Multi-omics characterization of partial chemical reprogramming reveals evidence of cell rejuvenation

Abstract

Partial reprogramming by cyclic short-term expression of Yamanaka factors holds promise for shifting cells to younger states and consequently delaying the onset of many diseases of aging. However, the delivery of transgenes and potential risk of teratoma formation present challenges for in vivo applications. Recent advances include the use of cocktails of compounds to reprogram somatic cells, but the characteristics and mechanisms of partial cellular reprogramming by chemicals remain unclear. Here, we report a multi-omics characterization of partial chemical reprogramming in fibroblasts from young and aged mice. We measured the effects of partial chemical reprogramming on the epigenome, transcriptome, proteome, phosphoproteome, and metabolome. At the transcriptome, proteome, and phosphoproteome levels, we saw widescale changes induced by this treatment, with the most notable signature being an upregulation of mitochondrial oxidative phosphorylation. Furthermore, at the metabolome level, we observed a reduction in the accumulation of aging-related metabolites. Using both transcriptomic and epigenetic clock-based analyses, we show that partial chemical reprogramming reduces the biological age of mouse fibroblasts. We demonstrate that these changes have functional impacts, as evidenced by changes in cellular respiration and mitochondrial membrane potential. Taken together, these results illuminate the potential for chemical reprogramming reagents to rejuvenate aged biological systems and warrant further investigation into adapting these approaches for in vivo age reversal.

Figure 1:. Functional effects of partial chemical reprogramming

A. Overview of the study. Tail and ear fibroblasts were isolated from young (4-month-old) and old (20-month-old) male C57BL/6 mice, and cryo-stocks were prepared once reaching ~80–90% confluency (passage number P1). All fibroblasts used in this study were ≤ P3. These cells were subjected to partial chemical reprogramming followed by indicated analyses. B. AP staining. Young and old fibroblasts were treated with 2c, 7c, or DMSO for 4 days, followed by visualization of cells positive for alkaline phosphatase activity with the StemAb^™ Alkaline Phosphatase Staining Kit II (4X objective). Scale bars: 126.2 μm (100 pixels). C. TMRM staining. Following treatment for 6 days with 2c, 7c, or DMSO, fibroblasts were stained with 250 nM TMRM and 10 μg/ml Hoechst 33342 for 20 minutes at 37°C, 5% CO₂, and 3% O₂. TMRM fluorescence intensity was normalized to the number of nuclei per field and quantified across 4–5 images from random fields for each independent biological replicate (n = 3). For CCCP treatment, cells were treated with 50 μM CCCP in DMSO for 15 minutes prior to TMRM staining. Error bars represent means ± standard deviations, and data were quantified based on percent change from control-treated fibroblasts. Scale bars: 31.1 μm (100 pixels). Statistical significance was determined by one-way ANOVA and Tukey’s post-hoc analysis. *p < 0.05, **p < 0.01, ***p < 0.001. D. Effects on oxygen consumption. Top: representative raw traces of oxygen consumption of cells subjected to the Mito Stress Test protocol (basal, followed by 1 μM oligomycin a, 5 μM FCCP, and 1 μM rotenone / antimycin a) following 6 days of partial chemical reprogramming. Error bars represent means ± standard deviations from 3 technical replicates per treatment. Bottom: quantified oxygen consumption rates across 4 independent biological replicates (n = 4, error bars represent means ± standard deviations). Statistical significance was determined by one-way ANOVA and Tukey’s post-hoc analysis. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2:. Effect of partial chemical reprogramming on gene expression

A. PCA of bulk RNA-seq samples. Fibroblasts were treated with 2c, 7c, or control for 6 days followed by RNA-seq analyses. B. Differentially expressed genes. Differentially expressed genes were determined using edgeR and considered statistically significant at a Benjamini-Hochberg false discovery rate (FDR) cut-off < 0.05. C. Expression of pluripotency markers. Effect of partial chemical reprogramming on the normalized expression of pluripotency markers and Klf4 and c-Myc (Myc). Statistical inference was performed with edgeR. *p.adjusted < 0.05, **p.adjusted < 0.01, ***p.adjusted < 0.001. D) Splicing damage. Splicing damage was determined by the proportion of alternative-splicing events that may disrupt protein function. Statistical significance was determined by one-way ANOVA and Tukey’s post-hoc analysis. ^p < 0.1, *p < 0.05. E. Association of gene expression changes induced by chemical reprogramming with signatures of aging and OSKM reprogramming. Signatures of aging are labelled in red, whereas signatures of OSKM reprogramming are labelled in cyan. NES: normalized enrichment score. Statistical inference was done with the fgsea R package. *FDR < 0.05, **FDR < 0.01, ***FDR < 0.001.

