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Gontan Group

Department                           Developmental Biology

Principal investigator           Cristina Gontan

E-mail address                       m.gontanpardo@erasmusmc.nl

 

Restoring X Chromosome Inactivation in Female iPSCs through Naive Resetting and Capacitation

Suitable as a BEP? No

Suitable as a MEP? Yes

Suitable as an Academic Research Project? No

Techniques:

  • Human iPSC culture, naive resetting, and neuronal differentiation,
  • Molecular and Imaging techniques to study X chromosome regulation.
  • qPCR and RNA sequencing
  • Immunofluorescence and Western blot.
  • RNA FISH will be used to assess X chromosome inactivation status
  • CRISPR-based approaches will allow manipulation of RNF12 and REX1

The discovery of human inducible pluripotent stem cells (iPSCs) has opened new possibilities for regenerative medicine and disease modeling. However, the loss of epigenetic integrity, particularly the erosion of X chromosome inactivation (XCI) in female cells, limits their use. In this project, we have established a culture pipeline to restore proper XCI by resetting female iPSCs to a naive state and then re-priming them. We aim to assess how this resetting affects gene expression during neuronal differentiation and to study the roles of two key XCI regulators, RNF12 and REX1, in this process. The goal is to enable the reliable use of female iPSCs for modeling X linked neurodevelopmental disorders and advancing personalized approaches to human disease.

Further reading (click to link to article)

DOI: 10.1038/nature11070

DOI: 10.1126/sciadv.adg1936

(Example) projects submitted by lab in past years

(2024-2025) How in RNF12 involved in the X-linked intellectual disability syndrome TOKAS?

Supervisor: Kyra Swildens, k.swildens@erasmusmc.nl

Previously, our lab has demonstrated that the X-linked E3 ubiquitin ligase RNF12 is implicated in X chromosome inactivation (XCI). In addition to its role in XCI, pathogenic variants in RNF12 in males have been shown to cause the neurodevelopmental disorder Tonne-Kalscheuer Syndrome (TOKAS), defined as X-linked intellectual disability (XLID) in combination with behavioral anomalies and/or congenital malformations. Heterozygous female carriers of RNF12 mutations display extremely skewed XCI, defined as >90% of the cells containing the same inactive X chromosome (Xi), with the RNF12 mutant allele on the Xi. As it remains unknown how RNF12 mutations induce TOKAS, we focus on elucidating the molecular basis of RNF12-mediated XLID in human induced pluripotent stem cell (iPSC)-derived neuronal networks.

Techniques

  • iPSC culture
  • Neural differentiations
  • CRISPR/Cas9 (cloning & nucleofection)
  • FACS
  • PCR
  • qRT-PCR
  • WB
  • Immunofluorescence

Further reading

Frints, S.G.M., et al. (2019). Molecular Psychiatry, 24, 1748–1768. DOI: 10.1038/s41380-018-0065-x.