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Production and Testing of humAn-derived Neurons and brain organoids: advanced model probing in neurodevelopmental disorders

Periodic Reporting for period 1 - ProTeAN (Production and Testing of humAn-derived Neurons and brain organoids: advanced model probing in neurodevelopmental disorders)

Reporting period: 2018-05-07 to 2020-05-06

The ProTeAN project was an innovative and ambitious project that combined expertise from academic and clinical partners to develop 3D models to study disorders that arise during brain development, at the Center for Neuroscience and Cell Biology, Portugal. Autism spectrum disorder (ASD), intellectual disability and attention-deficit/hyperactivity disorder, affect over 3% of children worldwide. ASD alone is estimated to afflict 1 in 150 children and its annual cost to society has been proposed to run as high as 400 billion euros in the EU. Clinical trials to test drugs for autism have repeatedly failed as previous studies using mouse models may not accurately replicate what occurs in the human body. Therefore, it is critical to develop realistic models that will advance knowledge on brain-related disorders and to test therapeutic strategies.
The goal of the ProTeAN project was to implement the required methodologies of developing 3D models of brain development, named “brain organoids”. This allows the neurons, the building blocks of the brain, to grow in a three dimensional environment and form complex structures as occurs in humans, instead of growing these cells on a plastic surface, that could have been hiding the actual biological processes that are affected during brain development. To generate the brain organoids, we collected tissue samples from deciduous teeth or third molars from ASD patients and controls, through a minimally invasive procedure, in comparison to other more invasive procedures, such as skin biopsies. By using samples directly from patients, we will be able to later test therapeutic strategies, in a personalised medicine approach.
The first milestones we achieved were the establishment of the first dental stem cell biobank in Portugal to study brain disorders and its approval by the Ethics Committees from the Pediatric Hospital of Coimbra and the University of Coimbra. We were able to optimize the required protocols to transform the dental stem cells into induced pluripotent stem cells that can become virtually any cell type of the body. Therefore, we used the induced pluripotent stem cells to obtain neurons arranged in a 3D conformation that would then become the more organized brain organoids. Brain organoids were then characterized to analyze any alterations in the morphology of their neurons and in the connectivity between them. We showed that dental stem cells are a suitable source to generate brain organoids and that these 3D brain models are realistic models that will allow us to understand the mechanisms behind brain disorders and test for personalized therapeutic approaches.
Regarding science communication and outreach, this project was widely disseminated through national and international media and also through outreach activities to raise autism awareness and to educate the community on the scientific advancements achieved in this research field.
In conclusion, the ProTeAN project represented a novel line of research at CNC, the host institution, and it was crucial for the implementation and optimization all the methodologies required for brain organoid development and characterization. We believe that now we are fully equipped to advance this project at a fast pace and be able to perform comparisons between brain organoids from patients and controls, and ultimately identify therapeutic strategies in a personalised medicine approach.
The first milestone we achieved was the establishment of the first dental stem cell biobank in Portugal to study brain disorders. This biobank now includes dental stem cells that were collected in a minimally invasive manner, both from ASD patients and controls. Stem cells from “milk teeth” (ages 5-12) may be collected in young children upon loss of their temporary teeth. Additionally, stem cells from apical papilla (SCAPs) can be obtained from “wisdom teeth” (ages 14-24). The participants’ data was protected through pseudonymization of samples and through the European GDPR guidelines, assured by collaborators at the Biomedical Rights Center, University of Coimbra. This biobank was also a critical resource for the ITN “Syn2Psy”, funded by the European Commission.
The dental stem cells were then expanded and characterized by flow cytometry, to identify proteins on the surface of the cells, and qRT-PCR, to detect the proteins that are being transcribed in the cells, and we identified the presence of stem cell markers. Besides, the dental stem cells displayed typical morphology - small and spherical. We optimized the required protocols to transform the dental stem cells into induced pluripotent stem cells and then used those to generate 3D brain organoids, according to literature. Brain organoids were characterized by their neuronal morphology and electric properties of neurons and circuits formed within the brain organoids. At 3 months, brain organoids expressed mature neuronal markers, indicating their potential to be used as robust models to study human neurodevelopment. Neurons in 6-month organoids showed complex electric activity and they are competent to fire action potentials when stimulated with current.
We showed that dental stem cells are a suitable source to generate brain organoids and that 3D brain cultures are realistic models that will allow to understand the mechanisms behind brain development disorders and test for personalised approaches.
This project was widely disseminated through the media. Besides, the fellow participated in science communication activities such as Brain Awareness Week, European Researchers Night, to raise autism awareness and to educate the community on the scientific advancements achieved in this research field, through oral communications and outreach activities. The fellow participated in interviews such as a science dissemination TV program from the national TV channel SIC, which reached an audience of 1.2 million people, in Portugal.
This project was highly innovative since we generated brain organoids from dental stem cells instead of using more invasive sources of stem cells. Overall, this approach presented several advantages:
1. Cell-based models that closely mimic human diseases
2. Minimally invasive
3. Allows for personalised therapeutic strategies
4. Ethical concerns with animal experimentation are avoided
The results of the ProTeAN project provided evidence that human-derived models represent realistic models of brain development to obtain translatable results into the clinical setting. This will pave the way for innovative diagnostic and treatment models which encourages personalised interventions to improve health outcomes. This will contribute to the improvement of health and well-being of all afflicted with these disorders and to significantly reduce overall social and economic costs. Besides, this project strengthened academic and clinical collaborations, promoting high quality research.
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