Mechanisms, pathways and strategies for rejuvenation through epigenetic reprogramming

Transcriptional remodeling studies utilizing OSKM occupancy data are limited to events at the beginning (fibroblasts) or end points (pluripotent stem cells), with everything in between still up for interpretation.

“Importantly, we must note that all of the transcriptional remodeling studies utilizing OSKM occupancy data are limited to the events that occur in the beginning (fibroblasts) or end points (pluripotent stem cells), with everything in between still up for interpretation 80,83,85 .”

• cites [80] Chronis, C. et al. Cooperative binding of transcription factors orchestrates reprogramming. Cell 168, 442–459 (2017). sourceDisputed

  The Results section, “Comprehensive mapping of TFs, chromatin features and expression at defined reprogramming stages” (page not provided), says the study measured OSKM occupancy not only in starting fibroblasts and pluripotent cells, but also at 48 hours and in pre-iPSCs as a proxy for a late intermediate stage. The Discussion likewise describes an analysis at four reprogramming stages, contrary to the claim that occupancy studies are limited to the endpoints. (LLM, conf=0.91)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured
• cites [83] Polo, J. M. et al. A molecular roadmap of reprogramming somatic cells into iPS cells. Cell 151, 1617–1632 (2012). sourcePartly supported

  In Results (‘Transcription dynamics predicts distinct reprogramming factor activities’; page not provided), the paper says it used published genome-wide OSKM occupancy data from pluripotent stem cells to analyze expression changes during reprogramming. But it also reports Oct4 and Sox2 occupancy measured directly in intermediate cells at days 3 and 12, so the claim that everything between the endpoints remains open to interpretation overstates the limitation. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [85] Schwarz, B. A. et al. Prospective isolation of poised iPSC intermediates reveals principles of cellular reprogramming. Cell Stem Cell 23, 289–305 (2018). sourcePartly supported

  In Results (‘Rapid Rewiring of Chromatin States During Reprogramming’; page unavailable), the paper says earlier bulk-culture studies left chromatin changes in rare cells poised to become iPSCs unknown. It also uses ESC ChIP-seq data to assess OKS binding (‘Critical Pluripotency Regions are Hyperaccessible in Poised Intermediates’; page unavailable). But it does not establish that all studies using OSKM occupancy data examined only fibroblasts or pluripotent endpoints; it reports its own analyses of intermediate cells’ transcription and chromatin accessibility. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

Later time points (days 15 and 17) failed to show an epigenetic rejuvenation effect.

“later time points (days 15 and 17) failed to show an epigenetic rejuvenation effect.”

• cites [74] Gill, D. et al. Multi-omic rejuvenation of human cells by maturation phase transient reprogramming. Elife 11, e71624 (2022). sourceDisputed

  In Results, under “Optimal transient reprogramming reverses age-associated changes in the epigenome,” the paper reports smaller reductions in DNA methylation age after longer transient reprogramming, not a complete absence of epigenetic rejuvenation at days 15 and 17. It identifies 13 days as the optimal duration. (LLM, conf=0.89)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured

Many aging-associated epigenetic marks are shed without the initiation of actual pluripotency programs.

“many aging-associated epigenetic marks are shed without the initiation of actual pluripotency programs.”

• cites [114] Olova, N., Simpson, D. J., Marioni, R. E. & Chandra, T. Partial reprogramming induces a steady decline in epigenetic age before loss of somatic identity. Aging Cell 18, e12877 (2019). sourceDisputed

  In Introduction, Results and Discussion (page not available), the paper reports that epigenetic age declines before full pluripotency, but says this decline occurs within the first wave of pluripotency gene expression. It also describes the early reprogramming phase as involving activation of pluripotency genes, contrary to the claim that the decline occurs without initiation of those programs. (LLM, conf=0.87)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured

Loss of epigenetic information has been proposed as a critical cause of aging that precedes many other aspects of age-related deterioration.

“Among the hallmarks of aging, loss of epigenetic information has been proposed as a critical cause of aging that precedes many other aspects of age-related deterioration 2,[5][6][7][8] .”

• cites [2] Booth, L. N. & Brunet, A. The aging epigenome. Mol. Cell 62, 728–744 (2016). sourceSupported
• cites [5] Alle, Q., Borgne, E. L., Milhavet, O. & Lemaitre, J. M. Reprogramming: emerging strategies to rejuvenate aging cells and tissues. Int. J. Mol. Sci. 22, 8 (2021). sourcePartly supported

  Section 2.2.1 identifies epigenetic alterations as one of several primary hallmarks that trigger accumulating harmful consequences; Section 2.2.2 describes later damage responses to primary hallmarks. The paper does not single out loss of epigenetic information as a critical cause that precedes many other aspects of deterioration, and instead presents aging as multifactorial. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [6] Oberdoerffer, P. & Sinclair, D. A. The role of nuclear architecture in genomic instability and ageing. Nat. Rev. Mol. Cell Biol. 8, 692–702 (2007). sourceNot checked — no full text of the source
• cites [7] Sen, P., Shah, P. P., Nativio, R. & Berger, S. L. Epigenetic mechanisms of longevity and aging. Cell 166, 822–839 (2016). sourceNot checked — no full text of the source
• cites [8] Zhang, W., Qu, J., Liu, G. H. & Belmonte, J. C. I. The ageing epigenome and its rejuvenation. Nat. Rev. Mol. Cell Biol. 21, 137–150 (2020). sourceNot checked — no full text of the source

Multiple interventions, acting at different levels, can delay or even reverse the aging process.

“In contrast to the view of aging as an irreversible and unidirectional process akin to an increase in cell entropy, recent studies have demonstrated that multiple interventions, acting at different levels, can delay or even reverse this process [10][11][12][13][14][15][16][17] .”

• cites [10] Cole, J. J. et al. Diverse interventions that extend mouse lifespan suppress shared age-associated epigenetic changes at critical gene regulatory regions. Genome Biol 18, 58 (2017). sourcePartly supported

  The Background (page unavailable) says genetic, dietary, and drug interventions can extend longevity and healthspan, supporting the claim that interventions at different levels can delay aging. The paper reports suppression of age-associated epigenetic changes, but does not demonstrate reversal of the aging process. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [11] Conboy, I. M. et al. Rejuvenation of aged progenitor cells by exposure to a young systemic environment. Nature 433, 760–764 (2005). sourceNot checked — no full text of the source
• cites [12] Martel, J. et al. Emerging use of senolytics and senomorphics against aging and chronic diseases. Med. Res. Rev. 40, 2114–2131 (2020). sourceNot checked — no full text of the source
• cites [13] Mehdipour, M. et al. Plasma dilution improves cognition and attenuates neuroinflammation in old mice. Geroscience 43, 1–18 (2021). sourcePartly supported

  The Introduction describes slowing or reversing aging as a prominent interpretation of heterochronic-parabiosis studies. The Discussion reports rejuvenative effects from plasma dilution and discusses drug-based approaches, supporting the point that interventions can improve age-related outcomes through different mechanisms. It does not demonstrate reversal of the aging process as a whole; its findings concern particular tissues and brain-related outcomes. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [14] Mehdipour, P. et al. Epigenetic therapy induces transcription of inverted SINEs and ADAR1 dependency. Nature 588, 169–173 (2020). sourceCould not verify

  The Abstract and Main section report effects of epigenetic therapy and ADAR1 depletion on cancer cells and tumour growth, not on aging. Searches for interventions that delay or reverse aging, including rejuvenation and lifespan, found no passage addressing the claim. No page numbers are available in our copy. (LLM, conf=0.76)

  Experimental — accuracy not yet measured
• cites [15] Ocampo, A. et al. In vivo amelioration of age-associated hallmarks by partial reprogramming. Cell 167, 1719–1733.e1712 (2016). sourceSupported
• cites [16] Ros, M. & Carrascosa, J. M. Current nutritional and pharmacological anti-aging interventions. Biochim. Biophys. Acta Mol. Basis Dis. 1866, 165612 (2020). sourceNot checked — no full text of the source
• cites [17] Sarkar, T. J. et al. Transient non-integrative expression of nuclear reprogramming factors promotes multifaceted amelioration of aging in human cells. Nat. Commun. 11, 1545 (2020). sourceSupported

Aging-associated DNA methylation patterns and biomarkers persisted when cells were transdifferentiated to different cell types.

