Reprogramming to recover youthful epigenetic information and restore vision

In the paper

“A proposed cause of ageing is the accumulation of epigenetic noise that disrupts gene expression patterns, leading to decreases in tissue function and regenerative capacity1–3.”

What the sources say

• ?Not checked — no full text of the sourcecites [1] Sinclair, D. A., Mills, K. & Guarente, L. Accelerated aging and nucleolar fragmentation in yeast sgs1 mutants. Science 277, 1313–1316 (1997). source
• ?Not checked — no full text of the sourcecites [2] Imai, S. & Kitano, H. Heterochromatin islands and their dynamic reorganization: a hypothesis for three distinctive features of cellular aging. Exp. Gerontol. 33, 555–570 (1998). source
• Partly supported

  The Introduction proposes that chromatin alterations and resulting gene-expression changes can drive aging, and the Discussion describes DNA-damage-induced chromatin reorganization as a possible contributor. Our copy does not establish the claimed decreases in tissue function or regenerative capacity; the Discussion says the physiological relevance of age-related expression changes remains debated. The cited passages have section labels but no page numbers. (LLM, conf=0.88)

  cites [3] Oberdoerffer, P. et al. SIRT1 redistribution on chromatin promotes genomic stability but alters gene expression during aging. Cell 135, 907–918 (2008). sourceExperimental — accuracy not yet measured

In the paper

“Over time, the central nervous system (CNS) loses function and regenerative capacity5–7.”

What the sources say

• ?Not checked — no full text of the sourcecites [5] Kennard, M. A. Relation of age to motor impairment in man and in subhuman primates. Arch. Neurol. Psychiatry 44, 377–397 (1940). source
• ?Not checked — no full text of the sourcecites [6] Goldberg, J. L., Klassen, M. P., Hua, Y. & Barres, B. A. Amacrine-signaled loss of intrinsic axon growth ability by retinal ganglion cells. Science 296, 1860–1864 (2002). source
• Supported

  Section 2.2 identifies the central nervous system among tissues affected by age-related degeneration and dysfunction. It also reports that adult neurogenesis and neural stem cells decline with age, reducing regenerated neurons and causing functional impairment. Page numbers are not provided in the supplied copy. (LLM, conf=0.92)

  cites [7] Yun, M. H. Changes in regenerative capacity through lifespan. Int. J. Mol. Sci. 16, 25392–25432 (2015). source

In the paper

“During ageing, for reasons that are unclear, these patterns change in ways that can be used to calculate DNA methylation (DNAm) age, a representation of biological age that can predict future health and lifespan4.”

What the sources say

• Partly supported

  The Background and Conclusions (pages not provided) describe age-related methylation changes and a DNAm-age biomarker derived from them. The paper demonstrates prediction of age, but the passages do not establish that DNAm age predicts future health or lifespan; the Conclusions say its role as a marker of aging needs further research. (LLM, conf=0.89)

  cites [4] Horvath, S. DNA methylation age of human tissues and cell types. Genome Biol. 14, 3156 (2013). sourceExperimental — accuracy not yet measured

In the paper

“Because Klf4 can repress rather than promote axonal growth24, we tested a dual-cistron of only Oct4 and Sox2 and observed no regenerative effect.”

What the sources say

• Supported

  The paper’s Abstract identifies KLF4 as a repressor of axon growth in retinal ganglion cells and other CNS neurons. It also reports that KLF4 overexpression reduced axon length in embryonic retinal ganglion cells, directly supporting the cited statement that KLF4 can repress axonal growth. (LLM, conf=0.94)

  cites [24] Moore, D. L. et al. KLF family members regulate intrinsic axon regeneration ability. Science 326, 298–301 (2009). source

In the paper

“If damaged, RGCs of embryos and neonates can regenerate axons, but this capacity is lost within days after birth, likely due to a molecular program intrinsic to the RGCs6,22.”

