The Impact of Exogenous Stimuli on hiPSC-Derived 3D Neural Models

Replication of biomimetic and clinically relevant neural in vitro models is necessary for physiological and pathophysiological understanding of the human brain. Despite recent advances in human pluripotent stem cell (hiPSC)-derived three-dimensional neural tissue modelling, particularly through organoids and bioprinted constructs, limitations around translation remain. While current models effectively incorporate biochemical stimuli, the specific influences of electrical and mechanical parameters on neural subtype development remain poorly understood. This thesis investigates how electrical stimulation parameters and mechanical cues influence striatal neural subtype development, with implications for both enhanced in vitro modelling and therapeutic applications.

To address this, a working model of a human iPSC-derived striatal organoid model was optimised for heterogeneity, containing medium spiny neurons (MSN) and interneurons of the lateral (LGE) and medial ganglionic eminences (MGE). This revealed that initial seeding density significantly impacts region-specific differentiation, with higher densities preferentially generating LGE-originating neurons. Notably, Activin A supplementation induced the first reported instance of morphogen-driven organoid gyrification, characterised by the expression of surface folds with increased cellular density.

This striatal model was deployed for experimental work in validating a high-throughput electrical stimulation platform and subsequent investigation of frequency-dependant impact on neural subtype development. Hereby, the influence of high and low frequencies (1 – 210 Hz) was assessed, in line with intrinsic neural oscillations and clinically applicable stimulation parameters. Distinct effects on neural organisation and subtype-specific signalling were observed at later developmental stages. Hereby, GABAergic signalling of the striatal interneurons was significantly enhanced after low-frequency (1 Hz) stimulation, combined with an increase in NMDA receptor expression. Higher frequency stimulation enhanced cellular organisation with distinct striatal marker clustering. These frequency-specific responses provide a molecular basis for understanding how varying stimulation parameters differentially affect neural circuits of neural subtypes with distinct electrophysiological profiles.

As a further aspect influencing the suitability and translatability of 3D neural models, the impact of mechanical modulus and biomaterial degradation on neural behaviour was investigated. This was achieved through optimisation of a GelMA/gelatin (4%/4%) hydrogel, and established a platform for examining how substrate properties affect neural development. The modifiable hydrogel composition demonstrated soft mechanical properties conducive to neural tissue development and network formation, while maintaining rheological characteristics compatible with 3D printing parameters. These results enable future studies of mechanical gradients on neural subtype specification.

Taken together, these findings advance the understanding of the biophysical and bioelectric influences on neural development and behaviour in vitro. The identification of frequency-dependent responses, particularly the enhancement of GABAergic signalling through low-frequency stimulation, demonstrates the need to adjust stimulation parameters to the unique cellular profiles of the target tissue. The behavioural impact from mechanical forces exerted on a cellular and molecular level, through the choice of modelling technique, further supports the integration of multiple parameters for enhanced in vitro modelling.

Overall, the integration of select electrical, mechanical, and biochemical stimuli guides the development of more clinically applicable and reproducible 3D neural tissue models. Combined with the evaluation of neural-subtype specific responses to frequency-dependent stimulation in patient-derived models, this offers a foundation for optimising clinical stimulation parameters towards a targeted therapeutic approach that is particularly valuable in neuropsychiatric disorders with high heterogeneity.