A Human Induced Pluripotent Stem Cell Model of Early Neural Development in Neurofibromatosis Type 1

Abstract

Neurofibromatosis type 1 is a monogenic disorder caused by loss-of-function pathogenic variants in the NF1 gene. NF1 codes for the protein Neurofibromin, a negative regulator of rat sarcoma virus (RAS): a critical upstream effector of the RAS-MAPK signalling cascade. Neurofibromatosis type 1 patients also have an increased risk of being diagnosed with autism spectrum disorder (ASD). ASD is a complex heterogenous neurodevelopmental disorder. It is characterised by deficits in social communication and restricted, repetitive behaviours. The presentations of ASD amongst neurofibromatosis type 1 cohorts vary greatly. So far, there have not been any genotype-phenotype correlations identified within in a UK cohort. Human induced pluripotent stem cells (hiPSCs) offer an unrivalled opportunity to study neurodevelopmental disorders in vitro due to their capacity for unlimited self-renewal and trilineage differentiation. Furthermore, they offer the ability to study a disorder within a patients’ genetic background. To date, there have not been any studies utilising neurofibromatosis type 1 patient-derived hiPSCs. Therefore, this project aimed to generate a patient-derived hiPSC model of neurofibromatosis type 1 early neural development. First, a genotype-phenotype correlation analysis was carried out using behavioural measures associated with ASD severity. The analysis did not reveal a significant association between genotypes and behavioural outcomes within a UK neurofibromatosis type 1 cohort. Next, hiPSCs were derived from two individuals diagnosed with neurofibromatosis type 1 and ASD, and an unaffected control relative. After several attempts, isogenic controls were not able to be generated using CRISPR/Cas9. Despite this, a GABAergic neural differentiation protocol was successfully optimised, through activation of Sonic Hedgehog signalling. Furthermore, the early GABAergic neural differentiation of NF1Δ26+/- hiPSCs was characterised. To do this, RNA-sequencing, and other molecular techniques such as western blotting, qPCR and immunofluorescence, were used. Transcriptomic changes that occurred during unaffected control and NF1Δ26+/- neural differentiation were highly correlated, suggesting that the hiPSCs were differentiating similarly. However, the expression of cell adhesion molecules, such as the Contactin and Protocadherin families, were dysregulated in NF1Δ26+/- cGABA rosettes. Furthermore, ZO1 localisation studies showed that NF1Δ26+/- cells organised themselves into rosettes that had significantly lumens. The work described in this thesis, suggested that the early neural development of NF1Δ26+/- cells is disrupted in this model. The findings of this thesis offer an initial glimpse into the in vitro differentiation of patient-derived NF1Δ26+/- hiPSCs and will prove informative for the neurofibromatosis type 1 and RASopathy fields.

Date of Award	1 Aug 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Susan Kimber (Supervisor), Richard Baines (Supervisor) & Shruti Garg (Supervisor)