Figure 3:. Effect of partial chemical reprogramming on protein expression

A. PCA of log₂ normalized protein abundances. Fibroblasts were treated for 6 days with 2c, 7c, or control followed by mass spectrometry-based proteomic analyses. B. Global effects of partial chemical reprogramming on the proteome. Scaled heatmap of normalized protein abundances with clustering. C. Comparisons between effects on protein and gene expression. Log₂ fold changes of protein (vertical axis) versus mRNA (horizontal axis) for 20-month-old fibroblasts treated with 7c. Spearman correlation coefficient is shown in bold. Labeled are the genes that are most strongly differentially expressed after 7c treatment (adjusted p-value < 0.05) at both the mRNA and protein levels (shown in green). Also depicted are genes significantly changing only at the protein level (shown in cyan), or at the mRNA level (shown in red). Spearman correlation coefficients between protein and mRNA levels were > 0.7 for all treatment conditions (refer to Figure 3 – figure supplement 1). D. Functional GSEA. Pathways enriched by gene expression changes induced by partial chemical reprogramming (blue: 7c, green: 2c) and aging in primary fibroblasts (red) from the current study, as well as by established signatures of OSKM reprogramming (cyan) and aging (red). ^Benjamini-Hochberg FDR < 0.1, *FDR < 0.05, **FDR < 0.01, ***FDR < 0.001. E. Effect of partial chemical reprogramming on the expression of protein complexes associated with aging. Protein abundances of mitochondrial OXPHOS complexes, chaperones, collagens, and the spliceosome are all affected by partial chemical reprogramming. Each datapoint represents the abundance of an individual protein. Significance was assessed with a linear regression model that included effects for gene, treatment, age group, and a treatment:age group interaction. p-values within each panel were corrected for multiple testing with the default Dunnett correction (“single-step”) method in the multcomp R package. ‘adjusted p-value < 0.1, *adjusted p-value < 0.05, **adjusted p-value < 0.01, ***adjusted p-value < 0.001, n.s. adjusted p-value ≥ 0.1.

Figure 4:. Correlation of gene and protein expression changes with signatures of aging and OSKM reprogramming

Spearman correlation of gene expression and protein abundance changes induced by partial chemical reprogramming (blue: 7c, green: 2c) with the signatures of OSKM reprogramming (cyan) and aging (red). Correlation coefficients ρ were calculated by the Spearman method based on the union of top 650 genes with the lowest p-value for each pair of signatures. Statistically significant pairwise correlations with Spearman ρ > 0.1 are labelled with asterisks. ***Benjamini-Hochberg FDR < 0.001.

Figure 5:. Effect of partial chemical reprogramming on the phosphoproteome

A. Targeted GSEA analysis of phosphorylation targets for four selected gene ontologies following partial chemical reprogramming. Bar lengths depict normalized enrichment scores. **Benjamini-Hochberg FDR < 0.01, ***FDR < 0.001. B. Kinase signaling pathways. z-values for a kinase enrichment analysis are shown for the effects of partial reprogramming with 2c (horizontal axis) vs. 7c (vertical axis) in young (top) and old (bottom) fibroblasts. Signaling pathways significantly affected (Benjamini-Hochberg FDR < 0.05) by both 7c and 2c treatment (Prkaca) are colored in red, and signaling pathways affected only by 2c in young fibroblasts (Makp1 and Akt1) are colored in green (highlighted in bold for old fibroblasts). Horizontal and vertical red lines indicate the 0.05 Benjamini-Hochberg FDR significance threshold cut-offs for the comparisons 2c vs. control and 7c vs. control, respectively. C. Role of Prkaca in partial chemical reprogramming. Left: knockdown of Prkaca during 7c partial chemical reprogramming in 20-month-old fibroblasts and effects on mitochondrial membrane potential, as assessed by TMRM fluorescence (n = 3 independent biological replicates). p-values were determined by one-way ANOVA and Tukey’s post-hoc analysis. ns p ≥ 0.1, ***p < 0.001. Right: staining of 20-month-old fibroblasts for cellular localization of Tom20 (red) and Prkaca (green) during 7c partial chemical reprogramming. Representative images are shown, and data was collected for n = 3 independent biological replicates. Scale bars: 20 μm.

Figure 6:. Effect of partial chemical reprogramming on the metabolome

A. Hierarchical clustering of polar metabolite samples. Fibroblasts were treated for 6 days with 2c, 7c, or control followed by cell scraping and collection in cold 0.9% saline solution (n = 3 independent biological replicates). Polar metabolites were isolated from the frozen cell pellets by chloroform-methanol extraction, and the upper polar phase was analyzed by hydrophobic interaction liquid chromatography (HILIC) coupled to a quadrupole mass spectrometer in both positive and negative ionization modes^,. All subsequent analyses were performed using MetaboAnalyst 5.0. B. Metabolites affected by partial chemical reprogramming. A total of 203 metabolites were identified by HILIC-positive and -negative methods, combined. Metabolite peak areas were normalized to the amount (μg) of protein in each cell pellet (determined by BCA assay) and log-transformed. In total, abundances of 109 metabolites were significantly altered by partial chemical reprogramming (Benjamini-Hochberg FDR < 0.05, colored in red). C. Global effects of partial chemical reprogramming on the metabolome. Scaled heatmap of normalized, log-transformed metabolite abundances. D. Abundances of top 50 metabolites significantly altered by partial chemical reprogramming with 2c or 7c. Scaled heatmap with labeled metabolites. E. Expression of metabolite-related proteins. Expression of proteins related to s-adenosylhomocysteine biosynthesis (top) and xanthine metabolism (bottom) following partial chemical reprogramming with 2c and 7c vs. control in young and old cells. ‘Benjamini-Hochberg FDR < 0.1, *FDR < 0.05, **FDR < 0.01, ***FDR < 0.001.

Figure 7:. Effect of partial chemical reprogramming on biological age

A. Effect of partial chemical reprogramming on transcriptomic age (tAge). tAge was assessed by using mouse multi-tissue transcriptomic clocks to analyze the bulk RNA-seq data presented in Figure 2 (n = 4 independent biological replicates). p-values were determined by one-way ANOVA and Tukey’s post-hoc analysis. ns p ≥ 0.1, ^p < 0.1, *p < 0.05, **p < 0.01. B. Effect of partial chemical reprogramming on epigenetic age (DNAmAge). Levels of mean DNA methylation (DNAm) was assessed by DNAm microarray on the Horvath mammal 320k chip (n = 5 independent biological replicates). p-values were determined by one-way ANOVA and Tukey’s post-hoc analysis. *p < 0.05, **p < 0.01, ***p < 0.001.