In Vivo Amelioration of Age-Associated Hallmarks by Partial Reprogramming

Abstract

Aging is the major risk factor for many human diseases. In vitro studies have demonstrated that cellular reprogramming to pluripotency reverses cellular age, but alteration of the aging process through reprogramming has not been directly demonstrated in vivo. Here, we report that partial reprogramming by short-term cyclic expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) ameliorates cellular and physiological hallmarks of aging and prolongs lifespan in a mouse model of premature aging. Similarly, expression of OSKM in vivo improves recovery from metabolic disease and muscle injury in older wild-type mice. The amelioration of age-associated phenotypes by epigenetic remodeling during cellular reprogramming highlights the role of epigenetic dysregulation as a driver of mammalian aging. Establishing in vivo platforms to modulate age-associated epigenetic marks may provide further insights into the biology of aging.

Keywords: aging; cellular reprogramming; epigenetics; lifespan.

Figure 1. Amelioration of Cellular Markers of Aging by Short-Term In Vitro Induction of Oct4, Sox2, Klf4, and c-Myc

(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 2. Epigenetic Remodeling during Short-Term In Vitro Induction of Oct4, Sox2, Klf4, and c-Myc Reverses Cellular Markers of Aging

(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 methyl-transferase 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 methyl-transferase inhibitor chaetocin. Scale bar, 10 μm. ****p < 0.0001 according to one-way ANOVA with Bonferroni correction. Data are presented as mean ± SEM.

Figure 3. Establishment of In Vivo Induction Protocol in 4F Mice

(A) Body weight of 4F mice upon continuous administration of doxycycline (−Dox n = 11; +Dox n = 26). (B) Survival of 4F mice upon continuous administration of doxycycline (−Dox n = 11; +Dox n = 26). ***p < 0.0005 according to log-rank (Mantel-Cox) test. (C) Schematic representation of cyclic doxycy-cline administration protocol. (D) qPCR analysis of Oct4, Sox2, Klf4, and c-Myc in blood samples of 4F mice after 2 days of doxycycline administration. **p < 0.005, ***p < 0.0005, and ****p < 0.0001 according to one-way ANOVA with Bonferroni correction. (E) Teratomas (arrows) in 4F mice carrying two copies of OSKM and rtTA cassette after 8 weeks of cyclic doxycycline administration. (F) Histological analysis of teratoma with ectoderm, mesoderm, and endoderm. Scale bar, 50 μm. Data are presented as mean ± SEM. See also Figure S3.

Figure 4. Extension of Lifespan and Prevention of Age-Associated Phenotypes by In Vivo Induction of Oct4, Sox2, Klf4, and c-Myc

(A) Body weight of LAKI and LAKI 4F mice upon cyclic doxycycline administration. LAKI (−Dox n = 20; +Dox n = 13) and LAKI 4F (−Dox n = 18; +Dox n = 15). (B) Survival of LAKI and LAKI 4F mice upon cyclic doxycycline administration. LAKI (−Dox n = 20; +Dox n = 13) and LAKI 4F (−Dox n = 18; +Dox n = 15). ****p < 0.0001 according to log-rank (Mantel-Cox) test. (C) Representative photograph of 16-week-old LAKI 4F mice upon cyclic doxycycline administration. (D) Necropsy analysis of 14-week-old LAKI 4F mice upon cyclic doxycycline administration. (E) Histological analysis of indicated organs of 14-week-old LAKI 4F mice upon cyclic doxycycline administration. Arrows depict decreased epidermal thickness and increased keratinization, small lymphoid nodules in the splenic white pulp, tubular atrophy in the kidney, and loss of parietal cells in the stomach of untreated LAKI 4F mice. Scale bar, 50 μm (skin and kidney) and 100 μm (spleen and stomach). *p < 0.05, ***p < 0.001, and ****p < 0.0001 according to one-way ANOVA with Bonferroni correction. (F) Histology of aortic arch of LAKI 4F mice upon cyclic doxycycline administration. Scale bar, 50 μm. *p < 0.05 and ****p < 0.0001 according to one-way ANOVA with Bonferroni correction. (G) ECG analysis in LAKI 4F mice upon cyclic doxycycline administration (−Dox n = 4; +Dox n = 4). Heart rate represented as beats per minute (bpm). *p < 0.05, **p < 0.001, and ****p < 0.0001 according to one-way ANOVA with Bonferroni correction. Data are presented as mean ± SEM. See also Figure S4.