What the sources say

• ?Not checked — no full text of the sourcecites [6] Goldberg, J. L., Klassen, M. P., Hua, Y. & Barres, B. A. Amacrine-signaled loss of intrinsic axon growth ability by retinal ganglion cells. Science 296, 1860–1864 (2002). source
• Partly supported

  The “Intrinsic factors that limit RGC regeneration” section (page not supplied) says RGC axon growth speed falls sharply from embryonic to postnatal ages, likely because of molecular programs intrinsic to RGCs. It does not say that embryonic and neonatal RGCs regenerate after damage or that this capacity is lost within days after birth. (LLM, conf=0.85)

  cites [22] Laha, B., Stafford, B. K. & Huberman, A. D. Regenerating optic pathways from the eye to the brain. Science 356, 1031–1034 (2017). sourceExperimental — accuracy not yet measured

In the paper

“In cell culture, the ectopic expression of the four Yamanaka transcription factors Oct4, Sox2, Klf4, and c-Myc (OSKM) can reprogram cultured somatic cells to become pluripotent stem cells14, a process that erases cellular identity and resets DNAm age4,15.”

What the sources say

• ?Not checked — no full text of the sourcecites [14] Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006). source
• Supported

  The section “DNAm age of induced pluripotent stem cells and stem cells” (page unavailable) describes iPS cells as pluripotent cells derived from non-pluripotent cells by inducing genes. It reports that their DNAm age is significantly younger than that of corresponding primary cells and indistinguishable from embryonic stem cells, supporting the attributed age reset. The specific OSKM factors are attached to a different citation in the citing sentence. (LLM, conf=0.90)

  cites [4] Horvath, S. DNA methylation age of human tissues and cell types. Genome Biol. 14, 3156 (2013). source
• Supported

  The STAR Methods describe reprogramming cultured mouse fibroblasts into iPSCs, consistent with a change from somatic-cell identity to pluripotency. The Results and Discussion report that these iPSCs had much younger estimated DNA-methylation ages than the fibroblasts, while cautioning that the estimates used a clock developed from mouse blood. (LLM, conf=0.88)

  cites [15] Petkovich, D. A. et al. Using DNA methylation profiling to evaluate biological age and longevity interventions. Cell Metab. 25, 954–960.e6 (2017). source

In the paper

“Many regenerative approaches that work well in young individuals often fail in older ones25,26.”

What the sources say

• Partly supported

  The Abstract and Discussion (page not provided) report that Pten deletion promotes axon regeneration in young mice but has a greatly diminished effect in older mice. The paper tests one approach in mice; it does not establish that many approaches often fail in older individuals, and it describes diminished regeneration rather than complete failure. (LLM, conf=0.85)

  cites [25] Geoffroy, C. G., Hilton, B. J., Tetzlaff, W. & Zheng, B. Evidence for an age-dependent decline in axon regeneration in the adult mammalian central nervous system. Cell Rep. 15, 238–246 (2016). sourceExperimental — accuracy not yet measured
• ?Could not verify

  The Abstract describes successful rod-photoreceptor regeneration in mature mouse retinas, with treatment beginning at four weeks of age. Our copy does not compare outcomes in young and older individuals or say that regenerative approaches often fail with age (searched: young versus older regeneration, age-dependent failure). (LLM, conf=0.76)

  cites [26] Yao, K. et al. Restoration of vision after de novo genesis of rod photoreceptors in mammalian retinas. Nature 560, 484–488 (2018). sourceExperimental — accuracy not yet measured

In the paper

“In a premature ageing mouse model of Hutchinson-Gilford progeria syndrome (HGPS), cyclic transgene-mediated expression of these four genes alleviates symptoms and extends lifespan, raising the possibility that OSKM might counteract normal ageing, too16.”