“Conversely, other works have shown that aging-associated DNA methylation patterns and biomarkers persisted when cells were transdifferentiated (Box 1) to different cell types, emphasizing the necessity of partial de-differentiation for rejuvenation (Box 1) to occur [22][23][24][25] .”

• cites [22] Ahlenius, H. et al. FoxO3 regulates neuronal reprogramming of cells from postnatal and aging mice. Proc. Natl Acad, Sci, USA 113, 8514–8519 (2016). sourceCould not verify

  The section “Several Aging-Associated Cellular Features Correlate with Reduced Reprogrammability” reports senescence markers in donor fibroblasts, while “Developmental Stage but Not Adult Aging Impairs iN Cell Reprogramming” reports their conversion into neurons. Neither section establishes that aging biomarkers persisted after conversion. Searches for DNA methylation patterns, epigenetic age, and retained markers found no passage addressing that claim. (LLM, conf=0.76)

  Experimental — accuracy not yet measured
• cites [23] Huh, C. J. et al. Maintenance of age in human neurons generated by microRNA-based neuronal conversion of fibroblasts. eLife 5, e18648 (2016). sourceSupported
• cites [24] Kim, Y. et al. Mitochondrial aging defects emerge in directly reprogrammed human neurons due to their metabolic profile. Cell Rep. 23, 2550–2558 (2018). sourceSupported
• cites [25] 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). sourcePartly supported

  The Results report that neurons directly converted from donor fibroblasts retained age-associated transcriptional and functional signatures. They do not establish that age-associated DNA methylation patterns persisted through direct conversion; the Discussion instead mentions methylation patterns being erased during iPSC reprogramming. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Partial de-differentiation is necessary for rejuvenation to occur.

“emphasizing the necessity of partial de-differentiation for rejuvenation (Box 1) to occur [22][23][24][25] .”

• cites [22] Ahlenius, H. et al. FoxO3 regulates neuronal reprogramming of cells from postnatal and aging mice. Proc. Natl Acad, Sci, USA 113, 8514–8519 (2016). sourceCould not verify

  The Developmental Stage section reports direct conversion of fibroblasts from aged mice into neurons, and the Discussion addresses how age affects reprogramming. Neither section addresses rejuvenation or establishes that partial de-differentiation is necessary for it (searched: partial de-differentiation, rejuvenation, and age-associated characteristics). (LLM, conf=0.77)

  Experimental — accuracy not yet measured
• cites [23] Huh, C. J. et al. Maintenance of age in human neurons generated by microRNA-based neuronal conversion of fibroblasts. eLife 5, e18648 (2016). sourcePartly supported

  The Introduction says that inducing pluripotency in adult fibroblasts reverts cellular age, while direct neuronal conversion bypasses stem-cell stages and retains the donor’s age signature. This supports a link between reprogramming through pluripotency and rejuvenation, but the paper does not establish that partial de-differentiation is necessary for rejuvenation. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [24] Kim, Y. et al. Mitochondrial aging defects emerge in directly reprogrammed human neurons due to their metabolic profile. Cell Rep. 23, 2550–2558 (2018). sourcePartly supported

  The Results section, “iPSC-Derived Neurons from Aging Donors Show No Age-Related Mitochondrial Defects” (page unavailable), reports mitochondrial rejuvenation after iPSC reprogramming and no age-related mitochondrial differences in the iPSC-derived neurons tested. The paper contrasts this with directly converted neurons that retain aging features, but it does not establish that partial de-differentiation is necessary for rejuvenation. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [25] 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). sourcePartly supported

  The Results section reports that neurons derived through iPSCs remained largely rejuvenated, whereas directly converted neurons retained aging signatures. This supports a link between passage through a pluripotent state and rejuvenation, but the paper does not test partial de-differentiation or establish that it is necessary. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

Aging probably stems from a failure of maintenance mechanisms that ultimately impact epigenetic regulation and gene expression.

“The precise reason for why we age is not completely understood, but the progressive nature and influence of external factors suggest that the process probably stems from a failure of maintenance mechanisms that ultimately impact epigenetic regulation and gene expression 1,2 .”

• cites [1] Lopez-Otin, C., Blasco, M. A., Partridge, L., Serrano, M. & Kroemer, G. Hallmarks of aging: an expanding universe. Cell 186, 243–278 (2023). sourcePartly supported

  Genomic Instability (p. 4) says DNA repair and maintenance mechanisms lose efficiency with age, while Epigenetic Alterations (p. 9) and Gene Expression Changes (p. 13) link age-related epigenetic changes to gene expression. The paper does not establish the claimed causal chain in which failure of maintenance mechanisms is the probable origin of aging and ultimately causes those epigenetic and expression changes. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [2] Booth, L. N. & Brunet, A. The aging epigenome. Mol. Cell 62, 728–744 (2016). sourceSupported

The long-term impact of interventions on whole organisms is still poorly understood.

“Such interventions have been successful in reducing cell damage and preserving tissue health, but the long-term impact on whole organisms is still poorly understood [11][12][13][14] .”

• cites [11] Conboy, I. M. et al. Rejuvenation of aged progenitor cells by exposure to a young systemic environment. Nature 433, 760–764 (2005). sourceNot checked — no full text of the source
• cites [12] Martel, J. et al. Emerging use of senolytics and senomorphics against aging and chronic diseases. Med. Res. Rev. 40, 2114–2131 (2020). sourceNot checked — no full text of the source
• cites [13] Mehdipour, M. et al. Plasma dilution improves cognition and attenuates neuroinflammation in old mice. Geroscience 43, 1–18 (2021). sourceCould not verify

  The Results describe cognitive and neuroinflammation measurements six days after a single plasma exchange in old mice; the proteomics comparison includes human samples collected one month after treatment (Results, Fig. 5). Our copy does not state that the long-term impact of interventions on whole organisms is poorly understood. Searches for long-term outcomes, lifespan, and longitudinal follow-up did not locate that point. (LLM, conf=0.72)

  Experimental — accuracy not yet measured
• cites [14] Mehdipour, P. et al. Epigenetic therapy induces transcription of inverted SINEs and ADAR1 dependency. Nature 588, 169–173 (2020). sourceCould not verify

  The Main section reports short-term tumour outcomes in mice, including measurements around days 14–15, and cellular responses up to 24 days. Neither those passages nor searches for long-term organism-level effects state that the long-term impact of interventions on whole organisms is poorly understood. (LLM, conf=0.74)

  Experimental — accuracy not yet measured

The external environment and the genetic background of an organism have pivotal roles in the aging process by affecting events that maintain epigenetic information.

“Several studies highlight how the external environment and the genetic background of an organism have pivotal roles in the aging process by affecting events that maintain epigenetic information 2,3 .”

• cites [2] Booth, L. N. & Brunet, A. The aging epigenome. Mol. Cell 62, 728–744 (2016). sourcePartly supported

  The Introduction says chromatin modifiers respond to extracellular signals and that epigenomic changes may contribute to aging; the Outlook says environmental stimuli can modulate epigenomic networks. These passages support an environmental link, but they do not establish that an organism’s genetic background affects aging through events that maintain epigenetic information. The Outlook also frames the consequences of environmental modulation as a subject for further study. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [3] Anderson, R. M., Bitterman, K. J., Wood, J. G., Medvedik, O. & Sinclair, D. A. Nicotinamide and PNC1 govern lifespan extension by calorie restriction in Saccharomyces cerevisiae. Nature 423, 181–185 (2003). sourcePartly supported

  The paper’s main text reports that calorie restriction and mild stress extend yeast lifespan through PNC1 and Sir2, and that manipulating nicotinamide metabolism changes rDNA silencing—an epigenetic process. This supports the environmental, genetic and epigenetic links in yeast, but the claim’s organism-wide wording goes beyond the paper’s direct findings; its Abstract only speculates about related regulation in higher organisms. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

The modulatory activities of OSK and MYC on each other are bidirectional.

“supporting the notion that the modulatory activities of OSK and MYC on each other are bidirectional.”

