Multi-omic rejuvenation of naturally aged tissues by a single cycle of transient reprogramming

Abstract

The expression of the pluripotency factors OCT4, SOX2, KLF4, and MYC (OSKM) can convert somatic differentiated cells into pluripotent stem cells in a process known as reprogramming. Notably, partial and reversible reprogramming does not change cell identity but can reverse markers of aging in cells, improve the capacity of aged mice to repair tissue injuries, and extend longevity in progeroid mice. However, little is known about the mechanisms involved. Here, we have studied changes in the DNA methylome, transcriptome, and metabolome in naturally aged mice subject to a single period of transient OSKM expression. We found that this is sufficient to reverse DNA methylation changes that occur upon aging in the pancreas, liver, spleen, and blood. Similarly, we observed reversion of transcriptional changes, especially regarding biological processes known to change during aging. Finally, some serum metabolites and biomarkers altered with aging were also restored to young levels upon transient reprogramming. These observations indicate that a single period of OSKM expression can drive epigenetic, transcriptomic, and metabolomic changes toward a younger configuration in multiple tissues and in the serum.

Keywords: OSKM; Yamanaka; aging; epigenetic clocks; pluripotency; reprogramming; transcriptomic clocks.

FIGURE 1

Transient OSKM reprogramming partially rejuvenates the methylation profile of old pancreas. (a) Principal component analysis (PCA) of aging‐related differentially methylated (DM) promoters of young (13 weeks, n = 5), old (55 weeks, n = 5), and old‐OSKM (55 weeks, n = 5) pancreas. (b) DM promoters were divided into hyper‐ and hypomethylated during aging, and shown is the number of these promoters that alters their methylation profile due to transient OSKM activation. Hnf1a promoter is a representative example of an age‐associated hypermethylated promoter that becomes demethylated in old‐OSKM pancreas. (c) PCA of aging‐associated DM enhancers of young, old, and old‐OSKM pancreas. (d) DM enhancers were classified into hyper‐ and hypomethylated during aging, and shown is the subset of these enhancers that alters their methylation profile due to transient OSKM activation. Arid5b enhancer is a representative example of an age‐related hypermethylated enhancer that becomes demethylated in old‐OSKM pancreas. (e) The methylation status of aging‐hypermethylated or hypomethylated promoters and (f) enhancers that were found to be OSKM‐demethylated or remethylated, respectively, was evaluated immediately after OSKM cessation (day 7) or after 14 days of recovery (day 21). (g) Methylation levels measured by bisulfite pyrosequencing of four CpGs, located in regions hypermethylated with aging in the pancreas (n = 4 to 6). Bars in b, d, and g represent the standard deviation (SD) of the data. Statistical significance was evaluated using one‐way ANOVA with Tukey's multiple comparison method, and comparisons are indicated as *p < 0.05 and **p < 0.01

FIGURE 2

Transient OSKM reprogramming rejuvenates the transcriptome of old pancreas. (a) Principal component analysis (PCA) of aging‐related differentially expressed genes (DEGs: fold change > 1.5 and raw pval < 0.01) including young, old, and old‐OSKM pancreas. (b) Representation of these aging‐DEGs (217 genes in total) colored by their alteration of expression induced by OSKM: OSKM‐upregulated genes are depicted in pink and OSKM‐downregulated genes are depicted in blue, while the names of the top ten genes, either upregulated or downregulated with aging, are also depicted (c) Enrichment analysis based on ROAST (Efron & Tibshirani, ; Wu et al., 2010) was performed comparing young versus old, and old versus old‐OSKM pancreas depicting: (c) mTOR signaling pathway (KEGG_04150) and (d) DNA Replication (KEGG_03030). Statistical significance in c and d was evaluated using Kolmogorov–Smirnov test. (e‐h) Z‐score representation of the expression profile with GS adjustment for the indicated gene‐sets among young, old, and old‐OSKM pancreas. Gene‐sets have been selected for following this pattern: Young ≠ Old & Young = Old OSKM after a Normal‐Normal hierarchical model (gaga) (Rossell, 2009) (5% FDR)

