UCL Great Ormond Street Institute of Child Health


Great Ormond Street Institute of Child Health


Developing 3D patient-derived neuronal models to elucidate neurodevelopmental defects

Project title 
Developing 3D patient-derived neuronal models to elucidate neurodevelopmental defects in Spinal Muscular Atrophy

Supervisors names
Giovanni Baranello
Serena Barral

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease due to the lack of SMN protein which leads to progressive degeneration of motor neurons in the spinal cord. The majority of SMA 1 children, the more severe end of the spectrum, may exhibit neurodevelopmental comorbidities1. Elevated SMN levels are required during foetal and early postnatal brain development2. Animal studies have shown that growth, development and function of specific brain regions are disrupted when SMN levels are reduced, in particular hippocampus and cerebellum3. Although novel treatments are becoming increasingly available to patients, it is still unclear whether the observed brain-related comorbidities may be equally targeted. 

Recent advances in induced pluripotent stem cells (iPSCs) and 3D culture systems have led to the generation of “brain” organoids that resemble several areas of the human brain. Organoids can recapitulate aspects of in vivo brain architecture and physiology and therefore offer new possibilities for modelling neurological disorders and for the development of new therapeutic approaches. 

The overarching objective of this project is to investigate the cellular and molecular mechanisms underlying the disruption in brain development in SMA using patient iPSC-derived neuronal models. 

The objects of this project are:

1. Generate SMA type 1 patient-derived iPSCs and CRISPR-Cas9 isogenic controls.
2. Develop SMA hiPSC-derived cortical assembloids and hippocampal spheroids from SMA type 1 patient-derived iPSC lines and investigate neurodevelopment disruption.  
3. Analyse neurophysiology neuronal maturation and network formation during development in the generated 3D patient derived models. 

1. SMA type 1 skin fibroblasts will be collected from three patients and reprogrammed into pluripotent cells using a Sendai Virus-based protocol. Derived iPSC lines will be fully characterized using a panel of established technologies (Sanger sequencing, single nucleotide polymorphism array, immunofluorescence technology, real time PCR, spontaneous in vitro differentiation). iPSC isogenic control lines will be generated using CRISPR/Cas9 technology and further characterized for chromosomal integrity. 
2. SMA patient-derived, healthy age-match control (already available) and isogenic iPSC lines will be differentiated in cortical assembloids and hippocampal spheroids. Cellular identity will be investigated using immunofluorescence and quantitative RT-PCR for a panel of neural progenitors and mature neurons markers. SMN protein expression will be analysed via immunofluorescence and western blotting.
3. Developmental defects will be evaluated using immunofluorescence analysis for proliferative and cell death markers, and neural progenitor/neuronal mature specific proteins. Synaptic maturation and neuronal network formation will be analysed using relevant pre- and postsynaptic proteins (e.g. VGAT, GEPHYRIN) and promoter-specific lentiviruses (LV-hSyn-RFP and LV-mdlx5/6-GFP) to evaluate neurite arborization complexity. Bulk RNA sequencing will be performed comparing patient to control derived organoids to identify alteration in gene expression. 
4. SMA and control-derived organoids will be analysed using electrophysiology, i.e. single cell patch-clamp to measure neuronal excitability, and local field potential to evaluate neuronal network formation.  

Months 0-9: Generation of SMA type 1 patient-derived induced pluripotent stem cells (iPSCs) and CRISPR-Cas9 isogenic controls.
Months 10-20: Development of SMA hiPSC-derived cortical assembloids and hippocampal spheroids from SMA type 1 iPSCs and characterization.
Months 20-36: Investigation of neurodevelopment disruption in SMA patient-derived cortical assembloids and hippocampal spheroids.

1.    Polido GJ. Dem Neuropsychol. 2019;13(4):436-443. 
2.    Ramos DM. J Clin Invest. 2019 1;129(11):4817-4831.
3.    Wishart TM. Hum Mol Genet. 2010

Giovanni Baranello: g.baranello@ucl.ac.uk; Serena Barral: s.barral@ucl.ac.uk