What the sources say

• Supported

  The Methods describe cyclic doxycycline induction of the OSKM transgene, and the Results report improved appearance and a dramatic increase in median and maximal lifespan in LAKI 4F mice. The paper identifies LAKI as a mouse model of HGPS. The suggestion about normal ageing is the citing authors’ inference, not a finding attributed to this source. (LLM, conf=0.94)

  cites [16] Ocampo, A. et al. In vivo amelioration of age-associated hallmarks by partial reprogramming. Cell 167, 1719–1733.e12 (2016). source

In the paper

“Among the Yamanaka factors, c-Myc is an oncogene that reduces mouse lifespan20 and is not required for the initiation of cellular reprogramming21.”

What the sources say

• Supported

  The Introduction identifies MYC as a cellular proto-oncogene. The Results (‘Myc+/− Mice Have Increased Longevity’; page unavailable) report that mice with reduced MYC expression lived longer than normal Myc+/+ mice, supporting the lifespan claim in that comparison. (LLM, conf=0.85)

  cites [20] Hofmann, J. W. et al. Reduced expression of MYC increases longevity and enhances healthspan. Cell 160, 477–488 (2015). source

In the paper

“Continuous expression of all four factors in normal mice, however, often induces teratomas17–19 or is fatal within days16, ostensibly due to tissue dysplasia18.”

What the sources say

• Partly supported

  The Results report teratomas after four weeks of factor induction in a small proportion of chimeric mice, and dysplastic lesions in all mice examined after 3–9 days. They also report that induced mice became morbid within days, but do not establish that dysplasia caused death; the sentence cites a different source for the fatality claim. The paper therefore supports the occurrence of teratomas and rapid dysplasia, but not the implied frequency of teratomas or the proposed cause of fatality. (LLM, conf=0.84)

  cites [17] Ohnishi, K. et al. Premature termination of reprogramming in vivo leads to cancer development through altered epigenetic regulation. Cell 156, 663–677 (2014). sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [18] Abad, M. et al. Reprogramming in vivo produces teratomas and iPS cells with totipotency features. Nature 502, 340–345 (2013). source
• Partly supported

  The Results report teratomas in wild-type mice given all four OKSM factors, with high-dose mice developing palpable teratomas after 6–13 weeks. The Introduction mentions dysplasia in a separate study of mice with muscle damage, but this paper does not establish tissue dysplasia as the cause of fatality within days. The within-days fatality clause is cited to a different source (16). (LLM, conf=0.86)

  cites [19] Senís, E. et al. AAV vector-mediated in vivo reprogramming into pluripotency. Nat. Commun. 9, 2651 (2018). sourceExperimental — accuracy not yet measured
• Supported

  The section “Cyclic In Vivo Induction of Reprogramming Factors Does Not Lead to Tumor Formation” reports high mortality after four days of continuous OSKM induction in 4F mice. The citing sentence attaches this source to the claim of death within days; its teratoma and dysplasia claims have other citations. (LLM, conf=0.93)

  cites [16] Ocampo, A. et al. In vivo amelioration of age-associated hallmarks by partial reprogramming. Cell 167, 1719–1733.e12 (2016). source

In the paper

“The DNAm age of RGCs was calculated based on a ribosomal DNA methylation clock33, which provided the best site coverage (67/72 CpG sites) and was highly correlated with chronological age (Extended Data Fig. 5g).”

What the sources say

• Partly supported

  The Results section ('CpGs from the rDNA are sufficient to accurately predict age within species'; page unavailable) reports a 72-CpG rDNA clock and a strong correlation between predicted and chronological age in mouse test subsets (median Spearman’s ρ = 0.92). It does not report coverage of 67 of 72 sites, establish that this clock had the best site coverage in the citing study, or test the correlation in RGCs; those appear to be results of the citing study. (LLM, conf=0.86)

  cites [33] Wang, M. & Lemos, B. Ribosomal DNA harbors an evolutionarily conserved clock of biological aging. Genome Res. 29, 325–333 (2019). sourceExperimental — accuracy not yet measured