FIGURE 3

Old livers present rejuvenated features after transient OSKM reprogramming. (a) Principal component analysis (PCA) of aging‐related differentially methylated (DM) promoters including young, old, and old‐OSKM livers. (b) DM promoters are divided into hyper‐ and hypomethylated during aging, and shown is the number of these promoters that alter their methylation profile due to transient OSKM activation. Hox10, Foxa3, and Thy1 promoters are representative examples of age‐associated hypermethylated promoters that become demethylated in old OSKM livers. (c) The expression of global aging genes identified by mouse Aging Cell Atlas (Zhang et al., 2021) was evaluated in very old livers (100 weeks; group 1 consists of 5 wild‐type mice as control, 5 reprogrammable mice activating OSKM for 1 week and recovering for 2 weeks, and 5 reprogrammable mice activating OSKM for 1 week and recovering for 4 weeks) compared to young (13 weeks; n = 5) control livers. (d) The expression of Nrf2 and ApoM, two aging‐associated genes, was measured in very old livers (100 weeks; group 1 as above together with group 2 consisting of other 5 wild‐type mice as control and 7 reprogrammable mice activating OSKM for 1 week and recovering for 4 weeks). (e) The levels of aspartate aminotransferase (AST/GOT) and alanine aminotransferase (ALT/GPT) were measured in the serum of wild‐type (n = 7) and reprogrammable (n = 6) mice before doxycycline treatment, as well as after 1 week of doxycycline and 2 weeks of recovery. Statistical significance was evaluated using one‐way ANOVA with Tukey's multiple comparison method, and comparisons are indicated as *p < 0.05, **p < 0.01 and ***p < 0.001

FIGURE 4

Evidences of OSKM‐induced rejuvenation in hematopoietic cells. (a) Correlation of the average methylation of 3 close CpGs located in the intragenic region of Hsf4 gene (Chr8; at the positions 105271000,105271005, and 105271015), as measured in the blood of mice, with their chronological age (in weeks) of the mice. (b) Δ change of the methylation levels in these CpGs of Hsf4 before and after a period of 5 weeks in very old (100 weeks) wild‐type mice (n = 4) and reprogrammable mice (n = 3). Both experimental groups were treated for 1 week with doxycycline and recovered for 4 weeks. Statistical significance was evaluated using Mann–Whitney nonparametric t‐test. (c) The expression of global aging genes identified by mouse Aging Cell Atlas (Zhang et al., 2021) was evaluated in very old spleens (100 weeks; 5 wild‐type mice as control, 7 reprogrammable mice activating OSKM for 1 week and 4 weeks of recovery) compared to young (13 weeks; n = 4) control spleens. (d) The expression of Nrf2, an aging‐associated gene, was measured in the same group of mice. Statistical significance was evaluated using one‐way ANOVA with Tukey's multiple comparison method, and comparisons are indicated as *p < 0.05, **p < 0.01, and ***p < 0.001

FIGURE 5

Metabolomic analysis in the serum of very old mice reveals systemic beneficial effects upon transient OSKM activation. (a) Metabolomic analyses were performed on the sera of very old (100 weeks) female reprogrammable mice from two independent experiments (each experiment analyzed separately by mass spectrometry); Group 1: n = 6 and Group 2: n = 6. The sera of these mice were analyzed longitudinally, that is, before and after a single cycle of reprogramming. The Δ change of the levels of 4 metabolites that were identified to change with aging, being either upregulated (4‐hydroxyproline, thymine, trimethyl‐lysine) or downregulated (indole‐3‐propionic acid), are depicted after activating OSKM for 1 week and recovering for 2 to 4 weeks. Statistical significance was evaluated using a paired t‐test as values were confirmed to follow a normal distribution using the Shapiro–Wilk test. Comparisons are indicated as *p < 0.05, **p < 0.01, and ***p < 0.001