Reprogramming Suppresses Premature Senescence Phenotypes of Werner Syndrome Cells and Maintains Chromosomal Stability over Long-Term Culture

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.

Generation of Endothelial and Smooth Muscle Cells from Werner Syndrome-Specific Induced Pluripotent Stem Cells

“…WRN plays a key role in chromatin homeostasis such as replication fork progression, base excision repair, and telomere maintenance 10) . Previous studies reported that dermal fibroblasts derived from WS patients could be reprogrammed into iPSCs 27) . Cells from patients with genetic disorders are an ideal tool for research; however, their application is limited in basic research, because it is difficult to obtain sufficient amounts of patientsʼ cells.…”

“…Because several reprogramming steps involve cell stress and extensive cell division, iPSCs may acquire chromosomal abnormalities during reprogramming. Previous reports showed that WS fibroblasts could be reprogrammed into iPSCs; however, the parental cultured fibroblasts already showed mosaicism with chromosomal abnormality 27) . We karyotyped the T lymphocyte-derived WS-iPSC lines, and observed that all the types of cells had normal karyotypes (Figure-2).…”

“…The cell clumps were plated onto type I collagen-coated dishes and cultured for 1 day in ReproStem (ReproCELL) without bFGF. On the next day, the medium was changed to DMEM/N2/B27 medium containing 5 μM BIO (Sigma-Aldrich) 27) . The cells were cultured for 3 days, after which the medium was changed to N2/B27 medium without BIO.…”

“…In this approach, exogenous genes are integrated into the host genome, and the dermal fibroblasts are obtained via skin biopsy. Previous reports have shown that WS fibroblasts could be reprogrammed into iPSCs by retroviral vectors, but the generated WS-specific iPSCs (WS-iPSCs) frequently showed abnormal karyotypes due to the abnormal karyotypes of the parental WS fibroblasts 27) . We previously established a method to generate integrationfree iPSCs from peripheral blood cells 28)-32) .…”

Absence of premature senescence in Werner's syndrome keratinocytes

“…For instance, ectopic expression of telomerase results in lifespan extension of WS fibroblasts (Wyllie et al 2000). Reprogramming WS cells to specific lineages that naturally express telomerase also result in lifespan extension as was demonstrated in induced pluripotent and neuronal progenitor cells (Shimamoto et al 2014;Cheung et al 2014). …”