Figure 5. Amelioration of Cellular Markers of Aging by In Vivo Induction of Oct4, Sox2, Klf4, and c-Myc

(A) Expression of Oct4 and Sox2 in the indicated organs of LAKI 4F upon cyclic doxycycline administration. *p < 0.05, **p < 0.005, ***p < 0.0005, and ****p < 0.0001 according to one-way ANOVA with Bonferroni correction. (B) Immunostaining and quantification of Ki67 positive cells in stomach, kidney, and skin of LAKI 4F mice upon cyclic doxycycline administration. Scale bar, 50 μm. (C and D) Immunofluorescence of H3K9me3 and H4K20me3 in kidney and spleen of LAKI 4F mice upon cyclic doxycycline administration. Scale bar, 10 μm. (E) Immunostaining of β-galactosidase in liver of LAKI 4F mice upon cyclic doxycycline administration. (F) Immunostaining and quantification of Pax7-positive cells in muscle of LAKI 4F mice upon cyclic administration of doxycycline. *p < 0.05, ***p < 0.001, and ****p < 0.0001 according to one-way ANOVA with Bonferroni correction. (G) Immunostaining and quantification of Cytokeratin-15 in skin of LAKI 4F mice upon cyclic doxycycline administration. Scale bar, 20 μm. **p < 0.005, ***p = 0.0001. Data are presented as mean ± SEM. See also Figure S5.

Figure 6. Amelioration of Aging Hallmarks in Wild-Type Mice and Human Cells by Short-Term In Vitro Induction of Oct4, Sox2, Klf4, and c-Myc

(A) Immunofluorescence of Oct4 and Sox2 in WT 4F TTFs. Scale bar, 5 μm. (B) Immunofluorescence and quantification of γH2AX foci in late-passage cells from WT 4F mice. 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 late-passage cells from WT 4F mice. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 according to one-way ANOVA with Bonferroni correction. (D) Immunofluorescence and quantification of H3K9me3 in late-passage WT 4F cells. Scale bar, 10 μm. *p < 0.05, and ****p < 0.0001 according to one-way ANOVA with Bonferroni correction compared to control. (E) Immunofluorescence and quantification of γH2AX foci in late-passage human 4F cells. Scale bar, 10 μm. ***p < 0.0005 and ****p < 0.0001 according to one-way ANOVA with Bonferroni correction compared to control. (F) Immunofluorescence and quantification of H3K9me3 in late-passage human 4F cells. Scale bar, 10 μm. *p < 0.05, **p < 0.005, and ****p < 0.0001 according to one-way ANOVA with Bonferroni correction compared to control. Data are presented as mean ± SEM. See also Figure S6 and Table S1.

Figure 7. Improved Resistance to Metabolic Disease and Skeletal Muscle Injury in Aged WT Animals by In Vivo Reprogramming

(A) Schematic representation of induction of pancreatic injury by low-dose (30 mg/kg) STZ following in vivo reprogramming in 12-month-old WT 4F mice. (B) Glucose tolerance test (GTT) in 12-month-old WT 4F mice following beta cell ablation by low dose STZ (−Dox n = 6; +Dox n = 6). **p < 0.01 and ***p = 0.0005 according to two-tailed Student’s t test. (C) Immunostaining of Insulin and quantification of pancreatic islet size in pancreas of 12-month-old WT 4F mice 2 weeks following STZ. **p < 0.005 according to two-tailed Student’s t test. (D) Schematic representation of induction of muscle injury by CTX following in vivo reprogramming in 12-month-old WT 4F mice. (E) Representative image of H&E staining of tibialis anterior (TA) muscle of 12-month-old WT 4F mice following muscle injury by CTX injection. Scale bar, 50 μm. (F) Immunostaining of Laminin in muscle sections of 12-month-old WT 4F mice. (G) Quantification of fiber cross-sectional area frequency distribution and percentage of central nucleated fibers in muscle sections of 12-month-old WT 4F mice following muscle injury by CTX injection (−Dox n = 4; +Dox n = 4). **p < 0.001 according to two-tailed Student’s t test. (H) Immunostaining and quantification Pax7-positive cells in muscle sections of 12-month-old WT 4F mice following muscle injury by CTX injection (−Dox n = 4; +Dox n = 4). *p < 0.05 according to two-tailed Student’s t test. Data are presented as mean ± SEM. See also Figure S7.