• cites [53] Soufi, A., Donahue, G. & Zaret, K. S. Facilitators and impediments of the pluripotency reprogramming factors’ initial engagement with the genome. Cell 151, 994–1004 (2012). sourceSupported

MYC primarily facilitates early gene induction for proliferation.

“First, MYC primarily facilitates early gene induction for proliferation, whereas OCT4 and SOX2 induce chromatin opening while also binding readily open chromatin enriched with KLF4 motifs, supporting the notion that KLF4 recruits OCT4 and SOX2 to somatic chromatin that later becomes closed 83 .”

• cites [83] Polo, J. M. et al. A molecular roadmap of reprogramming somatic cells into iPS cells. Cell 151, 1617–1632 (2012). sourceSupported

Repressive H3K27me3 is depleted only in enhancers that gain H3K4me2 toward a pluripotent state.

“By contrast, repressive H3K27me3 is depleted only in enhancers that gain H3K4me2 toward a pluripotent state.”

• cites [86] Koche, R. P. et al. Reprogramming factor expression initiates widespread targeted chromatin remodeling. Cell Stem Cell 8, 96–105 (2011). sourcePartly supported

  The Results section, “Repressive H3K27me3 is lost specifically at sites where H3K4 methylation is gained” (page unavailable), reports H3K27me3 depletion where H3K4me2 increases, but examines promoters, not enhancers. The enhancer Results section describes H3K4me2 changes toward an ES-cell-like state; it does not establish H3K27me3 depletion at those enhancers or the claim that depletion occurs only there. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

MYC is considered one of the most potent amplifiers of reprogramming.

“it is considered one of the most potent amplifiers of reprogramming 58 .”

• cites [58] Smith, Z. D., Sindhu, C. & Meissner, A. Molecular features of cellular reprogramming and development. Nat. Rev. Mol. Cell Biol. 17, 139–154 (2016). sourcePartly supported

  The section “Transcriptional changes during early reprogramming” (page unavailable) says MYC enhances transcription of responsive cell-cycle genes after OSKM induction, supporting a role in early reprogramming. It does not rank MYC among the most potent amplifiers of reprogramming; the amplification described is of transcription, not reprogramming itself. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

On nucleosomes, levels of H3K4me2 change rapidly at enhancers of pluripotency and development-related genes before gene expression changes are observed.

“On nucleosomes, levels of H3K4me2, a transcriptional activation mark, change rapidly at enhancers of pluripotency and development-related genes before gene expression changes are observed.”

• cites [86] Koche, R. P. et al. Reprogramming factor expression initiates widespread targeted chromatin remodeling. Cell Stem Cell 8, 96–105 (2011). sourcePartly supported

  The Results section reports rapid H3K4me2 changes at promoters of pluripotency and development-related genes before their transcriptional activation. Its enhancer section separately reports rapid H3K4me2 changes, but does not establish that those enhancers belong to the specified genes or that their changes precede gene-expression changes. Page numbers are unavailable in our copy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

OSK factors interact with the genome and each other differently in mice than in human cells.

“OSK factors interact with the genome and each other differently in mice than in human cells [80][81][82] , complicating the design and interpretation of studies.”

• cites [80] Chronis, C. et al. Cooperative binding of transcription factors orchestrates reprogramming. Cell 168, 442–459 (2017). sourcePartly supported

  The Results section, “Changes in OSK binding mirror enhancer re-organization” (page unavailable), says that early OSK binding in mouse cells predominantly targets initially open chromatin, unlike what has been reported for human-cell reprogramming. The paper also describes cooperation among OSK factors in mouse cells, but does not establish that their interactions with each other differ between mouse and human cells. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [81] Knaupp, A. S. et al. Transient and permanent reconfiguration of chromatin and transcription factor occupancy drive reprogramming. Cell Stem Cell 21, 834–845 (2017). sourcePartly supported

  The Introduction (page not available) describes conflicting reports of OSK chromatin targeting in mouse and human reprogramming studies, supporting a reported difference in genome interactions. It does not establish a species-specific difference in how OSK factors interact with one another; the Discussion (page not available) also reports mouse results consistent with pioneer-factor activity, so the contrast is less settled than the claim suggests. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [82] Narayan, S., Bryant, G., Shah, S., Berrozpe, G. & Ptashne, M. OCT4 and SOX2 work as transcriptional activators in reprogramming human fibroblasts. Cell Rep. 20, 1585–1596 (2017). sourcePartly supported

  The Results (‘Changing locations of DNA bound factors’; page not available) describe OCT4 and SOX2 binding to DNA and co-binding in human cells. The paper does not compare those interactions—or KLF4’s—with mouse cells, so it does not support the claimed mouse–human difference. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

OCT4 overexpression alone is sufficient to induce pluripotency when other canonical reprogramming factors are endogenously expressed or are in the presence of other chromatin remodeling chemical factors.

“as OCT4 overexpression alone is sufficient to induce pluripotency when other canonical reprogramming factors are endogenously expressed or are in the presence of other chromatin remodeling chemical factors 32,34,35 .”

• cites [32] Peskova, L., Cerna, K., Oppelt, J., Mraz, M. & Barta, T. OCT4-mediated reprogramming induces embryonic-like microRNA expression signatures in human fibroblasts. Nat. Sci. Rep. 9, 15759 (2019). sourcePartly supported

  The Introduction (page unavailable) says Oct4 overexpression combined with small molecules has been sufficient to reprogram fibroblasts into iPSCs. It does not establish the alternative condition that other canonical factors are endogenously expressed, or identify those small molecules as chromatin-remodeling agents. The Discussion (page unavailable) also distinguishes the plastic state observed with Oct4 alone from reprogramming toward iPSCs. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [34] Kim, J. B. et al. Direct reprogramming of human neural stem cells by OCT4. Nature 461, 649–653 (2009). sourceNot checked — no full text of the source
• cites [35] Zhu, S. et al. Reprogramming of human primary somatic cells by OCT4 and chemical compounds. Cell Stem Cell 7, 651–655 (2010). sourcePartly supported

  The paper’s introduction reports OCT4-only reprogramming of neural stem cells that endogenously express SOX2 and cMYC, and its results show reprogramming of primary human cells using OCT4 as the sole introduced factor plus a chemical cocktail (page unavailable in our copy). But the cocktail is not simply chromatin-remodeling chemicals: it includes a histone deacetylase inhibitor alongside compounds targeting signaling and metabolism. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

OCT4 binds regulatory regions of pluripotency network genes to create an autoregulatory pluripotency network.

“Third, OCT4 binds regulatory regions of pluripotency network genes to create an autoregulatory pluripotency network 42,43 .”

• cites [42] Jerabek, S. et al. Changing POU dimerization preferences converts OCT6 into a pluripotency inducer. EMBO Rep. 18, 319–333 (2017). sourcePartly supported

  The Introduction (page unavailable) says genome-wide studies identified Sox2–Oct4 binding elements in enhancers of pluripotency-related genes, including Pou5f1 (the Oct4 gene), Nanog, and Utf1. This supports binding to regulatory regions, including one associated with Oct4 itself, but the paper does not establish that this binding creates an autoregulatory pluripotency network. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [43] You, J. S. et al. OCT4 establishes and maintains nucleosome-depleted regions that provide additional layers of epigenetic regulation of its target genes. Proc. Natl Acad. Sci. USA 108, 14497–14502 (2011). sourceSupported

Alterations or additions of specific chromatin features are thought to prime the transient epigenetic landscape for reprogramming before gene regulation.

“Thus, alterations or additions of specific chromatin features are thought to 'prime' the transient epigenetic landscape for reprogramming before gene regulation 85 .”

• cites [85] Schwarz, B. A. et al. Prospective isolation of poised iPSC intermediates reveals principles of cellular reprogramming. Cell Stem Cell 23, 289–305 (2018). sourceSupported

OCT4 directly binds enhancers of Polycomb group-repressed genes to induce conversion of their associated promoters from monovalent to bivalent domains.

“Second, OCT4 directly binds enhancers of Polycomb group-repressed genes, which modify histones and silence target genes, to induce conversion of their associated promoters from monovalent to bivalent domains 40,41 .”

