Brain development in a dish

This thesis shows the path I took in the quest for modeling the brain, as the title says, in a dish. The introduction of induced pluripotent stem cells (iPSCs) as means to research biological processes has opened up whole new fields of study and unprecedented possibilities of generating human cells in vitro. iPSCs have due to their pluripotent state the theoretical ability to be an unlimited source of cells, capable to generate any cell of the human body. They can be derived from somatic tissue and therefore used to generate disease specific cells.

In paper I, we show the generation of disease specific neural stem cells from lissencephaly patients carrying a mutation in doublecortin (DCX). Lissencephaly is a disorder that affects cell migration, a phenotype we could replicate in our model. Furthermore, we show a defect in proper neurite outgrowth that we could rescue through the SLIT/ROBO pathway, and a prolonged proliferation. Together, showing the feasibility of using iPSC derived neural stem cells to model human neurodevelopmental disorders such as lissencephaly.

In paper II we explored the role of p53 in neurodevelopment using both iPSC derived neuroepithelial stem cells (NES) and 3D brain organoids.Here we used lentiviral knockdown of tumor protein (TP53) in both the NES and iPSCs to follow neural development. We show the importance of p53 in maintaining genomic stability of NES cells and the involvement in maintaining the metabolic balance, resulting in lower expression of oxidative phosphorylation (OXPHOS) genes, shifting the cells to a more glycolytic state. Further differentiation into neurons showed an increased pace of differentiating. When placing p53 in the context of brain organoids, we show the reduction of TBR2+ intermediate progenitor cells (IPCs) and TBR1+ neurons. Analyzing metabolic gene profile also revealed the downregulation of OXPHOS related genes, indicating the regulation by p53 of the metabolism in brain organoids.

In the manuscript, we explored amore metrological aspect for modeling neurodevelopmental diseases. By generating brain organoids and evaluating the neuronal activity we show the feasibility for future drug screening. Furthermore, we could perform an in vitro transplantation of NES cells and show their differentiation, making a small step towards an alternative for in vivo transplantation.

In summary, this thesis shows some of the potential of in vitro brain development and the applications these brain models can be used for.

List of scientific papers

I. An in vitro model of lissencephaly: expanding the role of DCX during neurogenesis. M Shahsavani, RJ Pronk, R Falk, M Lam, M Moslem, SB Linker, J Salma, K Day, J Schuster, B-M Anderlid, N Dahl, FH Gage, and A Falk. Molecular Psychiatry.
https://doi.org/10.1038/mp.2017.175

II. p53 controls genomic stability and temporal differentiation of human neural stem cells and affects neural organization in human brain organoids. Ana Marin Navarro#, Robin Johan Pronk#, Astrid Tjitske van der Geest, Ganna Oliynyk, Ann Nordgren, Marie Arsenian-Henriksson, Anna Falk, and Margareta Wilhelm. Cell Death and Disease. #Authors contributed equally.
https://doi.org/10.1038/s41419-019-2208-7

III. Generation of a 2D and 3D System to Understand Neuropsychiatric Disorders. Pronk R.J., Hauger P.C., Marin Navarro A, Wilhelm M, and Falk A. [Manuscript]