In the paper

“Evidence from yeast and mammals supports an Information Theory of Ageing10,11, in which the loss of epigenetic information disrupts youthful gene expression patterns1–3, leading to cellular dysfunction and senescence12,13.”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites [10] Sinclair, D. A. & LaPlante, M. D. Lifespan: Why We Age—and Why We Don’t Have To 13–23, 158–175 (Simon & Schuster, 2019).
• ?Not checked — no full text of the sourcecites [11] Shannon, C. E. A mathematical theory of communication. Bell Syst. Tech. J. 27, 379–423 (1948). source
• ?Not checked — no full text of the sourcecites [1] Sinclair, D. A., Mills, K. & Guarente, L. Accelerated aging and nucleolar fragmentation in yeast sgs1 mutants. Science 277, 1313–1316 (1997). source
• ?Not checked — no full text of the sourcecites [2] Imai, S. & Kitano, H. Heterochromatin islands and their dynamic reorganization: a hypothesis for three distinctive features of cellular aging. Exp. Gerontol. 33, 555–570 (1998). source
• Supported

  The Introduction describes chromatin changes causing aging phenotypes in yeast, and the Discussion says the paper presents evidence for a related response in mammals that may contribute to age-related gene-expression changes. This supports the yeast-and-mammal evidence attributed to this citation; the later claims about disrupted gene expression and senescence carry separate citations. (LLM, conf=0.86)

  cites [3] Oberdoerffer, P. et al. SIRT1 redistribution on chromatin promotes genomic stability but alters gene expression during aging. Cell 135, 907–918 (2008). source
• Partly supported

  The Epigenetic Alterations overview (page not provided) says aging is accompanied by epigenetic changes, and the Transcriptional alterations section reports age-related changes in gene expression. The paper also discusses longevity-related evidence from yeast and mammals. It does not set out an Information Theory of Ageing or establish the claimed causal chain from loss of epigenetic information through disrupted youthful expression to cellular dysfunction and senescence. (LLM, conf=0.85)

  cites [12] López-Otín, C., Blasco, M. A., Partridge, L., Serrano, M. & Kroemer, G. The hallmarks of aging. Cell 153, 1194–1217 (2013). sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [13] Yang, J.-H. et al. Erosion of the epigenetic landscape and loss of cellular identity as a cause of aging in mammals. Preprint at https://doi.org/10.1101/808642 (2019). source

In the paper

“IOPs were measured with a rebound TonoLab tonometer (Colonial Medical Supply, Espoo, Finland), as previously described40.”

What the sources say

• Supported

  The Materials and Methods section (‘IOP measurements’; page not provided) explicitly states that IOP was measured with a rebound TonoLab tonometer from Colonial Medical Supply and gives the same location. It also describes the measurement procedure. (LLM, conf=0.95)

  cites [40] Krishnan, A. et al. Overexpression of soluble Fas ligand following adeno-associated virus gene therapy prevents retinal ganglion cell death in chronic and acute murine models of glaucoma. J. Immunol. 197, 4626–4638 (2016). source

In the paper

“Visual acuity of mice was measured using an optomotor reflex-based spatial frequency threshold test58.”

What the sources say

• Supported

  The Materials and methods section ('Optomotor response') states the claim directly and describes how the spatial frequency threshold was determined in mice. (LLM, conf=0.95)

  cites [58] Sun, D., Moore, S. & Jakobs, T. C. Optic nerve astrocyte reactivity protects function in experimental glaucoma and other nerve injuries. J. Exp. Med. 214, 1411-1430 (2017). source

In the paper

“The number of regenerating axons in the optic nerve was estimated by counting the number of CTB-labeled axons at different distances from the crush site as described previously29.”

What the sources say

• Supported

  The paper’s main text (indexed under Abstract; page unavailable) says it assayed optic-nerve regeneration using CTB-labeled axons in sections across the lesion site. It also reports quantifying regeneration by distance from the lesion epicenter, supporting the described counting method. (LLM, conf=0.88)

  cites [29] Park, K. K. et al. Promoting axon regeneration in the adult CNS by modulation of the PTEN/mTOR pathway. Science 322, 963–966 (2008). source

In the paper

“or 248/582 and 77,342/ 193,651 sites (ridge) of two entire lifespan multi-tissue clocks62.”