• cites [40] Taberlay, P. C. et al. Polycomb-repressed genes have permissive enhancers that initiate reprogramming. Cell 147, 1283–1294 (2011). sourcePartly supported

  The Results section reports OCT4 occupancy at the MYOD1 enhancer before the MYOD1 promoter acquired a bivalent signature. But the Discussion says OCT4 may establish bivalency directly or may act through indirect global changes; the experiment does not establish a direct causal mechanism across Polycomb-repressed genes generally. (LLM, conf=0.88)

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• cites [41] Fu, K. et al. Comparison of reprogramming factor targets reveals both species-specific and conserved mechanisms in early iPSC reprogramming. BMC Genomics 19, 956 (2018). sourcePartly supported

  The Results section on syntenic conserved peaks reports OCT4 binding peaks in enhancer regions and a higher proportion of those peaks in bivalent promoters. It does not identify those enhancers as belonging to Polycomb-repressed genes or show that OCT4 binding converts associated promoters from monovalent to bivalent domains. Page numbers are unavailable in our copy. (LLM, conf=0.84)

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During full reprogramming, OCT4 directly recruits the BAF chromatin remodeling complex to promote a euchromatic chromatin state that enhances reprogramming factor binding.

“First, OCT4 directly recruits the BAF chromatin remodeling complex to promote a euchromatic chromatin state that enhances reprogramming factor binding [37][38][39] .”

• cites [37] Chen, K. et al. Heterochromatin loosening by the OCT4 linker region facilitates KLF4 binding and iPSC reprogramming. EMBO J. 39, 99165 (2020). sourceNot checked — no full text of the source
• cites [38] Singhal, N. et al. Chromatin-remodeling components of the baf complex facilitate reprogramming. Cell 141, 943–955 (2010). sourceNot checked — no full text of the source
• cites [39] Singhal, N. et al. BAF complex enhances reprogramming of adult human fibroblasts. J. Stem Cell Res. Ther. 6, 336 (2016). sourcePartly supported

  The Introduction (page unavailable) says Brg1 favors a euchromatic structure and thereby enhances Oct4 recruitment; the results section reports that Brg1 and Baf155 improve reprogramming. The source does not say that OCT4 directly recruits the BAF complex. That reverses the recruitment relationship described in the Introduction. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Electron transport chain proteins are downregulated in the first 3 days of reprogramming before a gradual metabolic switch to glycolysis, a characteristic typically seen in rapidly proliferating cells.

“Similar to gene expression changes, clustering analysis of proteins with temporal expression dynamics similar to gene expression changes reveals that electron transport chain proteins are downregulated in the first 3 days of reprogramming before a gradual metabolic switch to glycolysis, a characteristic that is typically seen in rapidly proliferating cells 84,97 .”

• cites [84] Hansson, J. et al. Highly coordinated proteome dynamics during reprogramming of somatic cells to pluripotency. Cell Rep. 2, 1579–1592 (2012). sourcePartly supported

  The Results section, “The metabolic switch to glycolysis is preceded by a stoichiometric change in the electron transport system,” supports an early decrease in some electron transport proteins followed by a gradual shift toward glycolysis, which it describes as characteristic of fast-proliferating cells. But it specifies that only complexes I and IV were downregulated by day 3; complexes II, III and V increased. The claim overgeneralizes the downregulation to electron transport chain proteins as a whole. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [97] Vander Heiden, M. G., Cantley, L. C. & Thompson, C. B. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324, 1029–1033 (2009). sourceSupported

MYC binding increases by 40-fold with OSK.

“and MYC binding increases by 40-fold with OSK, supporting the notion that the modulatory activities of OSK and MYC on each other are bidirectional.”

• cites [53] Soufi, A., Donahue, G. & Zaret, K. S. Facilitators and impediments of the pluripotency reprogramming factors’ initial engagement with the genome. Cell 151, 994–1004 (2012). sourceSupported

OCT4 is the master regulator of epigenetic reprogramming.

“Multiple studies have suggested OCT4 (Fig. 1a) as the master regulator of epigenetic reprogramming [31][32][33] , as OCT4 overexpression alone is sufficient to induce pluripotency when other canonical reprogramming factors are endogenously expressed or are in the presence of other chromatin remodeling chemical factors 32,34,35 .”

• cites [31] Kim, K. P., Han, D. W., Kim, J. & Schöler, H. R. Biological importance of OCT transcription factors in reprogramming and development. Nat. Exp. Mol. Med. 53, 1018–1028 (2021). sourceSupported
• cites [32] Peskova, L., Cerna, K., Oppelt, J., Mraz, M. & Barta, T. OCT4-mediated reprogramming induces embryonic-like microRNA expression signatures in human fibroblasts. Nat. Sci. Rep. 9, 15759 (2019). sourceSupported
• cites [33] Radzisheuskaya, A. & Silva, J. C. R. Do all roads lead to OCT4? The emerging concepts of induced pluripotency. Trends Cell Biol. 24, 275–284 (2014). sourcePartly supported

  The Concluding remarks call Oct4 an essential transcriptional regulator in reprogramming, and ‘Overcoming epigenetic barriers’ describes its role in opening repressed chromatin. But ‘Molecular mechanisms of Oct4 in reprogramming’ says its precise mechanism remains unclear, while the epigenetic-barrier section describes its possible role in overcoming a specific barrier. The paper does not establish Oct4 as the master regulator of epigenetic reprogramming. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

Distinct mechanisms observed among combinations of OCT4 and SOX2 (without KLF4) versus OSK may explain the controversy regarding whether OSK mainly function as pioneer factors or bind readily open chromatin in early reprogramming, or both.

“The distinct mechanisms observed among combinations of OCT4 and SOX2 (without KLF4) versus OSK may explain the controversy regarding whether OSK mainly function as the pioneer factors or bind readily open chromatin in early reprogramming, or both 80,82,83,85 .”

• cites [80] Chronis, C. et al. Cooperative binding of transcription factors orchestrates reprogramming. Cell 168, 442–459 (2017). sourceSupported
• cites [82] Narayan, S., Bryant, G., Shah, S., Berrozpe, G. & Ptashne, M. OCT4 and SOX2 work as transcriptional activators in reprogramming human fibroblasts. Cell Rep. 20, 1585–1596 (2017). sourcePartly supported

  The Results section (‘Putative enhancers at sites of bound transcription factors’; page unavailable) reports that OCT4 and SOX2 often bound sites where new putative enhancers formed, whereas KLF4-bound enhancers were almost all pre-existing. This supports a distinction between the factors in early reprogramming, but the paper does not compare an OCT4–SOX2 combination without KLF4 against OSK or establish that such a comparison explains the pioneer-factor controversy. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [83] Polo, J. M. et al. A molecular roadmap of reprogramming somatic cells into iPS cells. Cell 151, 1617–1632 (2012). sourcePartly supported

  The Results section ('Transcription dynamics predicts distinct reprogramming factor activities'; page not provided) reports gradual activation of OCT4/SOX2 targets, a distinct early and late role for KLF4, and a correlation between OS-target activation and chromatin state. It does not compare OCT4/SOX2-only reprogramming with OSK reprogramming or address whether OSK act as pioneer factors rather than bind open chromatin. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [85] Schwarz, B. A. et al. Prospective isolation of poised iPSC intermediates reveals principles of cellular reprogramming. Cell Stem Cell 23, 289–305 (2018). sourcePartly supported

  The Results section, “Rapid Rewiring of Chromatin States During Reprogramming” (page unavailable), describes OKS as pioneer factors that bind closed chromatin. “Critical Pluripotency Regions are Hyperaccessible in Poised Intermediates” reports binding by Oct4 and Sox2 with or without Klf4 at hyperaccessible sites. Neither passage compares distinct mechanisms for those combinations or proposes that they explain the stated controversy. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

Small molecule stimulation facilitates chemical reprogramming by inducing an intermediate plastic state.

“Recent studies have demonstrated the potential of small molecule stimulation in facilitating chemical reprogramming by inducing an intermediate plastic state [71][72][73] .”

