In vitro model of Fragile X Syndrome using naïve iPSCs

Project Information

NeuroStemX

Grant agreement ID: 839753

DOI

10.3030/839753

Project closed

EC signature date 12 April 2019

Start date 1 June 2019

End date 31 August 2021

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 183 473,28

EU contribution

€ 183 473,28

Coordinated by

UNIVERSITA DEGLI STUDI DI PADOVA
Italy

Periodic Reporting for period 1 - NeuroStemX (In vitro model of Fragile X Syndrome using naïve iPSCs)

Reporting period: 2019-06-01 to 2021-05-31

Summary of the context and overall objectives of the project

Fragile X Syndrome (FXS) is the leading cause of inherited mental retardation, intellectual disability, and is the single most common cause of autism spectrum disorders. The trinucleotide repeat expansion, methylation and epigenetic silencing of fragile mental retardation 1 (FMR1) gene promoter leads to the loss of the corresponding protein. However, the timing and cell type affected by FMR1 silencing in the brain of FXS patients are still unknown due to the absence of reliable in vivo and in vitro models.
The general objective of NeuroStemX project was the investigation of the genetic and epigenetic modifications of FMR1 locus during the ealy embrionic stage of neural development by means of an in vitro innovative patient-specific human model. Human model based on naïve iPSCs (which represent the in vitro counterpart of pre-implantation pluripotent stem cells) has the potential to reproduce the human neural development from an early pluripotency stage to a late neural differentiation in neurons and brain organoids, reproducing in vitro the molecular events that lead to FMR1 silencing in vivo in human FXS embryos.
NeuroStemX project addressed some of the most crucial problems related with FXS pathogenesis and modelling using an original approach based on the use of a new innovative technique to derive naïve iPSCs from somatic cells (skin fibroblasts) developed in Prof. Elvassore group and the generation of FXS neurons with various approaches that I learned during my PhD and PostDoc periods, in combination with the micro technologies based on microfluidics mastered in Prof. Elvassore’s team.
The NeuroStemX project focused on 2 specific aims: 1) identifying the timing of FMRP silencing during neural development using naïve iPSCs and 2) establishing a reliable in vitro system to model neuronal defects observed in FXS patients.
We hypothesize that the investigation of the early phase of human neural development in human model of Fragile X Syndrome (FXS) are fundamental for the establishment of a targeted therapeutic strategy.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

During this action we successfully collected human fibroblasts from 4 FXS male patients and generated both naive and primed iPSCs. I have generated 12 primed iPSC lines (11 from FXS and 1 control) and 9 naïve iPSCs (5 from FXS and 4 from control) which will be available to other labs upon Material Transfer Agreement (MTA).
I have assessed the expression of FMR1 in naïve iPSCs and found that, starting from FXS fibroblasts not expressing FMR1, reprogramming them to primed iPSCs still not expressing FMR1, we can reset these cells toward naive pluripotent state and re-establish the expression of the FMR1 protein.
I set up an innovative expansion method for naïve cells in feeder free conditions and in 3D, which is functional for any further application of human naïve cells and in particular to their differentiation into 3D cortical brain organoids, which is functional to their differentiation into brain organoids.
I have followed FMR1 expression during naïve differentiation into 3D cortical brain organoids and assessed that during the naive to primed transition the FMR1 gene is still expressed, confirming that the silencing of FMR1 occurs later during development. I followed cortical brain organoids up to 30 days confirming that the silencing should occur later during development. Longer time point are under evaluation.Then I set up a differentiation strategy based on transcriptional programming to obtain neurons from primed iPSCs using a non integrating mRNA based overexpression of NGN2, which has been described in an OpenAccess peer reviewed paper (Tolomeo A., Laterza C. et al., 2020).
All these results have been disseminated through the participation to one international workshop and two conferences as invited and selected speaker, respectively.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The generation of a naive based 3D brain organoid in vitro model is a beyond the state of the art achievement, since the maintenance and differentiation of naive iPSCs represent still an opened issue and their differentiation into 3D cortical brain organoids has never been reported so far from naive iPSCs. In addition the dynamic of FMR1 expression from naive stage to primed and to 3D cortical brain organoids is something that was still missing in the field. The discovery of the timing and cell specificity of FMR1 silencing that this model will allow, will be instrumental to provide crucial knowledge aimed at finding new pharmaceutical compounds able to revert the disease phonotype associated with FXS.

Outline of NeuroStemX project