ILANIT 2020

Generation of induced neurons from isogenic clones of Fragile X human embryonic stem cells


Lital Gildin 1,2 Dalit Ben-Yosef 1,2
1Wolfe PGD Stem Cell Lab, Racine IVF Unit, Lis Maternity Hospital Tel-Aviv Sourasky Medical Center, Israel
2Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Israel

Introduction – Fragile X syndrome (FXS), a common cause of intellectual disability and autism, results from epigenetic silencing of the FMR1 gene in full mutation patients. During neural differentiation FMR1 silencing leads to impaired cognition. However, the effect of FMR1 mutation on neuronal function is still unclear.

Methods – Clones of male FX-hESC (LisFX6) were isolated by low density seeding. PCR based CGG repeat number assay was performed. hESC clones were directly differentiated into induced neurons (iNs) by overexpress of NEUROG-1. Neuronal identity of the iNs was determined by the expression of neuronal markers and FMRP (by IF and qRT-PCR).

In addition, morphology was analyzed by imageJ and Incucyte analysis software in order to characterize the difference between wild type and full mutation iN clones.

Results – A total of 40 subclones were isolated, which express FMR1 in the undifferentiated stage. iNs clones were positive for all neuronal markers, but while wild type and permutated iNs express FMRP, the full mutation iNs silenced the gene. Significant changes in soma area, soma perimeter, soma roots and neurite branch point were seen already at day 7 after differentiation.

Conclusions – Wild-type and full-mutated subclones which share the same genetic background and differentiated into matured iNs mimic the natural phenotype and serve as a great platform for studying the effect of FMR1 loss on the development of mental retardation.