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.
Highlights
Rett syndrome (RTT) is a debilitating autism spectrum developmental disorder that predominantly affects females [1,2]
Three female RTT patient fibroblast lines, as well as 1 female fibroblast line from an unrelated healthy individual, were obtained from the Coriell Institute for Medical Research. Among these Coriell fibroblast lines, GM17880 was derived from 5-year-old female RTT patient carrying the T158M mutation (Threonine 158 to Methionine, missense, present in 9–11% of RTT patients), GM07982 was derived from a 25-year-old female patient carrying the V247X mutation (Valine 247 to stop codon, nonsense, very rare in RTT patients), GM11270 was derived from a 8-year-old female patient carrying the R306C mutation (Arginine 306 to Cysteine, missense, present in 4–7% of RTT patients), and AG07306 was derived from a 28-year old healthy female
Detailed characterization revealed that these induced pluripotent stem cells (iPSC) lines had the characteristic human embryonic stem cell morphology (Figure 1, leftmost column), carried the parental RTT mutations at the DNA level (Figure S2), expressed the same set of pluripotency markers as the hESCs did (Figure 1), had normal karyotypes (Figure S3), showed extensive demethylation in the promoter of the endogenous Oct4 locus (Figure S4), and formed teratomas when injected into NOD-SCID mice (Figure S5)
Summary
Rett syndrome (RTT) is a debilitating autism spectrum developmental disorder that predominantly affects females [1,2]. Male mutant mice that lack either the entire Mecp gene [10] or the essential methyl-DNA binding domain [9] develop normally until 5 weeks of age when RTT-like symptoms are first observed, including reduced brain weight, hindlimb clasping and impaired locomotor function. These mice later develop respiratory abnormalities [12] and die prematurely around 8–10 weeks of age. Female mutant mice heterozygous for these deletions display RTT-like symptoms, yet the onset is typically much later than in their male counterparts. The most noticeable pathologies in these mice are the decrease in the size of neuronal nuclei [9] and the reduced complexity of neuronal dendritic arborization [13,14,15,16], which are observed in human autopsy samples [17]
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