What the sources say

• Partly supported

  Table 1 in Results (page not provided) lists 582 CpGs for the all-CpGs elastic-net clock and 193,651 for the ridge clock. The paper does not report that 248 or 77,342 of those sites were covered; searches for both coverage figures found no matching passage. (LLM, conf=0.86)

  cites [62] Thompson, M. J. et al. A multi-tissue full lifespan epigenetic clock for mice. Aging (Albany NY) 10, 2832–2854 (2018). sourceExperimental — accuracy not yet measured

In the paper

“The mathematical algorithm and available software underlying the skin & blood clock for in vitro studies (based on 391 CpGs) was previously published57.”

What the sources say

• Supported

  The Results section, “The DNAm age estimator for skin and blood,” says the clock was constructed from 391 CpGs selected by elastic net regression and describes how their weighted average forms the estimator. The Methods section says the R software code underlying the clock is in the Supplement. (LLM, conf=0.92)

  cites [57] Horvath, S. et al. Epigenetic clock for skin and blood cells applied to Hutchinson Gilford Progeria Syndrome and ex vivo studies. Aging (Albany NY) 10, 1758–1775 (2018). source

In the paper

“We chose to use differentiated human neurons that would maintain ageing signatures53.”

What the sources say

• Partly supported

  The Summary reports that directly converted human induced neurons retain aging-associated signatures. It also says that iPSC-derived neurons do not, and the Results section (‘iPSC-Derived iNs Remain Transcriptionally Rejuvenated’) confirms this distinction. The claim is supported only if ‘differentiated human neurons’ means directly converted neurons, not differentiated neurons generally. (LLM, conf=0.88)

  cites [53] Mertens, J. et al. Directly reprogrammed human neurons retain aging-associated transcriptomic signatures and reveal age-related nucleocytoplasmic defects. Cell Stem Cell 17, 705–718 (2015). sourceExperimental — accuracy not yet measured

In the paper

“Interestingly, the signature CpG sites were associated with genes involved in synaptic and neuronal processes and enriched for the binding of polycomb repressive complex 2 (PRC2) and its histone methyltransferase product H3K27me3, both of which are known to recruit Tet enzymes47,48 (Extended Data Fig 10g–i).”

What the sources say

• Partly supported

  The section on maintenance of hypomethylated promoters says that PRC2 and TET proteins can recruit each other, and Fig. 1 explicitly depicts PRC2 recruiting TETs to bivalent promoters. It describes H3K27me3 as a histone mark at these regions, but does not say that H3K27me3 itself recruits TET enzymes. (LLM, conf=0.88)

  cites [47] Wu, X., Li, G. & Xie, R. Decoding the role of TET family dioxygenases in lineage specification. Epigenetics Chromatin 11, 58 (2018). sourceExperimental — accuracy not yet measured
• Partly supported

  The Conclusions state that PRC2 recruits Tet1 to H3K27me3 regions in embryonic stem cells (page not specified). The paper does not establish that H3K27me3 itself recruits Tet enzymes, or that this recruitment applies to Tet enzymes generally or to other cell types. (LLM, conf=0.88)

  cites [48] Neri, F. et al. Genome-wide analysis identifies a functional association of Tet1 and Polycomb repressive complex 2 in mouse embryonic stem cells. Genome Biol. 14, R91 (2013). sourceExperimental — accuracy not yet measured

In the paper

“compared to 102/435 sites of the whole lifespan multi-tissue clock61”

What the sources say

• Partly supported

  The Results (Construction of a multi-tissue clock; page unavailable) state that the whole-lifespan multi-tissue clock contains 435 CpG sites. The Discussion (page unavailable) reports coverage in other datasets, but neither passage reports 102 covered sites; the cited paper therefore supports the denominator, not the claimed coverage count. (LLM, conf=0.89)

  cites [61] Meer, M. V., Podolskiy, D. I., Tyshkovskiy, A. & Gladyshev, V. N. A whole lifespan mouse multi-tissue DNA methylation clock. eLife 7, e40675 (2018). sourceExperimental — accuracy not yet measured

In the paper

“In agreement with previous studies57,63,64, DNAm age estimates of neurons tend to be lower than their chronological age but remain correlated.”

