strategies have been developed to select specific neuronal subpopulations.
Some ESRs have also been working on genetic constructs such as lentiviruses for calcium sensors to detect neuronal activity and modified rabies viruses to analyze neuronal connectivity. Molecular constructs for cell reprograming have also been developed and tested for converting glial cells into in cell culture. Molecular constructs have also been developed to follow the astrocyte-to-neuronal conversion or to expand neural stem cells in the brain.
To obtain more complex brain models, some ESRs have been working on developing brain-on-chip systems and 3D brain organoids. Two different brain on chip designs have been developed: the first consists on a chip composed of four open chambers, two for cell culture connected by microchannels and two media reservoirs; and the second microfluidic chip has been design to hold organoid cultures. Cerebral organoids we have also successfully generated and improved by adjusting the culture conditions; elongating the neural induction state, reducing the oxygen tension and subjecting the tissue to an electrical field during the early stage of the organoid development. In addition, a tunable hydrogel was also produced for the 3D neuronal cultures and bioprinting.
For in vivo approaches, mouse/rat models of AD, HD and multiple sclerosis (MS), were successfully established in the respective labs. Expansion of neural stem cells with molecular tools was performed in the AD mouse model and preliminary data suggest a rescue in some AD-associated cognitive deficits. Similarly, we have shown that the overexpression of 3 transcription factors by adeno-associated viruses (AAV) is able to reprogram astrocytes into neuronal cells in the brain.
Based on the above objectives and developments, we envisage a future scenario beyond the project duration (see project timeline in Fig. 1) that enables trained ESRs to create and develop their own ideas and advanced stem cell technologies for NDs.
Through ASCTN-Training, the ESRs gain an impressive set of skills and expertise in a field that will be in desperate need of highly capable scientists:
• Innovative and translational research.
• Highly individualized and monitored skill development plans.
• Trans-sectorial knowledge.
• Extensive team building, project management and communication skills.
The ASCTN-Training education is undoubtedly made deeper by high level of interaction and complementarity of interests between the academic and non-academic partners and develop a blueprint for structuring doctoral training at the European level. All ESRs receive research training in both academia and industry to ensure significant impact on public-private sector collaboration. ASCTN-Training ensures technological and commercial exploitation of the project results. European Brain Council (EBC) provides ESRs with inter-disciplinary training that will help them to grasp how their future NDs research can be potentially translated into practice and policy that will address unmet societal needs.
The knowledge to be generated in ASCT-Training falls within the global regeneration market, mainly driven by the rapidly ageing population and associated neurological diseases. This is where some participating SMEs’ activities fall within. The interest for new investment in tissue engineering and regenerative medicine is considerable, both among private investors and industrial players. The technology to be generated by the consortium has the potential to drive the further development of a company to bring the final product on the market and become the in vitro benchmark.
Timeline for ASCTN-Training with major outcomes during the project duration and beyond.