Abstract 14666: Treating Aging Model of Werner Syndrome Specific Induced Pluripotent Stem Cells by Crisper/cas9 Systems
Backgrounds: Werner syndrome (WS) is a rare autosomal recessive disorder characterized by premature onset of several aging-associated diseases, such as atherosclerosis, diabetes, cancer, and early death. The aging phenotypes of WS resemble to those of normal aging. WS is caused by mutations in WRN gene belonging to the RecQ DNA helicase which plays a role in genomic stability. But some of WS phenotypes are hardly explained by genomic instability. Thus, we aimed to model WS by patient-specific induced pluripotent stem cells (iPSCs) to elucidate the mechanisms of WS and aging.
Methods and Results: We sampled T lymphocytes from a patient with WS. Then we transduced with Yamanaka factors (OCT4, SOX2, KLF4, and MYC) by Sendai virus, and iPSC colonies were derived. We confirmed that WS-iPSCs expressed pluripotent markers, could differentiate into all three germ-layer derived tissues, and retained a normal karyotype. We could culture WS-iPSCs over 2 years with pluripotent status. Then, we differentiated WS-iPSCs into fibroblast-like cells. WS-iPSC-derived fibroblast-like cells (WS-iPSC-fibroblasts) showed characteristics of cellular senescence, the poorly proliferation rate and increased positive cells of β-galactosidase activity and γ-H2AX a marker for DNA damage, subjected to cellular stress substance. Singled WS-iPSC-fibroblasts showed other characters of excessive blebbing of plasma membrane and increased apoptosis. WS-iPSC-fibroblasts also showed the decreased migration capacity in wound healing assay (WHA). We performed microarray analysis, WS-iPSC-fibroblasts reproduced the global gene expression pattern of physiological aging. To confirm whether the phenotypes of WS-iPSCs are induced by WRN mutation, we generated isogenic control of WS-iPSC (corrected-WS-iPSC) by homologous recombination using the CRISPR/Cas9 system. We succeeded to generate heterozygous and homozygous recombinant corrected-WS-iPSCs. Both of corrected-iPSC-fibroblasts completely lost the aging-associated phenotypes and showed gene expressions resembling to control-iPSC-fibroblasts.
Conclusion: We modeled aging phenotypes by WS-specific iPSCs. And we successfully treated such phenotypes by CRISPER/Cas9 systems.
Author Disclosures: G. Yozu: None. S. Yuasa: None. H. Daida: None. K. Fukuda: None.
- © 2016 by American Heart Association, Inc.