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.
