Reprogramming suppresses premature senescence phenotypes of Werner syndrome cells and maintains chromosomal stability over long-term culture.

Akira Shimamoto,Kazumasa Zensho,Keiichi Fukuda,Hirofumi Koyama,Hidetoshi Tahara,Yukihiro Sera,Kyotaro Hirashima,Takahiko Shimizu,Koutaro Yokote,Shinsuke Yuasa,Makoto Goto,Mitsuhiko Osaki,Yasuhito Ishigaki,Minoru Takemoto,Hiroyuki Seimiya,Harunobu Kagawa,Mitsuo Oshimura,Yasuhiro Kazuki,Koji Hamasaki ,Y Kodama

doi:10.1371/journal.pone.0112900

Abstract

Werner syndrome (WS) is a premature aging disorder characterized by chromosomal instability and cancer predisposition. Mutations in WRN are responsible for the disease and cause telomere dysfunction, resulting in accelerated aging. Recent studies have revealed that cells from WS patients can be successfully reprogrammed into induced pluripotent stem cells (iPSCs). In the present study, we describe the effects of long-term culture on WS iPSCs, which acquired and maintained infinite proliferative potential for self-renewal over 2 years. After long-term cultures, WS iPSCs exhibited stable undifferentiated states and differentiation capacity, and premature upregulation of senescence-associated genes in WS cells was completely suppressed in WS iPSCs despite WRN deficiency. WS iPSCs also showed recapitulation of the phenotypes during differentiation. Furthermore, karyotype analysis indicated that WS iPSCs were stable, and half of the descendant clones had chromosomal profiles that were similar to those of parental cells. These unexpected properties might be achieved by induced expression of endogenous telomerase gene during reprogramming, which trigger telomerase reactivation leading to suppression of both replicative senescence and telomere dysfunction in WS cells. These findings demonstrated that reprogramming suppressed premature senescence phenotypes in WS cells and WS iPSCs could lead to chromosomal stability over the long term. WS iPSCs will provide opportunities to identify affected lineages in WS and to develop a new strategy for the treatment of WS.

Highlights

Werner syndrome (WS) is a rare human autosomal recessive disorder characterized by early onset of aging-associated diseases, chromosomal instability, and cancer predisposition [1,2]
Direct sequencing analysis of WS induced pluripotent stem cells (iPSCs) identified compound heterozygous Mut4/Mut6 mutations in the WRN gene similar to those observed in parental cells, and the derivation of WS iPSCs from parental cells was confirmed by Short tandem repeat (STR) analysis (Figures S2B and S2C)
We demonstrated that WS fibroblasts could be reprogrammed into iPSCs using Yamanaka factors, and the resulting iPSCs showed unlimited proliferative capacity that was sufficient for self-renewal over a period of 2 years

Summary

Introduction

Werner syndrome (WS) is a rare human autosomal recessive disorder characterized by early onset of aging-associated diseases, chromosomal instability, and cancer predisposition [1,2]. Fibroblasts from WS patients exhibit premature replicative senescence [3], and WRN, a gene responsible for the disease, encodes a RecQ-type DNA helicase [4,5,6,7], that is involved in maintenance of chromosome integrity during DNA replication, repair, and recombination [8,9]. It is reported that telomere loss caused by a defect in WRN helicase involves chromosome end fusions that are suppressed by telomerase [11]. These observations suggest that premature senescence in WS cells reflects defects in telomeric lagging-strand synthesis followed by accelerated telomere loss during DNA replication

Methods

Results

Conclusion