• cites [71] Guan, J. et al. Chemical reprogramming of human somatic cells to pluripotent stem cells. Nature 605, 325–331 (2022). sourceNot checked — no full text of the source
• cites [72] Hou, P. et al. Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds. Science 341, 651–654 (2013). sourceNot checked — no full text of the source
• cites [73] Liuyang, S. et al. Highly efficient and rapid generation of human pluripotent stem cells by chemical reprogramming. Cell Stem Cell 30, 450–459 (2023). sourceSupported

MYC does not exert pioneer factor function unlike the OSK factors.

“In contrast with the OSK factors, which function synergistically as pioneer factors during reprogramming, MYC (Fig. 1d) does not exert this function 55 .”

• cites [55] Soufi, A. et al. Pioneer transcription factors target partial DNA motifs on nucleosomes to initiate reprogramming. Cell 161, 555–568 (2015). sourceSupported

KLF4 and MYC are believed to drive the first wave of transcriptional activation.

“and KLF4 (as well as MYC, described in detail below) is believed to drive the first wave of transcriptional activation 42 .”

• cites [42] Jerabek, S. et al. Changing POU dimerization preferences converts OCT6 into a pluripotency inducer. EMBO Rep. 18, 319–333 (2017). sourceCould not verify

  The Introduction (page unavailable) identifies Klf4 and c-Myc as reprogramming factors, but does not say they drive a first wave of transcriptional activation. Searches for that phrase and for early transcriptional activation found no passage in our copy addressing the claimed timing or role. (LLM, conf=0.72)

  Experimental — accuracy not yet measured

OCT4-SOX2 binding increases KLF4 binding by several folds, mostly in chromatin regions that are closed in human fibroblasts.

“Co-immunoprecipitation and chromatin immunoprecipitation followed by sequencing studies reveal that OCT4-SOX2 binding increases KLF4 binding by several folds, mostly in chromatin regions that are closed in human fibroblasts 41,53,54 .”

• cites [41] Fu, K. et al. Comparison of reprogramming factor targets reveals both species-specific and conserved mechanisms in early iPSC reprogramming. BMC Genomics 19, 956 (2018). sourcePartly supported

  The Results section on general OSKM binding reports that OCT4, SOX2 and KLF4 co-bind more often than expected. The Results section on starting-cell chromatin states reports that about 40–50% of their human binding peaks lie in low-signal regions. Neither passage shows that OCT4–SOX2 binding causes a several-fold increase in KLF4 binding, or that this increase occurs mostly in closed chromatin. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [53] Soufi, A., Donahue, G. & Zaret, K. S. Facilitators and impediments of the pluripotency reprogramming factors’ initial engagement with the genome. Cell 151, 994–1004 (2012). sourcePartly supported

  The Results section, “Oct4, Sox2, and Klf4 bind as pioneer factors to closed chromatin” (page unavailable), reports that most initial binding events occur in closed chromatin in human fibroblasts. But the reported several-fold increase is c-Myc enhancing Klf4 occupancy, not OCT4-SOX2 increasing KLF4 binding. The paper does not establish the claimed direction of that effect. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [54] Wei, Z. et al. KLF4 organizes long-range chromosomal interactions with the OCT4 locus inreprogramming andpluripotency. Cell Stem Cell 13, 36–47 (2013). sourceCould not verify

  The Results report that Oct4, Sox2, and Klf4 bind the Oct4 distal enhancer and that Klf4 occupancy is higher in SSEA1+ than SSEA1− cells. Neither those passages nor searches for increased Klf4 binding and closed chromatin in human fibroblasts establish that OCT4-SOX2 binding increases KLF4 binding several-fold, or where such an increase occurs. (LLM, conf=0.72)

  Experimental — accuracy not yet measured

Vitamin C, IL-6, TGFβ and bone morphogenetic proteins regulate cellular reprogramming by affecting DNA methylation levels, thereby adjusting the expression of certain microRNAs or facilitating the mesenchymal-to-epithelial transition (MET).

“In addition to cell intrinsic transcription factors for cellular reprogramming, modulators such as vitamin C [62][63][64][65] , IL-6 [66][67][68] , TGFβ 69 and bone morphogenetic proteins 70 have been shown to regulate cellular reprogramming by affecting DNA methylation levels, thereby adjusting the expression of certain microRNAs or facilitating the mesenchymal-to-epithelial transition (MET).”

• cites [62] Esteban, M. A. et al. Vitamin C enhances the generation of mouse and human induced pluripotent stem cells. Cell Stem Cell 6, 71–79 (2010). sourcePartly supported

  The Discussion reports that vitamin C enhances reprogramming of mouse and human cells. It only proposes histone demethylation as a possible mechanism; it does not establish that vitamin C acts through DNA methylation, microRNA expression, or MET. This verdict concerns the vitamin C citations, not the separately cited modulators. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [63] Lee Chong, T., Ahearn, E. L. & Cimmino, L. Reprogramming the epigenome with vitamin C. Front. Cell Dev. Biol. 7, 128 (2019). sourceSupported
• cites [64] Stadtfeld, M. et al. Ascorbic acid prevents loss of Dlk1–Dio3 imprinting and facilitates generation of all-iPS cell mice from terminally differentiated B cells. Nat. Genet. 44, 398–405 (2012). sourcePartly supported

  The “Serum replacement and ascorbic acid prevent Gtl2 silencing” section reports that vitamin C prevented abnormal DNA hypermethylation during fibroblast reprogramming and increased reprogramming efficiency. The Discussion only raises the possibility that maintaining Dlk1-Dio3 imprinting could affect a microRNA involved in reprogramming; it does not establish that mechanism or report facilitation of MET. This verdict addresses the vitamin C portion to which this paper’s citation is attached, not the separately cited modulators. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [65] Wang, T. et al. The histone demethylases JHDM1A/1B enhance somatic cell reprogramming in a vitamin-C-dependent manner. Cell Stem Cell 9, 575–587 (2011). sourceNot checked — no full text of the source
• cites [66] Li, L. et al. Kupffer-cell-derived IL-6 is repurposed for hepatocyte dedifferentiation via activating progenitor genes from injury-specific enhancers. Cell Stem Cell 30, 283–299 (2023). sourcePartly supported

  The Introduction and the section “IL-6/STAT3 signaling is repurposed for hepatocyte reprogramming” support the IL-6 portion: Kupffer-cell-derived IL-6 promotes hepatocyte reprogramming through STAT3 and progenitor-gene expression. They do not support the attributed mechanism of altered DNA methylation, microRNA expression, or mesenchymal-to-epithelial transition; the paper instead describes STAT3 binding to injury-specific enhancers. Page numbers were not available in our copy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [67] Zhu, H. et al. IL-6 coaxes cellular dedifferentiation as a pro-regenerative intermediate that contributes to pericardial ADSC-induced cardiac repair. Stem Cell Res. Ther 13, 44 (2022). sourcePartly supported

  The Discussion section (‘Cellular dedifferentiation as a pre-regenerative intermediate’; page not provided) reports that IL-6/STAT3 signaling induces cardiomyocyte dedifferentiation, supporting the IL-6 reprogramming component. It does not link that effect to DNA methylation, altered microRNA expression, or mesenchymal-to-epithelial transition; instead, the Background describes JAK/STAT and other signaling pathways. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [68] Mosteiro, L. et al. Tissue damage and senescence provide critical signals for cellular reprogramming in vivo. Science 354, aaf4445 (2016). sourceNot checked — no full text of the source
• cites [69] Li, W., Wei, W. & Ding, S. TGF-β signaling in stem cell regulation. Methods Mol. Biol. 1344, 137–145 (2016). sourceNot checked — no full text of the source
• cites [70] Chen, J. et al. BMPs functionally replace KLF4 and support efficient reprogramming of mouse fibroblasts by OCT4 alone. Cell Res 21, 205–212 (2011). sourceNot checked — no full text of the source

Early transient events evoked by OSKM factors, such as somatic silencing and chromatin remodeling while reprogramming is initiated, seem to be required for both cellular rejuvenation and full reprogramming.

“Despite these different contexts, 'early transient events' evoked by OSKM factors, such as somatic silencing and chromatin remodeling while reprogramming is initiated, seem to be required for both cellular rejuvenation and full reprogramming [74][75][76] .”

