Isolation of MECP2-null Rett Syndrome patient hiPS cells and isogenic controls through X-chromosome inactivation

Abstract

Rett syndrome (RTT) is a neurodevelopmental autism spectrum disorder that affects girls due primarily to mutations in the gene encoding methyl-CpG binding protein 2 (MECP2). The majority of RTT patients carry missense and nonsense mutations leading to a hypomorphic MECP2, while null mutations leading to the complete absence of a functional protein are rare. MECP2 is an X-linked gene subject to random X-chromosome inactivation resulting in mosaic expression of mutant MECP2. The lack of human brain tissue motivates the need for alternative human cellular models to study RTT. Here we report the characterization of a MECP2 mutation in a classic female RTT patient involving rearrangements that remove exons 3 and 4 creating a functionally null mutation. To generate human neuron models of RTT, we isolated human induced pluripotent stem (hiPS) cells from RTT patient fibroblasts. RTT-hiPS cells retained the MECP2 mutation, are pluripotent and fully reprogrammed, and retained an inactive X-chromosome in a nonrandom pattern. Taking advantage of the latter characteristic, we obtained a pair of isogenic wild-type and mutant MECP2 expressing RTT-hiPS cell lines that retained this MECP2 expression pattern upon differentiation into neurons. Phenotypic analysis of mutant RTT-hiPS cell-derived neurons demonstrated a reduction in soma size compared with the isogenic control RTT-hiPS cell-derived neurons from the same RTT patient. Analysis of isogenic control and mutant hiPS cell-derived neurons represents a promising source for understanding the pathogenesis of RTT and the role of MECP2 in human neurons.

Neuronal maturation defect in induced pluripotent stem cells from patients with Rett syndrome

“…Female fibroblast cell lines with no MeCP2 mutations (Detroit 551 or PGP9f) were also shown to become both monoallelic and biallelic iPSCs, suggesting that both the retention and reactivation of X chromosome status during reprogramming are not restricted to MeCP2 mutant cell lines. Recently, the Ellis group generated iPSCs from patients with RTT and similarly observed retention of the inactive somatic X chromosome in iPSCs (39), independently confirming our findings. Pluripotent ESCs derived from inner cell mass are considered to be in the "naïve" state and have two active X chromosomes, whereas pluripotent EpiESCs derived from epiblasts have undergone random XCI and considered to be in the "primed" state (28).…”

A review of Rett syndrome (RTT) with induced pluripotent stem cells

“…Several mutations introduce premature stop codons throughout the gene and are predicted to result in a null allele. Cheung et al and Schanen et al have reported that phenotypic analysis of same genotype controls and neurons obtained from altered hiPS cells are the source to know the development of RTT and denotes the importance of MECP2 in human neurons (33,67). Consistent with RTT animal models and RTT post-mortem human brain tissue (68), both groups detected a decrease in cell soma size of RTT neurons compared with non-affected controls.…”

“…The generation of hiPSCs from RTT patients represents an inexhaustible source for in vitro derived patient-specific neurons, assuming that RTT-hiPSCs can be expanded indefinitely with a normal karyotype and stable genome and the generation of patient-specific hiPSCs from RTT girls has been an area of intense research as several groups have reported the generation of such cells (14,(32)(33)(34)(35)84,85). RTT-hiPSCs generated by different groups have similar properties as they carry pathogenic mutations in MECP2 or CDKL5 and are pluripotent in vitro and in vivo.…”

“…Analysis of isogenic control and mutant hiPS cell-derived neurons represents a promising source for understanding the pathogenesis of RTT and the role of MECP2 in human neurons. These neurons are useful for investigating the pathogenesis of RTT and have potential for use in drug screens and identification of novel compounds for therapy (14,(33)(34)(35)84,85). For this impending to be realized, efficient protocols that direct differentiation into adult stage neurons of defined subtypes may be required (89).…”

“…The first neurodegenerative diseases modelled using hiPSCs were monogenetically inherited, rare and fatal disorders: smooth muscle atrophy (SMA) and familial dysautonomia (FD) (29). iPSCs have been used to model human neuronal diseases, including spinal muscular atrophy, FD, and amyotrophic lateral sclerosis (29)(30)(31) including RTT (14,(32)(33)(34)(35).…”