Periodic Reporting for period 1 - NINTCORDEV (NIPBL and Integrator function and dysfunction in human cortical development)
Reporting period: 2018-05-01 to 2020-04-30
In most cases, ID is caused by a gene mutation, i.e. the DNA of the patient is altered at a specific location. These mutations often arise spontaneously prior to or shortly after fertilization and cause the affected gene to malfunction. To date, alterations in around 700 different genes have been linked to ID, but it remains unclear when, where and why malfunction of these genes results in cognitive impairment. In addition, it is thought that mutations in many more, not yet identified genes can cause ID. To provide patients and their families with the most accurate diagnosis, prognosis and treatment plan, it is important to be able to accurately pinpoint the causative gene mutation. In addition, recent studies suggest that some aspects of neurodevelopmental disorders may be treatable by drug intervention. Memory defects, repetitive behaviour and seizures were all reduced upon drug treatment in animal models of human disorders, suggesting that such a therapeutic avenue could have important consequences for the quality of life of ID patients.
For a better, faster diagnosis and potential treatment, it is important that we understand which developmental processes and which cells are affected by ID-causing gene mutations. Until recently, it was difficult to study these processes due to lack of an appropriate model system for human brain development. This has changed with the enormous progress that has been made in growing human brain organoids from pluripotent stem cells. These three-dimensional (3D) structures, also called ‘mini-brains’, generate similar cell types along a comparable timeline to embryonic and fetal human brains and thus can be used to study the effects of disease-causing mutations.
In our work, we focused on two genes, NIPBL and INTS8. Mutations in these genes cause related neurodevelopmental disorders, where patients display moderate to severe cognitive delay, seizures and difficulty communicating. We generated human neural stem cells and 3D brain organoids from cells in which NIPBL and INTS8 were mutated and used these to identify the misregulated pathways that can contribute to symptoms displayed by patients.
We analysed the genetic pathways that are disrupted in these mutant NSCs and found that misregulation of important developmental signals, as well as abnormal contacts between cells are potentially responsible for the inability of mutant neural stem cells to generate nerve cells. We also tested whether disrupting INTS8 in the developing mouse brain has an effect on the stem cells or migrating nerve cells, similar to what we found previously for two related genes, INTS1 and INTS11. Unfortunately, these experiments were inconclusive due to our inability to reduce the amount of INTS8.
We set up human brain organoid differentiation, first with control pluripotent stem cells. We let these cells aggregate to form so-called embryoid bodies and directed them towards the neural lineage. After about two weeks, organoids were transferred to an orbital shaker in order to optimally supply them with oxygen and nutrients. Over the course of several weeks to months, the organoids continued to grow and mature. Because success of our initial efforts proved to be quite variable, we adapted our procedure to more consistently generate brain organoids containing neocortical tissue by directly stimulating this fate. We analysed the composition of the brain organoids at different timepoints and found that they contain large, radially organised regions of neural stem cells, surrounded by differentiating nerve cells that were correctly specified according to their birth date. From day 70 onwards we also detected significant amounts of supporting cells, so-called astrocytes. In line with normal fetal development, these cells arise after the majority of nerve cells have been generated. Having successfully established human brain organoid cultures, we are now in the process of generating and analysing brain organoids from stem cells carrying mutations in NIPBL and INTS8.
Overview and dissemination of results
• Addition of patterning factors enables robust and reproducible brain organoid generation.
• Brain organoids contain radially organised stem cell zones surrounded by different subtypes of nerve cells and, at later stages, supporting glial cells.
Results have provided teaching materials for secondary school biology pupils and Medicine degree students.
Results have been communicated to a broader scientific audience through poster and oral presentations at (inter-)national conferences and workshops.
• Mutations in the INTS8 gene render neural stem cells unable to generate nerve cells.
Results have been communicated to local stakeholders through regular internal meetings and workshops.
Results have been communicated to a broader scientific audience through poster and oral presentations at (inter-)national conferences and workshops.