• cites [74] Gill, D. et al. Multi-omic rejuvenation of human cells by maturation phase transient reprogramming. Elife 11, e71624 (2022). sourcePartly supported

  The Results section on cell identity describes somatic-expression repression during the initiation phase and reports a reduction in DNA methylation age during reprogramming. The Discussion reports epigenetic rejuvenation with transient reprogramming, particularly after longer exposure. The paper does not establish that early somatic silencing or chromatin remodeling is required for either rejuvenation or full reprogramming. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [75] Guo, L. et al. Generation of induced progenitor-like cells from mature epithelial cells using interrupted reprogramming. Stem Cell Rep. 9, 1780–1795 (2017). sourcePartly supported

  The Introduction describes early OSKM-driven reprogramming, and the Results report temporary suppression of the Club-cell marker CCSP during induction. The paper does not establish chromatin remodeling or show that these early events are required for cellular rejuvenation and full reprogramming; the Discussion says the mechanism underlying its induced progenitor-like state remains to be clarified. The indexed copy provides section labels but no page numbers. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [76] Hishida, T. et al. In vivo partial cellular reprogramming enhances liver plasticity and regeneration. Cell Rep. 39, 110730 (2022). sourcePartly supported

  The Results section (page unavailable) reports that short-term 4F expression transiently downregulated mature-hepatocyte markers and changed chromatin accessibility. The Introduction also reports that short-term 4F induction ameliorated aging processes in an earlier mouse study. This paper does not establish that those early changes are required for either cellular rejuvenation or full reprogramming. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Reprogramming to pluripotency appears to require cell division.

“In addition to overhauling the epigenetic landscape via somatic silencing and chromatin remodeling, reprogramming to pluripotency appears to require cell division 93 so that cells can progress through the cell cycle-associated transcriptional dynamics 91 .”

• cites [93] Hanna, J. et al. Direct cell reprogramming is a stochastic process amenable to acceleration. Nature 462, 595–601 (2009). sourceSupported

The time point around day 15 represents an optimal point for achieving epigenetic rejuvenation without losing cell identity.

“These observations led to the conclusion that this time point represents an optimal point for achieving epigenetic rejuvenation without losing cell identity.”

• cites [114] Olova, N., Simpson, D. J., Marioni, R. E. & Chandra, T. Partial reprogramming induces a steady decline in epigenetic age before loss of somatic identity. Aging Cell 18, e12877 (2019). sourcePartly supported

  In Results and Discussion (page not provided), the paper reports a substantial decline in epigenetic age by day 15 while some fibroblast-specific genes still had high expression. It suggests a window for rejuvenation with partial maintenance of identity, but explicitly says its analysis cannot define precise safe boundaries. It therefore does not establish day 15 as an optimal point without loss of cell identity. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Some studies have not seen any detriment to tissues after more than a year of overexpression.

“some studies have not seen any detriment to tissues after more than a year of overexpression 92”

• cites [92] Lu, Y. et al. Reprogramming to recover youthful epigenetic information and restore vision. Nature 588, 124–129 (2020). sourceSupported

The OSK-induced regenerative phenotype in retinal ganglion cells is mediated by TET1 and TET2 DNA demethylases and TDG DNA glycosylase.

“the observed regenerative phenotype is mediated by the active contribution of TET1 and TET2 DNA demethylases and the TDG DNA glycosylase”

• cites [92] Lu, Y. et al. Reprogramming to recover youthful epigenetic information and restore vision. Nature 588, 124–129 (2020). sourceSupported

The observed rejuvenation effects were transient and diminished after stopping the DOX cycle in vitro.

“However, the observed rejuvenation effects were transient and diminished after stopping the DOX cycle in vitro 15 .”

• cites [15] Ocampo, A. et al. In vivo amelioration of age-associated hallmarks by partial reprogramming. Cell 167, 1719–1733.e1712 (2016). sourceSupported

Single-cell RNA sequencing showed that reprogramming events are consistently partial.

“the resolution of single-cell RNA sequencing here showed that reprogramming events are consistently partial.”

• cites [113] Roux, A. E. et al. Diverse partial reprogramming strategies restore youthful gene expression and transiently suppress cell identity. Cell Syst 13, 574–587 (2022). sourcePartly supported

  The Discussion (page unavailable) says single-cell profiles revealed transient reprogramming states and that RNA-velocity analysis suggested cells return toward their original states. It does not establish that reprogramming events are consistently partial: the authors describe a continuum of states and say further time-point measurements are needed to confirm the proposed trajectory. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

Expressing only MYC could not induce the loss of hepatocyte markers.

“expressing only MYC, known as a hepatocyte proliferation accelerator, could not induce the loss of hepatocyte markers.”

• cites [76] Hishida, T. et al. In vivo partial cellular reprogramming enhances liver plasticity and regeneration. Cell Rep. 39, 110730 (2022). sourceSupported

Whole-body overexpression of OSK for a year had no observable negative effects.

“whole-body overexpression of OSK for a year was found to have no observable negative effects”

• cites [92] Lu, Y. et al. Reprogramming to recover youthful epigenetic information and restore vision. Nature 588, 124–129 (2020). sourcePartly supported

  The “OSK stimulates axon regeneration” section reports no tumors or structural changes in the retina after 15 months of OSK induction, but that was retinal expression, not whole-body expression. The Methods (“Systemic delivery of AAV9”; page unavailable) say systemic OSK induction continued for only three weeks. The paper therefore does not support the claim that whole-body expression for a year had no observable negative effects. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Ectopic and constant expression of OSK in retinal ganglion cells in vivo restores vision by promoting axon regeneration in aged and glaucomatous mice without increasing cell proliferation.

“the ectopic and constant expression of OSK in retinal ganglion cells in vivo is able to restore vision by promoting axon regeneration in aged and glaucomatous mice without increasing cell proliferation (Fig. 3g).”

• cites [92] Lu, Y. et al. Reprogramming to recover youthful epigenetic information and restore vision. Nature 588, 124–129 (2020). sourcePartly supported

  The 'Recovery of vision in mice with glaucoma' section reports improved visual acuity and restored axon density after four weeks of OSK induction, with no evidence of RGC proliferation. The 'Restoration of vision in old mice' section also reports improved visual function, but says there was no obvious increase in axon density in those mice and proposes transcriptomic changes instead. Thus, axon regeneration is not established as the mechanism of vision restoration in aged mice; the vision experiments used four weeks of induction, not constant expression. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

There is a synergistic function between the Yamanaka factors that directly impacts reprogramming potential.

“it provided evidence of a synergistic function between the factors that directly impacted reprogramming potential.”

• cites [113] Roux, A. E. et al. Diverse partial reprogramming strategies restore youthful gene expression and transiently suppress cell identity. Cell Syst 13, 574–587 (2022). sourcePartly supported

  In “Subsets of the Yamanaka Factors are sufficient to elicit transient reprogramming effects” (page unavailable), the paper reports that reprogramming markers increased with the number of factors and notes that higher-order combinations are more effective at reprogramming. That supports an effect of factor combinations, but the paper does not establish a synergistic interaction or show that synergy directly determines reprogramming potential. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

In local OSKM wound treatment, the cell types responsible for rejuvenation cannot be uniquely identified because of the heterogeneous composition of the skin at the cellular level.

“despite the induction being local, the cell types responsible for the rejuvenation cannot be uniquely identified because of the heterogeneous composition of the skin at the cellular level.”

• cites [109] Doeser, M. C., Schöler, H. R. & Wu, G. Reduction of fibrosis and scar formation by partial reprogramming in vivo. Stem Cells 36, 1216–1225 (2018). sourcePartly supported

  The Abstract and Discussion (page unavailable) describe local OSKM induction in cutaneous wounds and reduced scarring. The paper does not say that skin-cell heterogeneity prevents identification of the cells responsible for rejuvenation; its in vitro results instead examine OSKM effects on fibroblasts specifically. (LLM, conf=0.78)

  Experimental — accuracy not yet measured

Bulk-based analyses could not consider transcriptional heterogeneity during reprogramming.

