Studies on Induced Pluripotent Stem Cell Models of Cortical Gyrification and Neurodevelopmental Disorders: Novel Generation of a Ferret Cerebral Organoid
Creators: name: Hickmott, Ryan
Publisher: name: RMIT University
Year: 2025
Subjects: classificationCode: 320606
subject: Regenerative medicine (incl. stem cells)
classificationCode: 320903
subject: Central nervous system
classificationCode: 320902
subject: Cellular nervous system

description: One of the most distinctive and instantly recognisable characteristics of the human brain is its highly folded cortex. Indeed, these folds – or ‘gyri’ (the outer folds) and ‘sulci’ (the inner folds) – are a critical feature of larger mammalian brains, allowing for greater neuronal density and more complex neural networks to manifest. Consequently, this has facilitated the evolution of brains with greater processing power and the emergence of higher cognitive functions. The process of cortical folding is termed ‘gyrification’, and despite the undeniable importance of this process, the genetic and cellular mechanisms that govern it remain largely unknown. Significantly, the shape, size and structure of folds are highly conserved between individuals of the same species, with the primary cortical folds (which emerge first in development) also being highly similar between gyrencephalic species (e.g. dogs, cats, sheep, and ferrets). As such, gyrification must arise due to tightly regulated genetic mechanisms. Moreover, perturbations in the corticodevelopmental process that impact cortical morphology are associated with numerous neurological disorders, ranging in severity. Notable examples include Rett syndrome – a condition associated with acquired microcephaly, disordered cortical folding and major loss of cognitive function.

Historically, the field of neuroscience (and bioscience as a whole) has learned much from the use of mice and rats as model organisms. However, while these species have been useful in the study of corticodevelopment and gyrification, the insights they can provide are ultimately limited due to their brains being lissencephalic (i.e. smooth/lacking in cortical folds). As such, scientists have turned their attention to the study of a naturally gyrencephalic mammal – the ferret. This creature is one of the smallest mammals known to develop a gyrified brain, thus making it ideal from a housing and welfare standpoint. Furthermore, unlike the vast majority of larger mammals that commence gyrification in utero around the start of the second trimester, the ferret is born smooth-brained before undergoing gyrification shortly after. This, along with its generally short gestational period of 42 days and generally contracted corticogenesis timeline, make it an ideal model of gyrification.

While many incredible insights concerning gyrification have been gained from the study of the ferret, the advent of novel technologies in recent years has raised the possibility of alternative models that do not require the ongoing sacrifice of animal life. Specifically, our discovery of the genetic factors required to reprogramme highly specialised cells back into the undifferentiated state – named ‘induced pluripotent stem cells’ (iPSCs) – has revolutionised biomedical research. IPSCs have proven to be invaluable laboratory models as they can be differentiated into virtually any cell type in vitro and used to study development. Furthermore, iPSCs generated from the tissue of patients carrying a genetic disorder can be used to create a highly specific model of that disorder. Strikingly, the advent of iPSCs has greatly advanced our ability to generate highly complex 3D in vitro models (known as ‘organoids’). Indeed, brain – or ‘cerebral’ organoids have already proved incredibly accurate at modelling early corticodevelopment and have also been instrumental in unravelling previously unknown mechanisms of neurodevelopmental disorders. However, it is important to note that this technology is still in its infancy and, as a result, has several limitations. In particular, brain organoids are notoriously poor at recapitulating the late-stage features of cortical development that are typically seen in vivo. This may be due to a variety of factors, including a lack of vascularisation (and subsequent hypoxia-induced core necrosis) and a complete intrauterine environment, the impact of which becomes increasingly evident as organoid development progresses. Furthermore, it has been widely shown that cerebral organoids conserve their species-specific in vivo developmental timeline, as exemplified by the rapid maturation of mouse organoids relative to human organoids. Consequently, a human cerebral organoid that can faithfully recapitulate the cortical folds has not yet been achieved. More broadly, given the rapid speed at which the field of iPSC research has evolved, guidelines on the best practices concerning their generation, validation, and overall use to ensure scientific validity have struggled to keep up. As such, the following studies were conducted as part of this thesis:

Study 1 of this thesis comprises a systematic review of the methodological practices and reporting standards surrounding the generation and use of iPSC models of Rett syndrome.

Study 2 aimed to conduct a spatiotemporal characterisation of key neural progenitor and neuronal markers throughout the developing ferret cortex, with the intention of this data serving as a useful resource for the wider research community. Moreover, this data was used as an in vivo benchmark for the characterisation of the novel model generated in Study 3.

Study 3 aimed to address the limitations of human cerebral organoids by creating for the first time a novel-species cerebral organoid derived from ferret-iPSCs. It was hypothesised that the highly contracted corticogenesis timeline of the in vivo ferret brain would result in a rapidly developing ferret cerebral organoid. It was further hypothesised that a cerebral organoid with a contracted developmental timeline would recapitulate more advanced corticodevelopmental features before factors arising from the in vitro environment impact development, thus enabling the manifestation of cortical folds.