Partial Reprogramming Ameliorates Cellular Phenotypes Associated with Aging

Figure 1 Amelioration of Cellular Markers of Aging by Short-Term In Vitro Induction of Oct4, Sox2, Klf4, and c-Myc Show full caption (A) Immunofluorescence of Oct4 and Sox2 in LAKI 4F TTFs. Scale bar, 10 μm. (B) Immunofluorescence and quantification of γH2AX foci in LAKI 4F TTFs. Scale bar, 10 μm. ∗∗∗∗ p < 0.0001, according to one-way ANOVA with Bonferroni correction. (C) qPCR analysis of stress response genes in the p53 pathway, senescence-associated metalloprotease MMP13 and interleukin-6 in LAKI 4F TTFs. ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 according to one-way ANOVA with Bonferroni correction. (D and E) Immunofluorescence of H3K9me3 and H4K20me3 in LAKI 4F TTFs. Scale bar, 10 μm. ∗∗p < 0.005 and ∗∗∗∗p < 0.0001 according to one-way ANOVA with Bonferroni correction. (F) Immunofluorescence of Lamin A/C and quantification of nuclear abnormality in LAKI 4F TTFs. Arrows indicate blebbing in the nuclear envelope in Oct4 negative cells. Scale bar, 10 μm. ∗∗p < 0.005 according to one-way ANOVA with Bonferroni correction. Data are presented as mean ± SEM. See also Figures S1 and S2

Figure S1 Characterization of LAKI 4F TTFs upon Short-Term Induction of OSKM, Related to Figure 1 Show full caption (A) qPCR analysis of Oct4, Sox2, Klf4 and c-Myc in LAKI 4F tail tip fibroblasts (TTFs). ∗∗p < 0.005, ∗∗∗p < 0.0005 and ∗∗∗∗p < 0.0001, according to one-way ANOVA with Bonferroni correction. (B) Analysis of cellular identity of LAKI 4F TTFs following partial reprogramming analyzed by flow cytometry using Thy1 and SSEA1 staining. (C) qPCR analysis of Nanog expression in LAKI 4F TTFs after short-term induction of OSKM by doxycycline treatment. (D) Quantification of foci intensity and foci volume of γH2AX staining in LAKI 4F TTFs. ∗∗∗∗p < 0.0001, according to one-way ANOVA with Bonferroni correction. (E) Immunostaining of 53BP1 in LAKI 4F TTFs following doxycycline treatment. The number, volume, and intensity of 53BP1 foci in LAKI 4F TTFs were quantified in Oct4 positive and negative cells. Scale bar, 10 μm. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.0005 and ∗∗∗∗p < 0.0001, according to one-way ANOVA with Bonferroni correction. (F) β-galactosidase activity in WT and LAKI 4F TTFs. Quantification of cells with β-galactosidase staining. Scale bar, 100 μm. ∗∗∗p = 0.001 and ∗∗∗∗p < 0.0001, according to one-way ANOVA with Bonferroni correction. (G) Levels of mitochondrial ROS in LAKI 4F TTFs after doxycycline treatment. ∗∗p < 0.01, according to one-way ANOVA with Bonferroni correction. Data are presented as mean ± SEM.

Liu et al., 2005 Liu B.

Wang J.

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et al. Genomic instability in laminopathy-based premature aging. Liu et al., 2005 Liu B.

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et al. Genomic instability in laminopathy-based premature aging. López-Otín et al., 2013 López-Otín C.

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Kroemer G. The hallmarks of aging. Nuclear lamins maintain proper architecture of the nuclear envelope (). Defects in the nuclear envelope resulting from the accumulation of progerin are the main drivers of premature aging in HGPS. Similar nuclear envelope abnormalities have been reported during physiological aging (). Importantly, short-term induction of OSKM in LAKI 4F cells significantly improved nuclear envelope architecture compared to untreated cells ( Figure 1 F). Lastly, in order to investigate the maintenance of the ameliorated aging-associated phenotypes after OSKM induction, we analyzed the level of DNA damage, H3K9me3, and nuclear envelope abnormalities 4 and 8 days after termination of doxycycline treatment. These analyses revealed a slow re-acquisition of age-associated phenotypes in LAKI 4F TTFs compared to untreated control cells ( Figures 2 A and 2B). Importantly, short-term re-induction of OSKM for an additional 4 days was able to revert the re-accumulation of aging-associated phenotypes ( Figures 2 A and 2B). These results suggest that cyclic induction of OSKM following an “on and off” scheme may have the capacity to prevent or reset the accumulation of age-associated phenotypes.