“it was not possible to consider transcriptional heterogeneity during reprogramming as bulk-based analyses were used”

• cites [113] Roux, A. E. et al. Diverse partial reprogramming strategies restore youthful gene expression and transiently suppress cell identity. Cell Syst 13, 574–587 (2022). sourceSupported

Analysis of the epigenetic clock after in vitro partial reprogramming showed approximately 30 years of rejuvenation.

“analysis of the epigenetic clock after in vitro partial reprogramming showed approximately 30 years of rejuvenation.”

• cites [74] Gill, D. et al. Multi-omic rejuvenation of human cells by maturation phase transient reprogramming. Elife 11, e71624 (2022). sourceSupported

Work in mice identified a maturation phase window during reprogramming where cells were most probable to experience rejuvenation without becoming iPS cells.

“work in mice identified a window, known as the maturation phase, during reprogramming where cells were most probable to experience rejuvenation without crossing the threshold and become iPS cells 96 .”

• cites [96] Samavarchi-Tehrani, P. et al. Functional genomics reveals a BMP-driven mesenchymal-to-epithelial transition in the initiation of somatic cell reprogramming. Cell Stem Cell 7, 64–77 (2010). sourcePartly supported

  The Introduction and Discussion describe a mouse fibroblast reprogramming model and identify initiation, maturation, and stabilization phases. They do not identify maturation as a window in which rejuvenation is most likely without cells becoming iPSCs; a search for rejuvenation found no passage making that connection. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Cellular damage accelerates epigenetic aging.

“this study is also the first to show that cellular damage accelerates epigenetic aging”

• cites [92] Lu, Y. et al. Reprogramming to recover youthful epigenetic information and restore vision. Nature 588, 124–129 (2020). sourcePartly supported

  In “OSK counters injury-induced DNAm ageing,” the paper reports that mouse retinal ganglion cells showed accelerated DNA-methylation age four days after injury. This supports the finding for that specific injury and cell type, but not the broader claim about cellular damage generally. The passage also does not establish that this was the first study to show the effect. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

After 9 days of reprogramming with LIN28 added to OSKM, HP1b mobility was restored to levels found in young fibroblasts without sacrificing cell identity.

“After 9 days of reprogramming, HP1b mobility, an indicator of active cell cycling, was restored to the same levels found in young fibroblasts without sacrificing cell identity 100 .”

• cites [100] Manukyan, M. & Singh, P. B. Epigenome rejuvenation: HP1β mobility as a measure of pluripotent and senescent chromatin ground states. Nat. Sci. Rep. 4, 4789 (2014). sourceSupported

Local and transient exposure of cutaneous wounds to OSKM significantly reduced fibrosis.

“local and transient factor exposure to cutaneous wounds significantly reduced fibrosis 109 .”

• cites [109] Doeser, M. C., Schöler, H. R. & Wu, G. Reduction of fibrosis and scar formation by partial reprogramming in vivo. Stem Cells 36, 1216–1225 (2018). sourceSupported

A single pulse of continuous OSKM induction for 7 days in naturally aged mice had no effect on the number of senescent cells.

“(interestingly, no effect on the number of senescent cells was detected)”

• cites [106] Chondronasiou, D. et al. Multi-omic rejuvenation of naturally aged tissues by a single cycle of transient reprogramming. Aging Cell 21, e13578 (2022). sourcePartly supported

  The Results section ('Evidence of rejuvenation in tissues with low reprogramming'; page unavailable) reports similar numbers of γH2AX-positive cells in the livers of very old mice with or without one OSKM cycle. These cells were measured as a marker of senescence, not as a direct count of all senescent cells across tissues, so the claim is broader than the finding. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Early experiments in human cells provided evidence that some biomarkers of aging (telomere lengths) were responding to epigenetic reprogramming.

“Early experiments in human cells were somewhat encouraging as they provide evidence that some of the biomarkers of aging (that is telomere lengths) were responding to the epigenetic reprogramming”

• cites [20] Marión, R. M. et al. Common telomere changes during in vivo reprogramming and early stages of tumorigenesis. Stem Cell Rep. 8, 460–475 (2017). sourcePartly supported

  The Results section (‘Telomere Elongation during Reprogramming-Induced Tissue Dedifferentiation’; page unavailable) reports longer telomeres in reprogrammed areas, supporting the claim that telomere length responds to reprogramming. But those experiments used mice, not human cells. The Introduction also discusses earlier in vitro findings without establishing the cited paper’s experiments as early human-cell experiments. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [26] Lapasset, L. et al. Rejuvenating senescent and centenarian human cells by reprogramming through the pluripotent state. Genes Dev. 25, 2248–2253 (2011). sourceSupported
• cites [28] Suhr, S. T. et al. Telomere dynamics in human cells reprogrammed to pluripotency. PLoS ONE 4, e8124 (2009). sourceSupported

Induction of OSK factors both before and after optic nerve injury led to increased regeneration capability of retinal ganglion cells.

“induction of the factors both before and after the optical nerve injury led to increased regeneration capability of retinal ganglion cells.”

• cites [92] Lu, Y. et al. Reprogramming to recover youthful epigenetic information and restore vision. Nature 588, 124–129 (2020). sourceSupported

Continuous overexpression of OSKM with a lower dosage of DOX (0.2 mg ml-1) in LAKI/+;4F/+ mice produced a near-identical increase in lifespan compared with the standard Ocampo protocol.

“Specifically, they compared continuous overexpression of OSKM with a lower dosage of DOX (0.2 mg ml -1 ) in LAKI/+;4F/+ mice to the standard protocol of Ocampo and colleagues and observed a near-identical increase in the lifespan of mice treated with both protocols.”

• cites [104] Alle, Q. et al. A single short reprogramming early in life initiates and propagates an epigenetically related mechanism improving fitness and promoting an increased healthy lifespan. Aging Cell 21, e13714 (2022). sourceSupported

Early human cell reprogramming protocols relied upon rejuvenation through re-differentiation after iPS cell formation.

“but protocols relied upon rejuvenation through re-differentiation after iPS cell formation 20,26,28 .”

• cites [20] Marión, R. M. et al. Common telomere changes during in vivo reprogramming and early stages of tumorigenesis. Stem Cell Rep. 8, 460–475 (2017). sourcePartly supported

  The Introduction reports that adult cells’ telomeres can lengthen during in vitro reprogramming into iPSCs, supporting an aspect of rejuvenation. It does not say that early human-cell protocols relied on re-differentiation after iPSC formation; the Results instead study in vivo reprogramming in mice. No page numbers are available in our copy. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [26] Lapasset, L. et al. Rejuvenating senescent and centenarian human cells by reprogramming through the pluripotent state. Genes Dev. 25, 2248–2253 (2011). sourceSupported
• cites [28] Suhr, S. T. et al. Telomere dynamics in human cells reprogrammed to pluripotency. PLoS ONE 4, e8124 (2009). sourcePartly supported

  The Introduction and Results describe forming human iPSCs and then re-differentiating them, so the sequence in the claim is supported. But the paper reports telomere elongation during iPSC conversion and shortening after re-differentiation; it does not say that early protocols relied on re-differentiation itself to produce rejuvenation. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

These findings validate a nonintegrative clinical method for reprogramming.

“These findings validate a nonintegrative clinical method for reprogramming”

• cites [17] Sarkar, T. J. et al. Transient non-integrative expression of nuclear reprogramming factors promotes multifaceted amelioration of aging in human cells. Nat. Commun. 11, 1545 (2020). sourcePartly supported

  The Results (page not available) describe an optimized non-integrative mRNA reprogramming protocol and report rejuvenation effects in cultured human cells. The paper calls the work a step toward potential therapeutic applications; it does not establish that the method has been clinically validated. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

The mechanism of OSK-induced axon regeneration is evolutionarily conserved.

“suggesting an evolutionarily conserved mechanism 92 .”

• cites [92] Lu, Y. et al. Reprogramming to recover youthful epigenetic information and restore vision. Nature 588, 124–129 (2020). sourceSupported

Cell proliferation is not the only driver of partial reprogramming.

“This suggests that cell proliferation is not the only driver of partial reprogramming 76 .”

• cites [76] Hishida, T. et al. In vivo partial cellular reprogramming enhances liver plasticity and regeneration. Cell Rep. 39, 110730 (2022). sourceSupported

Cyclic expression of OKSM improved cognitive functions in mice by increasing H3K9me3 levels in dentate gyrus adult neurons.

