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

<div><p>Werner syndrome (WS) is a premature aging disorder characterized by chromosomal instability and cancer predisposition. Mutations in <i>WRN</i> 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 <i>WRN</i> 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.</p></div

Suppression of Senescence-Associated Gene Expression in Reprogrammed WS iPSCs.

<p>(A) Expression of CDKI genes in parental fibroblasts and iPSCs. White columns show relative expression levels in the parental fibroblasts TIG-3, TIG-114, A0031, and WSCU01, and gray columns show those of their derived iPSC clones. Numbers under the horizontal axis in each graph show relative values in mRNA expression compared with that in TIG-3 fibroblasts. Values represent means of three technical replicates ± SD. (B) Expression of SASP genes in parental fibroblasts and iPSCs. Each graph is shown as in (A).</p

Recapitulation of Premature Senescence Phenotypes in Differentiated Cells from WS iPSCs.

<p>(A) Differentiation of EBs from normal (TIG-3) and WS (WSCU01 #02 and #13) iPSCs. Differentiated cells from WS iPSCs showed premature senescence. SA-β-gal staining was performed on day 25 of differentiation. Bars  = 100 µm. (B) Percentage of senescent cells after 25 days of differentiation. SA-β-gal-positive cells were counted in three randomly selected fields with 40× magnification. Values represent means of the three fields ± SD. (C) Expression of hTERT and p21 mRNAs in undifferentiated iPSCs (“U,” red columns), EBs after 12 days of formation (“E,” green columns), and differentiated cells after 25 days of differentiation (“D,” blue columns). Values represent means of three technical replicates ± SD. (D) Expression of SASP genes in differentiated cells from normal (TIG-3) and WS (WSCU01 #02 and #13) iPSCs after 25 days of differentiation. Graphs shows fold changes relative to undifferentiated iPSCs. Values represent means of three technical replicates ± SD.</p

Results of chromosome analysis of WS iPSC clones and their parenral cells.

<p>Abbreviations: t, translocation; del, deletion; der, derivative chromosome; p, short arm; q, long arm.</p><p>Results of chromosome analysis of WS iPSC clones and their parenral cells.</p

Sustained ESC-like characteristics of WS iPSCs after Long-Term Culture.

<p>(A) Expression of pluripotency genes in A0031-derived WS iPSC clones in early and late passages. (B) Expression of hESC markers in A0031-derived WS iPSC clone #23 in early and late passages. Bars  = 100 µm. (C) EB formation in A0031-derived WS iPSC clones from early and late passages. Bars  = 100 µm. (D) Immunocytochemical analysis of differentiation of EBs into 3 germ layers for A0031-derived iPSC clone #23 in early and late passages. β-III tubulin (ectoderm), desmin (mesoderm), vimentin (mesoderm and parietal endoderm), and α-fetoprotein (Afp, endoderm). Bars  = 100 µm. (E) Hematoxylin and eosin histology of teratomas from A0031-derived iPSC clone #23. Formation of all 3 germ layers is shown including melanin-producing cells (ectoderm), cartilage (mesoderm), and tracheal epithelium (endoderm).</p

Karyotype Analysis of WS iPSCs.

<p>Chromosomal profiles of G-band analysis. (A) Parental A0031 fibroblast and (B) A0031-derived iPSC clone #34. Arrows indicate translocation breakpoints. Chromosomal profiles of M-FISH analysis. A0031-derived iPSC clones (C) #23, (D) #34, and (E) #64 and WSCU01-derived iPSC clones (F) #02, (G) #13, and (H) #14. Arrows indicate translocation breakpoints or an extra chromosome.</p

Reprogramming of the SASP gene loci is mediated by factors other than activated telomerase.

<p>(A) Expression of CDKI genes in WS fibroblasts and their hTERT-transduced derivatives. White columns show relative expression levels in A0031 and WSCU01 fibroblasts, and gray columns show those of their hTERT-transduced derivatives. Numbers under the horizontal axis in each graph show relative values in mRNA expression compared with that in parental fibroblasts. Values represent means of three technical replicates ± SD. (B) Expression levels of SASP genes in WS fibroblasts and their hTERT-transduced derivatives. Each graph is shown as in (C).</p