What the sources say

• Supported

  The Results section, “Skin & blood clock applied to brain, liver, bone, and other body parts,” directly reports both findings for sorted neurons: DNAm age correlated strongly with chronological age (r=0.83), while its mean estimate was 6 years in people with a mean chronological age of 31 years. (LLM, conf=0.95)

  cites [57] Horvath, S. et al. Epigenetic clock for skin and blood cells applied to Hutchinson Gilford Progeria Syndrome and ex vivo studies. Aging (Albany NY) 10, 1758–1775 (2018). source
• Partly supported

  The Introduction and Methods state that DNAm age correlates with chronological age in sorted neurons. The Results report lower age acceleration for cerebellar brain samples, particularly in the oldest subjects, but do not establish that neurons generally have DNAm age estimates below chronological age. No page numbers are available in our copy. (LLM, conf=0.88)

  cites [63] Horvath, S. et al. The cerebellum ages slowly according to the epigenetic clock. Aging (Albany NY) 7, 294–306 (2015). sourceExperimental — accuracy not yet measured
• Partly supported

  The Epigenetic clock analysis section (page unavailable) says DNAm age correlates with chronological age in sorted neurons. It does not say neuronal DNAm age estimates tend to be lower than chronological age; the paper’s own reported correlation concerns fetal retinal tissue, not isolated neurons. (LLM, conf=0.86)

  cites [64] Hoshino, A., Horvath, S., Sridhar, A., Chitsazan, A. & Reh, T. A. Synchrony and asynchrony between an epigenetic clock and developmental timing. Sci. Rep. 9, 3770 (2019). sourceExperimental — accuracy not yet measured

In the paper

“That said, we do not wish to imply that DNA methylation is the only epigenetic mark involved in this process. It likely involves other transcription factors and epigenetic modifications, such as the PRC2 complex and H3K27me3, both of which are involved in maintaining stem cell plasticity and are associated with DNAm clock sites4,49,50.”

What the sources say

• Partly supported

  The section comparing the multi-tissue predictor with other age predictors reports that age-increasing clock CpGs are enriched near Polycomb-group target genes; the Additional file 9 description also links them to SUZ12, EED and H3K27me3 occupancy. The paper does not establish that PRC2, H3K27me3 or other transcription factors participate in the process the citing sentence proposes, or that their role in maintaining stem-cell plasticity explains the clock-site association. (LLM, conf=0.85)

  cites [4] Horvath, S. DNA methylation age of human tissues and cell types. Genome Biol. 14, 3156 (2013). sourceExperimental — accuracy not yet measured
• Partly supported

  The sections on genome-wide localization and PRC2 pluripotency and differentiation link PRC2 and H3K27me3 to stem-cell chromatin states and plasticity, though they qualify PRC2’s role in maintaining pluripotency. The paper discusses DNA methylation alongside these marks, but it does not address DNAm clock sites or establish their involvement in the citing paper’s specific process. (LLM, conf=0.82)

  cites [49] Margueron, R. & Reinberg, D. The Polycomb complex PRC2 and its mark in life. Nature 469, 343–349 (2011). sourceExperimental — accuracy not yet measured
• Partly supported

