Modelling cerebellar development using human induced pluripotent stem cells

Thesis

The cerebellum is an important brain region for coordinating a range of motor and non-motor functions, including language. Central to cerebellar function are Purkinje cells, large neurons with complex dendritic trees, that can be identified by high expression of transcription factor FOXP2 from early in development. However, little is known about the function of FOXP2 in Purkinje cell development and previous studies have relied on animal models. Human induced pluripotent stem cell (iPSC)-derived cerebellar organoids provide an accessible model for early human cerebellar development. However, current methods show limited Purkinje cell maturity, and it is challenging to track individual cell populations.

This thesis describes the generation of a fluorescent Purkinje cell reporter line based on FOXP2 expression, to successfully visualise the developing human Purkinje cell population live in cerebellar organoids. In addition, a method for 2D feeder-free dissociated growth of iPSC-derived cerebellar cultures was developed, generating more mature Purkinje cells and widening the application of human cerebellar models to a range of assays.

iPSC-derived cerebellar models were then applied to understanding the role of language-associated gene FOXP2 in the early human cerebellum. Transcriptomic analysis of isolated FOXP2-expressing cells identified putative targets and pathways regulated by FOXP2 in the developing human cerebellum. These targets were further investigated using FOXP2 knock-out organoids, identifying a role of FOXP2 in regulating expression of ZFHX4, a gene associated with speech and intellectual disability. In addition, FOXP2 was found to influence the intracellular calcium response to depolarisation in cerebellar neurons.

Together this work expands the capability of iPSC-derived models for studying early human cerebellar development with a specific focus on Purkinje cells, and highlights potential functions of FOXP2 in the developing human cerebellum.

Authors: Apsley, EJ

• DOI: 10.5287/ora-0nx8pnzek
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: cerebellum; FOXP2; purkinje cells; organoids
• Subjects: Cerebellum; Developmental biology; Induced pluripotent stem cells