Poster Presentation 26th ACMM “2020 Visions in Microscopy”

Immunofluorescent and light sheet characterisation of Rett syndrome cerebral in-vitro models (#202)

Florencia Haase 1 2 3 , David Duruchukwu 3 , Ian Alexander 4 5 , Leszek Lisowski 6 , Laurence Cantrill 4 7 , Patrick Tam 2 8 , Wendy Gold 1 2 3 4 5
  1. Kids Neuroscience Centre, Kids Research, Sydney Children’s Hospitals Network, Westmead, NSW, Australia
  2. The University of Sydney, School of Medical Sciences and Discipline of Child and Adolescent Health, Faculty of Medicine and Health, Sydney, NSW, Australia
  3. Molecular Neurobiology Research Lab, Kids Research, Sydney Children’s Hospitals Network, Westmead, NSW, Australia
  4. Discipline of Child and Adolescent Health, Sydney Medical School, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia
  5. Gene Therapy Research Unit, Children’s Medical Research Institute and The Children’s Hospital at Westmead, Westmead, NSW, Australia
  6. Translational Vectorology Group, Children’s Medical Research Institute, University of Sydney, Sydney, NSW, Australia
  7. Microscope Facility, Kids Research, Sydney Children’s Hospitals Network, Westmead, NSW, Australia
  8. Embryology Research Unit, Children’s Medical Research Institute, University of Sydney, Sydney, NSW, Australia

Introduction: Rett syndrome (RTT) is a severe neurodevelopmental disorder that mainly affects girls and is predominantly caused by mutations in the X-linked MECP2 gene. Despite over 40 clinical trials, no effective cure has yet been found. Gene therapy is an obvious next choice of treatment option. Delivery of the therapy may be achieved using Adeno Associated Viruses (AAVs) with selective tropism for genetically defective cells over wild type cells. In this project, brain organoids from patient cells with MECP2 mutations are used to model Rett syndrome and this model will be used to characterise and validate the efficacy of the vector delivery system for gene therapy.

Methods: Induced pluripotent stem cells (iPSCs) reprogrammed from cells sourced from a male RTT patient were cultured in 2D format and 3D organoids. The neuronal and glial cell populations were characterised by immunofluorescence, light sheet microscopy and RT-qPCR, comparing the mutant cell types to their isogenic counterparts.

Results: iPSCs have been characterised by Immunofluorescence and RT-qPCR. Neurons were generated from the iPSCs in 2D culture. Brain organoid protocols were optimised and cell types in the organoids were characterised. Methods of imaging cellular morphology and fluorescent protein marker by light sheet microscopy are being validated.

Discussion: The outcome of this project will enable the disease modelling of neurological diseases that impact on the function of neurons and glia in the brain and provides an effective platform for developing gene therapy to Rett syndrome patients.