Cellular models of human brain disorders from skin to brain

Abstract

Studying human brain development and disorders is very challenging. In the absence of comparable model organisms, human-related models, and limitations to obtain live cells from the human brain, induced Pluripotent Stem Cell (iPSC) technology, in particular, provided a unique tool to study the disease mechanisms and investigate potential treatments. The main goal of this thesis was to study neurological disorders and explore novel mechanisms underlying the diseases.

We have generated and characterised patient and healthy control iPSCs using Sendai virus as a safe non-integrating method to keep the host genome intact. We have then shown an example of a standardised culture condition by using recombinant spider silk coating for iPSCs and Embryonic Stem Cells (ESCs) cultivation in 2D and 3D formats. Healthy Pluripotent Stem Cell lines cultured on recombinant spider silk displayed the typical stem cell morphology with the expression of pluripotent stem cell markers. Considering the xeno-free culture coating and compatibility with the host immune system, the spider silk, can provide an optimal routine culture system for pluripotent stem cells and future iPSC based therapies.

Patient and healthy iPSCs were neurally induced to generate intermediate, expandable Neuroepithelial Stem Cell (NESC) lines. Morphologically, the derived NESCs displayed rosette structures in culture and expressed key neural stem cell markers. Also, the transcriptomic profile of derived lines displayed similarity that proposes the homogeneity of our NESC population despite patient genomic background variation. We have used a healthy control NESC line to model Alzheimer’s Disease (AD) in a dish by exogenous application of amyloid beta oligomers in differentiating culture. Interestingly, AD-related phenotype, dis-localisation of phosphorylated P21-activated kinases (pPAK) protein, was recapitulated only in 3D culture.

We further attempted to identify mechanisms underlying Type 1 Lissencephaly from patients carrying Doublecortin (DCX) mutations. Differentiating patients’ cells with dis-regulation of DCX expression exhibited a migrational defect, aberrant neurite outgrowth, and fewer dendrite bundles. In addition, we have dissected a proliferation phenotype of DCX mutant cells upon differentiation. Data suggests an indispensable role of DCX expression at an early stage of neural development which allows proper differentiation and migration.

Here we have shown that it is possible to make a robust cellular model to study human brain disorders using patient-specific cells. Identification and verification of disease phenotypes and exploring the underlying mechanisms could provide valuable insights into these complex disorders. These insights may offer novel approaches to therapeutic applications taking scientists one step closer to treating the patients. This study underlines the importance of cellular-based models, 2D and 3D, that can be used to study typical development as well as disease mechanisms.