  The Introduction and Discussion §4.4 identify PRC2 as a potential regulator of age-related DNA methylation, and Results §3.5 reports that PRC2 members associate with age-hypermethylated regions. The passages do not establish the attributed role of H3K27me3 or the claim that both PRC2 and H3K27me3 maintain stem-cell plasticity and are associated with DNA-methylation clock sites. No page numbers are available in our copy. (LLM, conf=0.85)

  cites [50] Mozhui, K. & Pandey, A. K. Conserved effect of aging on DNA methylation and association with EZH2 polycomb protein in mice and humans. Mech. Ageing Dev. 162, 27–37 (2017). sourceExperimental — accuracy not yet measured

In the paper

“Another 196 genes that were up-regulated during ageing are involved in ion transport or neuronal projection development, including Efemp1 (Extended Data Fig. 10b–d), a Tet1- and Tet2-regulated gene associated with glaucoma45 and age-related macular degeneration (AMD)46.”

What the sources say

• Supported

  The citation to this paper follows “glaucoma,” so the relevant attribution is EFEMP1’s association with glaucoma, not the citing authors’ Tet1/Tet2 finding or the separately cited AMD association. The Discussion reports an EFEMP1 variant co-segregating with primary open-angle glaucoma in an affected family. (LLM, conf=0.93)

  cites [45] Mackay, D. S., Bennett, T. M. & Shiels, A. Exome sequencing identifies a missense variant in EFEMP1 co-segregating in a family with autosomal dominant primary open-angle glaucoma. PLoS ONE 10, e0132529 (2015). source
• ?Not checked — no full text of the sourcecites [46] Marmorstein, L. Y. et al. Aberrant accumulation of EFEMP1 underlies drusen formation in Malattia Leventinese and age-related macular degeneration. Proc. Natl Acad. Sci. USA 99, 13067–13072 (2002). source

In the paper

“In 12-month-old mice, OSK treatment doubled RGC survival, similar to what was observed in 1- and 3-month-old mice (Extended Data Fig. 4a).”

In the paper

“Even without c-Myc, expression of OSK for five days promoted youthful mRNA profile, with no apparent loss of cellular identity or the induction of Nanog, a transcription factor indicative of pluripotency and oncogenicity (Extended Data Fig. 1a–c).”

In the paper

“After 10–18 months of continuous OSK induction, no increase in tumor incidence or negative effects on overall health were observed, indicating that cellular identity was maintained in OSK-expressing cells (Extended Data Fig.1 j–n).”

In the paper

“The longer the duration of the OSK induction, the greater the distance the axons extended, with no sign of RGC proliferation (Fig. 2b, c, Extended Data Fig. 5f).”

In the paper

“The mRNA levels of Stat3, a pro-regeneration gene28, robustly increased in response to OSK and was maintained post-injury (Extended Data Fig. 5a, Supplementary Table 1).”

In the paper

“Even after 15 months of continuous induction, OSK expression did not induce any tumors or structural changes in the retina (Extended Data Fig. 2c–i, Supplementary Videos).”

In the paper

“67/72 sites with at least 10 reads per site were covered for the ribosomal DNAm clock”

In the paper

“OSK-mediated recovery of neurite area, as well as both the number and length of axons, were blocked by Tet2 knockdown, but not by mTOR-S6K inhibition (Extended Data Fig. 8j–s).”

In the paper

“Of the 703,583 CpGs that were considered, 1,226 were selected (723 that trended towards hypermethylation with age and 503 that trended towards hypomethylation with age, see Supplementary Table 3).”

In the paper

“This resulted in a total of 703,583 sites, covered by uniquely mapped reads.”

In the paper

“The OSK-treated eyes also experienced a significant increase in visual acuity relative to the pre-treatment function baseline, restoring about half of visual acuity in the optomotor response (OMR) assay (Fig. 3c, d).”

In the paper

“Interestingly, the signature CpG sites were associated with genes involved in synaptic and neuronal processes and enriched for the binding of polycomb repressive complex 2 (PRC2) and its histone methyltransferase product H3K27me3, both of which are known to recruit Tet enzymes47,48 (Extended Data Fig 10g–i).”

