Optimized clonal isolation and immortalization of Rett syndrome patient fibroblasts for in vitro modeling of MECP2 mutations

TL;DR

Researchers developed a method to create long-lasting fibroblast models of Rett syndrome, allowing better study of MECP2 mutations and their effects.

Contribution

A scalable workflow for isolating and immortalizing clonal fibroblasts with specific MECP2 mutations is introduced.

Findings

Immortalized fibroblasts retained clonality and skewed X-chromosome inactivation.

Mutant clones showed RTT-like features such as histone hyperacetylation and oxidative stress dysregulation.

The method supports in vitro modeling of X-linked disorders and drug screening.

Abstract

Rett syndrome (RTT) is a severe neurodevelopmental disorder primarily caused by mutations in the X-linked MECP2 gene. Patient-derived fibroblasts serve as a practical system to study systemic aspects of RTT, however, their limited proliferative capacity due to cellular senescence poses a significant challenge. In this study, we establish a robust workflow to isolate and immortalize clonal fibroblast lines from female RTT patients heterozygous for two distinct MECP2 mutations (c.705delG and 1155del32). By employing single-cell cloning prior to hTERT-mediated immortalization, we generated stable, proliferative fibroblast clones with verified clonality and severely skewed X-chromosome inactivation. Wildtype clones exclusively expressed full-length MeCP2 protein, whereas mutant clones exhibit truncated or absent MeCP2. Immortalized lines retained elevated hTERT expression and sustained proliferation even at late passages. Notably, mutant clones recapitulated key molecular features of RTT, including histone hyperacetylation, dysregulation of oxidative stress markers, and aberrant expression of key signaling genes. Our approach provides a scalable and renewable in vitro model that faithfully captures critical aspects of RTT pathology and offers a complementary platform to existing animal and iPSC based systems. Moreover, the approach is adaptable for studying other X-linked genetic disorders and supports applications in mechanistic research and preclinical drug screening.

The online version contains supplementary material available at 10.1038/s41598-025-19619-x.

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