Generation of functional iPSC-derived astrocytes to model the neurodevelopmental disorder, Rett Syndrome

Abstract

Rett Syndrome (RTT) is one of the leading causes of mental disability in girls. It is a neurodevelopmental disorder caused by mutations in the MeCP2 gene, a ubiquitously expressed transcriptional repressor whose expression is particularly enriched in the central nervous system (CNS). It affects around 1 in 10,000-15,000 girls. It currently has no cure and treatments focus on symptom management; therefore, it is imperative that new methods of modelling RTT are developed that provide translational data. Primary human brain tissue is difficult to obtain, but in order to study neurological disorders it is vital that human brain cells can be studied. Human induced pluripotent stem cells (iPSCs) could provide a possible solution, as it is possible to generate neural cell types from healthy and patient donors. In this thesis two healthy iPSCs lines and a RTT patient iPSC line were used to generate astrocytes, and one healthy iPSC line was used to generate neurons. Astrocytes were consistently generated as highly enriched populations with minimal neuronal contamination. iPSC-derived astrocytes demonstrated spontaneous calcium events and could also respond to glutamate and ATP. In addition, they could remove glutamate from their surroundings and release lactate in response. Thus, this thesis has potentially demonstrated the first astrocyteneuron lactate shuttle in an iPSC-derived system. RTT iPSC-derived astrocytes removed significantly less glutamate from their surroundings compared to astrocytes from a healthy control iPSC line, showing that this model is capable of showing differences in astrocyte function in disease. RTT iPSC-derived astrocytes secreted significantly less lactate in response to glutamate uptake compared to healthy lines. Astrocyte-condition media (ACM) was also generated from the two healthy iPSC and RTT iPSC-derived astrocytes which was used to treat healthy iPSC-derived neurons. It was noted that this had an effect on the neuronal calcium response to glutamate, with healthy ACM potentially increasing this response. RNA sequencing performed on healthy and RTT iPSC-derived astrocytes revealed differentially expressed genes which were mostly enriched in the extracellular matrix organisation pathway. This thesis has shown that functional, enriched of astrocytes can be generated from healthy and RTT iPSCs. These astrocytes demonstrate functions typical of astrocytes in vivo and also reveal differences between healthy and RTT conditions. Therefore, it is feasible that iPSCderived astrocytes are an ideal tool to model the astrocytic component of RTT.