Figure 2 Epigenetic Remodeling during Short-Term In Vitro Induction of Oct4, Sox2, Klf4, and c-Myc Reverses Cellular Markers of Aging Show full caption (A) Immunofluorescence of γH2AX and Lamin A/C and quantification of γH2AX and nuclear abnormalities in LAKI 4F TTFs subjected to cyclic expression of OSKM for the indicated days. Scale bar, 10 μm. ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0005, and ∗∗∗∗p < 0.0001 according to one-way ANOVA with Bonferroni correction compared to control. (B) Immunofluorescence and quantification of H3K9me3 in LAKI 4F TTFs subjected to cyclic expression of OSKM for the indicated days. Scale bar, 10 μm. ∗∗∗p < 0.0005 and ∗∗∗∗p < 0.0001 according to one-way ANOVA with Bonferroni correction. (C) Immunofluorescence and quantification of H3K9me3 in LAKI 4F TTFs subjected to short-term expression of OSKM for 12 hr and 24 hr. Scale bar, 10 μm. ∗∗p < 0.005 and ∗∗∗∗p < 0.0001 according to one-way ANOVA with Bonferroni correction. (D) Immunofluorescence of γH2AX and Lamin A/C, and quantification of γH2AX and nuclear abnormalities in LAKI 4F TTFs subjected to short-term expression of OSKM for 12 hr and 24 hr. Scale bar, 10 μm. (E) Immunofluorescence and quantification of H3K9me3 in LAKI 4F TTFs subjected to short-term expression of OSKM in the presence of the H3K9 methyltransferase inhibitor chaetocin. Scale bar, 10 μm. ∗∗p < 0.01 and ∗∗∗∗p < 0.0001 according to one-way ANOVA with Bonferroni correction. (F) Immunofluorescence and quantification of γH2AX foci in LAKI 4F TTFs subjected to short-term expression of OSKM in the presence of the H3K9 methyltransferase inhibitor chaetocin. Scale bar, 10 μm. ∗∗∗∗p < 0.0001 according to one-way ANOVA with Bonferroni correction. Data are presented as mean ± SEM.

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et al. Premature termination of reprogramming in vivo leads to cancer development through altered epigenetic regulation. To gain further insight into the mechanism behind our observations, we first decided to evaluate the effect of short-term OSKM induction in the expression of lamin A/C and the accumulation of progerin. Although we have previously shown that reprogramming HGPS cells to pluripotency causes a switch in nuclear lamin composition (), the improvement of aging phenotypes in LAKI 4F cells after short-term induction of OSKM did not depend on changes in the expression of nuclear lamin A/C or B1 or on reduced accumulation of progerin ( Figures S2 A and S2B). Moreover, selective induction of programmed cell death upon short-term OSKM induction in cells with high levels of age-associated phenotypes was not responsible for the amelioration of aging hallmarks in LAKI 4F cells ( Figure S2 C). Furthermore, none of the cellular aging hallmarks analyzed were ameliorated in LAKI cells not carrying an OSKM polycystronic cassette, indicating that OSKM expression was responsible for the rescue of age-associated phenotypes ( Figures S2 D–S2H). In addition, infection of LAKI cells with single reprogramming factors (i.e., Oct4, Sox2, or c-Myc alone) was insufficient to improve nuclear envelope architecture ( Figure S2 I), suggesting that cellular reprogramming, but not the induction of proliferation alone, restored the aberrant nuclear envelope in these cells. Finally, based on the epigenetic nature of the reprogramming process, we decided to confirm the role of epigenetic remodeling as a driver of the amelioration of age-associated phenotypes by short-term induction of OSKM. For this purpose, we carried out a detailed time-course analysis of the amelioration of DNA damage and nuclear envelope abnormalities in LAKI 4F TTFs, as well as the restoration of H3K9me3. Analysis of LAKI 4F TTFs at 12 and 24 hr after induction of OSKM revealed that restoration of H3K9me3 precedes the amelioration of DNA damage and nuclear envelope defects, suggesting that epigenetic remodeling during cellular reprogramming acts as a driver of the improvement of age-associated phenotypes ( Figures 2 C and 2D). In addition, in order to further confirm this hypothesis, we analyzed the amelioration of aging phenotypes in LAKI 4F TTFs upon short-term induction of OSKM in the presence of 10nM chaetocin, an H3K9 methyltransferase inhibitor. Importantly, induction of OSKM, in the presence of 10 nM chaetocin, prevented the restoration of H3K9me3 and abolished the amelioration of DNA damage and defects in nuclear envelope induced by cellular reprogramming ( Figures 2 E and 2F). These observations reinforce the role of epigenetic remodeling during cellular reprogramming as a driver of the improvement of age-associated phenotypes by short-term expression of OSKM.

Figure S2 Characterization of LAKI 4F and LAKI TTFs following Doxycycline Treatment, Related to Figure 1 Show full caption (A) Detection of Lamin A/C and progerin levels in WT 4F and LAKI 4F TTFs after short-term doxycycline treatment by western blot. Arrowheads denote Lamin A and C. Asterisk denotes progerin. (B) qPCR analysis of Lamin A and Lamin B1 levels in LAKI 4F TTFs. (C) Analysis by flow cytometry of programmed cell death in LAKI 4F TTFs following doxycycline treatment. (D) Immunofluorescence of γH2AX in LAKI TTFs following doxycycline treatment. (E) Immunofluorescence of H3K9me3 in LAKI TTFs following doxycycline treatment. (F) qPCR analysis of stress response genes in the p53 pathway, senescence-associated metalloprotease MMP13 and interleukin-6 in LAKI TTFs following doxycycline treatment. (G) Immunofluorescence of Lamin A/C in LAKI TTFs following doxycycline treatment. (H) Levels of mitochondrial ROS in LAKI TTFs after doxycycline treatment. (I) Immunofluorescence of Oct4, Sox2, c-Myc and Lamin A/C in LAKI TTFs infected with GFP, Oct4, Sox2 and c-Myc retroviruses. Scale bar, 10 μm.

Collectively, these results demonstrate that short-term in vitro induction of OSKM in cells derived from a premature aging mouse model ameliorates multiple age-associated hallmarks observed during physiological aging, including the accumulation of DNA damage, cellular senescence, epigenetic dysregulation, and nuclear envelope defects. Thus, epigenetic remodeling induced by partial reprogramming may potentially modify the aging process.