Building Neural in vitro Models with Human Pluripotent Stem Cells: Neuronal Functionality and the Role of Astrocytes in the Networks

Our understanding of human brain development and function is still incomplete. Unfortunately, the field of neuroscience lacks representative human-specific models to accompany the animal studies. Access to human brain tissue for research purposes is limited, encouraging the utilization of novel approaches such as human pluripotent stem cell (hPSC)-derived neurons. Within the last few decades, research on hPSCs has undergone enormous expansion. Growing expectations are aimed at the application of stem cells and their derivatives in regenerative medicine, drug screening and disease modeling.

The main focus of this thesis was to evaluate the potential of hPSC-derived neural cultures in mimicking certain characteristics of central nervous system (CNS) development and functionality. The results were intended to help validate the utility of stem cell models for translational neuroscience applications. For this purpose, the differentiation capacities of hPSC-derived neuronal cells generated with two generally used differentiation methodologies were compared. The functional maturation of neurons following a prolonged differentiation time and optimization of culture conditions was assessed in network-level analyses. The results were complemented with a functional comparison to the widely used rodent in vitro model. Since astrocytes are the cells surrounding neurons and supporting neuronal functionality in the CNS, special focus was also placed on their role in both normal and neuroinflammatory conditions, the latter of which is typical of CNS insults.

The results of this thesis suggest that hPSC-derived neuronal cultures recapitulate many of the characteristics of CNS development in vivo. Specific chemical induction accelerated neural differentiation, leading to high cell purity and yield. Prolongation of the differentiation time increased the proportion of endogenously formed astrocytes and promoted the functionally mature activity type of neurons. Furthermore, the emergence of robust neuronal activity and the long-term maintenance of functional networks were achieved with the selection of defined laminin isoform as a culture substrate. With this improvement, the hPSC-derived networks exhibited time frames and stages of activity development similar to those of their rodent counterparts. However, marked variability was detected in the activity patterns between the rodent and human networks, which could relate to differences in their maturation stage or interspecies dissimilarities. Finally, hPSC-derived astrocytes were exposed to specific inflammatory stimuli. Their response showed distinct characteristics of astrogliosis observed in CNS diseases, and the studied neuronal effects suggested polarization into a neurosupportive phenotype. Established controlled co-cultures with human neurons and astrocytes provide an alternative hPSC-based platform for modeling cell interactions in the context of health and disease.

In conclusion, the work presented in this thesis advances the development of functional hPSC-derived neuronal networks, confirms the role of astrocytes as significant partners in these networks, and encourages their translation into human- specific models for neuroscience research.