Isogenic Pairs of Wild Type and Mutant Induced Pluripotent Stem Cell (iPSC) Lines from Rett Syndrome Patients as In Vitro Disease Model

Abstract

Rett syndrome (RTT) is an autism spectrum developmental disorder caused by mutations in the X-linked methyl-CpG binding protein 2 (MECP2) gene. Excellent RTT mouse models have been created to study the disease mechanisms, leading to many important findings with potential therapeutic implications. These include the identification of many MeCP2 target genes, better understanding of the neurobiological consequences of the loss- or mis-function of MeCP2, and drug testing in RTT mice and clinical trials in human RTT patients. However, because of potential differences in the underlying biology between humans and common research animals, there is a need to establish cell culture-based human models for studying disease mechanisms to validate and expand the knowledge acquired in animal models. Taking advantage of the nonrandom pattern of X chromosome inactivation in female induced pluripotent stem cells (iPSC), we have generated isogenic pairs of wild type and mutant iPSC lines from several female RTT patients with common and rare RTT mutations. R294X (arginine 294 to stop codon) is a common mutation carried by 5–6% of RTT patients. iPSCs carrying the R294X mutation has not been studied. We differentiated three R294X iPSC lines and their isogenic wild type control iPSC into neurons with high efficiency and consistency, and observed characteristic RTT pathology in R294X neurons. These isogenic iPSC lines provide unique resources to the RTT research community for studying disease pathology, screening for novel drugs, and testing toxicology.

Pluripotent stem cells reveal erythroid-specific activities of the GATA1 N-terminus

“…These findings occurred in male and female iPSCs that were hemizygous and heterozygous for the X chromosome-linked GATA1s mutation, respectively. Thus, female T21/GATA1s iPSC clones retained the same inactive X chromosome as the somatic cells from which they were derived, consistent with prior reports that X chromosome inactivation is maintained during human iPSC reprogramming (19)(20)(21). During the course of this study, serial Western blotting for GATA1 showed that female GATA1s iPSC lines did not undergo reactivation of the lyonized X chromosome, which can occur during iPSC passage (19,22).…”

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

“…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).…”

“…However, with the generation of RTT-hiPSCs from multiple groups, the XCI status of RTT-hiPSCs, and more generally, female hiPSCs, has been variable. Some researchers (33,34,84,85) reported the generation of RTT-hiPSCs that retain the Xi (post-XCI) from the founder somatic cell it was derived from, while others (14,90) reported the generation of some RTT-hiPSCs that reactivate the Xi of the founder somatic cell and hence carry two active X-chromosomes (pre-XCI).…”

“…Similarly, Renieri and colleagues and Chang and colleagues isolated isogenic control and mutant RTT-hiPSCs from some female patients carrying heterozygous mutations in the X-linked CDKL5 gene and MECP2, respectively (34,84). RTThiPSCs were post-XCI as they displayed non-random XCI skewing by AR assay and expressed CDKL5 or MECP2 in a non-random monoallelic manner allowing the isolation of isogenic control and mutant RTT-hiPSCs.…”