In the paper

“DNA demethylation is catalyzed by ten-eleven-translocation methylcytosine dioxygenases (TET1–3), either passively through DNA replication or actively, even in non-replicating cells, by reversion to cytosine via thymine DNA glycosylase (TDG)34.”

What the sources say

• ?Not checked — no full text of the sourcecites [34] Wu, X. & Zhang, Y. TET-mediated active DNA demethylation: mechanism, function and beyond. Nat. Rev. Genet. 18, 517–534 (2017). source

In the paper

“Thus, the ability of OSK to increase survival appears to be mediated within the RGC and is largely cell-autonomous.”

In the paper

“Attempts to reverse optic nerve damage have been only moderately successful, with no treatments able to restore eyesight either in late-stage glaucoma or in old age23.”

What the sources say

• ?Not checked — no full text of the sourcecites [23] Roska, B. & Sahel, J. A. Restoring vision. Nature 557, 359–367 (2018). source

In the paper

“Cells were confirmed as negative for mycoplasma”

In the paper

“RNA integrity was determined using TapeStation (Agilent Technologies, Palo Alto, CA, USA).”

• Also stated in: § Ultra-low input RNA library preparation and multiplexing · plain

  “RNA integrity was ascertained using a 2100 TapeStation (Agilent Technologies, Palo Alto, CA, USA).”

In the paper

“All animal procedures were approved by the IACUC of the respective institutions and according to appropriate animal welfare regulations.”

In the paper

“Any mice that developed signs of inflammation (clouding or an edematous cornea) were excluded.”

In the paper

“A sensitivity analysis was conducted by performing TF and histone enrichment for five sets of 1,226 random CpGs from the 702,357 unselected sites, which was then compared against the enrichment for our selected set in in order to identify TFs and histone marks that were specific to the selected set but not CpGs in general.”

In the paper

“All cell lines used were mycoplasma negative.”

In the paper

“PI-stained samples were analyzed on a BD LSR II analyzer and only single cells were gated for analysis.”

In the paper

“Two outliers were excluded by low intercorrelation (< 0.93) within the batch (Supplementary Table 5).”

In the paper

“Two individuals blinded to the experimental groups performed all RGC counts.”

In the paper

“A blinded observer, unable to see the direction of the moving bars, monitored the tracking behavior of the mouse.”

In the paper

“Mice that had intravitreal bleeding or developed signs of inflammation (clouding or an edematous cornea) during or post intravitreal injection were excluded from OMR and histological analyses.”

In the paper

“The final library was assessed by a Qubit 2.0 Fluorometer and an Agilent TapeStation.”

In the paper

“Only CpG sites covered in all samples were considered for analysis (BioProject PRJNA655981).”

In the paper

“All IOPs were taken at the same time of day (between 10:00 and 12:00 hrs) due to the variation of IOP throughout the day.”

In the paper

“All animal work was approved by the Institutional Animal Care and Use Committees (IACUCs) at Harvard Medical School, Boston Children’s Hospital, and the Mass Eye and Ear Institution.”

In the paper

“Exclusion criteria were pre-determined before experimentation.”

In the paper

“Individuals performing axon counts were blinded to the experimental groups.”

In the paper

“Using the eye as a model system, we present evidence that ectopically expressed OSK safely induces in vivo epigenetic restoration of aged CNS neurons, without causing a loss of cell identity or pluripotency.”

In the paper

“Here, we show that it is possible to safely reverse the age of a complex tissue and restore its biological function in vivo.”

In the paper

“Due to the repetitive nature of ribosomal DNA sequence, it is not suitable to whole-genome bisulfite mapping.”

In the paper

“Responses were measured before and after treatment by individuals blinded to the group of the animal and the treatment.”

In the paper

“To avoid batch effects, subsamples were evaluated separately as 6 mo and 18 mo in Batch 1, and 1 mo, 12 mo, and 30 mo in Batch 2 (Supplementary Table 5).”