Human iPSC derived neural cells as models of brain development and as tools in pharmaceutical drug discovery

Abstract

Human brain evolution has resulted in a cognitive superiority compared to all other animals. Unique cortical structures and expanding progenitor populations have been associated with the possibility for developing a highly folded neocortex and expanded surface area, which is linked to cognitive function. Alongside the development of the neuronal population there has been a remarkable evolution of a second population of brain cells called astrocytes. Astrocytes, which historically have been viewed as the glue of the brain, are now considered as a major regulator of brain homeostasis and neuron communication. Hypothesized to meet the increased complexity of neuronal sub-populations astrocytes have become highly diversified. Specific astrocytes can only be observed in higher primates and generally comprises a more advance form and structure enabling a single astrocyte to support a higher number of neurons. Additionally, it has been shown that human astrocytes can improve cognitive function in mice, an observation signifying the importance of astrocytes in human brain evolution. However, increased complexity is accompanied by biological errors resulting in human specific diseases. Disease mechanisms linked to human biological traits poses challenges when trying to uncover and develop treatments against its pathological conditions using animal models. With decreasing drug developmental programs in the pharmaceutical industry targeting neurological and psychiatric diseases there is a need to improve and accelerate drug discovery in this area.

Studying cellular functions of the human brain is challenging partly due to limited accessibility of brain tissue. Historically, the main source of cells was derived from healthy tissue following surgical procedures as well as post-mortem and fetal tissue. However, since the discovery of induced pluripotent stem cells, having the potential to generate any cell type in the body, accessibility to neural like cells has changed dramatically. Common strategies for acquiring neurons and astrocytes from pluripotent stem cells are to try and mimic the naturally occurring embryonic development. However, this requires the establishment of defined and detailed protocols instructing the cells how to develop and becoming the cell type of interest. Neurons follow a step-wise development program which have been uncovered and in great parts mimicked in the lab. However, whether this step-wise developmental progression holds true for astrocytes is yet to be defined.

The aim of this thesis was to develop a protocol to derive astrocytes from human induced pluripotent stem cells (hiPSC) and benchmark them against current models available for the pharmaceutical industry. Moreover, the project aimed to establish hiPSC derived neuronal and astrocyte models in a pharmaceutical setting to investigate their potential contribution in drug development. The characterization of four astrocytic models in comparison to a neural stem cell and nonneural model showed expected astrocyte specific characteristics. However, large differences in gene expression and astrocyte associated functions indicated a large heterogeneity among models which was also demonstrated in drug response stimulations. This clearly implies that discovery of new chemical compounds for further drug development will be context dependent, having identification bias towards the model of choice. Moreover, thorough characterization and diverse applications demonstrated a very robust and reproducible protocol for the generation of hiPSC derived astrocytes, a feature naturally critical if utilized in pharmaceutical assays. Finally, in addition to improved functionality compared to conventional models, hiPSC derived astrocytes show developmental traits linked to embryonic development increasing translability and model relevance. Furthermore, in a proof of principle study hiPSC derived neurons were shown to be able to predict unwanted side effect of a drug used to prevent excessive blood loss from major trauma or surgery. The drug is believed to affect specific neurons resulting in involuntary seizures. Besides demonstrating receptor activity of the drug, human iPSC derived neurons were shown to be applicable in the development of new drugs lacking this side effect. Finally, this was performed using a label-free and simple method which is highly applicable for drug screening.

In conclusion this thesis presents a protocol for the derivation of an astrocytic model having translatability to the embryonic development and possesses several cellular functions observed by astrocytes in vivo. The application of hiPSC derived neurons and astrocytes in a pharmaceutical setting demonstrate that they can make a significant contribution in drug discovery.