“Mechanistic studies were later complemented from Rodríguez-Matellán et al., who demonstrated that the cyclic expression of OKSM improved cognitive functions in mice by increasing H3K9me3 levels in dentate gyrus adult neurons.”

• cites [103] Rodríguez-Matellán, A., Alcazar, N., Hernández, F., Serrano, M. & Ávila, J. In vivo reprogramming ameliorates aging features in dentate gyrus cells and improves memory in mice. Stem Cell Rep. 15, 1056–1066 (2020). sourcePartly supported

  The Summary (p. 1) reports that cyclic induction of Yamanaka factors prevented an age-related reduction in dentate-gyrus H3K9me3 and was accompanied by improved object-recognition performance. The Discussion (p. 6) locates the H3K9me3 increase mainly in the layer containing mature neurons. The paper does not demonstrate that the memory improvement occurred *because of* the H3K9me3 increase; it presents the cellular changes and improved performance as accompanying findings. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Cells can be effectively rejuvenated by employing different regimens of overexpression.

“it was demonstrated in these studies that cells can be effectively rejuvenated by employing different regimens of overexpression.”

• cites [105] Browder, K. C. et al. In vivo partial reprogramming alters age-associated molecular changes during physiological aging in mice. Nat. Aging 2, 243–253 (2022). sourceNot checked — no full text of the source
• cites [106] Chondronasiou, D. et al. Multi-omic rejuvenation of naturally aged tissues by a single cycle of transient reprogramming. Aging Cell 21, e13578 (2022). sourcePartly supported

  The Discussion reports that one cycle of transient OSKM expression in naturally aged mice partially restored younger epigenetic, transcriptomic, and metabolomic patterns. It explicitly says the study focused on a single cycle, so it does not demonstrate rejuvenation using different overexpression regimens or establish that cells were broadly rejuvenated. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

The effects of reprogramming factors are tissue-specific.

“providing additional evidence for the tissue-specific nature of these effects.”

• cites [110] Hochedlinger, K., Yamada, Y., Beard, C. & Jaenisch, R. Ectopic expression of OCT4 blocks progenitor-cell differentiation and causes dysplasia in epithelial tissues. Cell 121, 465–477 (2005). sourceSupported

Maintenance of somatic profiles makes cells more probable to revert toward the original and previously established cell fate when expression of reprogramming factors is stopped.

“Maintenance of somatic profiles makes these cells more probable to revert toward the original and previously established cell fate when the expression of reprogramming factors is stopped.”

• cites [114] Olova, N., Simpson, D. J., Marioni, R. E. & Chandra, T. Partial reprogramming induces a steady decline in epigenetic age before loss of somatic identity. Aging Cell 18, e12877 (2019). sourcePartly supported

  In the Introduction, Results and Discussion, the paper says maintained fibroblast gene expression, together with increased senescence-gene expression, likely contributes to partially reprogrammed cells' high propensity to revert to a somatic phenotype. It separately reports that mouse fibroblasts revert when OSKM expression is discontinued. The claim presents maintenance alone as the cause of reversion after factors are stopped, which is stronger and more specific than the paper's account. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Long-term reprogramming can be achieved using a specific subset of reprogramming factors.

“and that long-term reprogramming can be achieved using a specific subset of reprogramming factors came from Lu et al. in 2020 (ref.92).”

• cites [92] Lu, Y. et al. Reprogramming to recover youthful epigenetic information and restore vision. Nature 588, 124–129 (2020). sourceSupported

Specific levels of OSK expression, in addition to cell identity, are essential to exert optimal function.

“further supporting the idea that specific levels of OSK expressions of the elements in addition to cell identity are essential to exert optimal function.”

• cites [92] Lu, Y. et al. Reprogramming to recover youthful epigenetic information and restore vision. Nature 588, 124–129 (2020). sourcePartly supported

  In “OSK stimulates axon regeneration” (page unavailable), OSK promoted regeneration when expressed in retinal ganglion cells but not amacrine cells, supporting a role for cell identity. The section also reports the greatest effect when all three factors were expressed together and suggests that inconsistent stoichiometry may explain why separate delivery failed. It does not establish specific expression levels as essential for optimal function. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

MYC might be responsible for some of the toxicity of reprogramming factors.

“suggesting that MYC might be responsible for some of the toxicity.”

• cites [92] Lu, Y. et al. Reprogramming to recover youthful epigenetic information and restore vision. Nature 588, 124–129 (2020). sourceSupported

Adeno-associated viruses encoding an inducible OSK system improved several health parameters, including frailty, in extremely old mice.

“and improved several health parameters, including frailty 108 .”

• cites [108] Macip, C. C. et al. Gene therapy mediated partial reprogramming extends lifespan and reverses age-related changes in aged mice. Preprint at bioRxiv, https://doi.org/10.1101/2023.01.04.522507 (2023). sourceSupported

Single-cell transcriptomic monitoring of reprogramming in the pancreas identified intermediate reprogramming states.

“the authors, in a related study, monitored the roadmap of reprogramming in the pancreas and identified intermediate reprogramming states by using a single-cell transcriptomic”

• cites [107] Chondronasiou, D. et al. Deciphering the roadmap of in vivo reprogramming toward pluripotency. Stem Cell Rep 17, 2501–2517 (2022). sourceSupported

Sarkar et al. obtained the first evidence of partial reprogramming-induced rejuvenation in old human cells in vitro.

“Sarkar et al. obtained the first evidence of partial reprogramming-induced rejuvenation in old human cells in vitro 17 .”

• cites [17] Sarkar, T. J. et al. Transient non-integrative expression of nuclear reprogramming factors promotes multifaceted amelioration of aging in human cells. Nat. Commun. 11, 1545 (2020). sourceSupported

Maturation phase partial reprogramming achieved more substantial rejuvenation than had been described before, at levels comparable to Guo et al.'s work in mice.

“they achieved more substantial rejuvenation than had been described before at levels comparable to Guo et al.'s work in mice 74,75 .”

• cites [74] Gill, D. et al. Multi-omic rejuvenation of human cells by maturation phase transient reprogramming. Elife 11, e71624 (2022). sourcePartly supported

  The Introduction and Discussion report that MPTR produced roughly 30 years of rejuvenation, apparently more than earlier transient reprogramming approaches. The Discussion also compares its magnitude with mouse studies, but names Chondronasiou et al. and Browder, not Guo et al.; our copy does not support the specific Guo comparison. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [75] Guo, L. et al. Generation of induced progenitor-like cells from mature epithelial cells using interrupted reprogramming. Stem Cell Rep. 9, 1780–1795 (2017). sourcePartly supported

  The Discussion describes interrupted reprogramming of mature Club cells from mice into proliferating progenitor-like cells. It does not report a measure of rejuvenation or a level that could substantiate the claimed comparison; the Introduction describes the work as exploring early reprogramming states. No page numbers are available in our copy. (LLM, conf=0.80)

  Experimental — accuracy not yet measured

All combinations of the Yamanaka factors at least partially suppressed the cell identity program and restored youthful gene expression at a magnitude correlated with the number of factors used rather than their identities.

“all combinations of the Yamanaka factors at least partially suppressed the cell identity program and restore youthful gene expression at a magnitude that correlated with the number of factors used rather than their identities.”

• cites [113] Roux, A. E. et al. Diverse partial reprogramming strategies restore youthful gene expression and transiently suppress cell identity. Cell Syst 13, 574–587 (2022). sourceSupported

Global transcriptomic analysis showed that chromatin accessibility was actively changed by 4F.

“the global transcriptomic analysis showed that chromatin accessibility was actively changed by 4F”

• cites [76] Hishida, T. et al. In vivo partial cellular reprogramming enhances liver plasticity and regeneration. Cell Rep. 39, 110730 (2022). sourceSupported

Perturbing epigenetic networks leads to extensive changes in gene expression that directly impact proteostasis, nutrient sensing, senescence and several other hallmarks of aging.

“Perturbing these epigenetic networks leads to extensive changes in gene expression that directly impact proteostasis, nutrient sensing, senescence and several other hallmarks of aging, suggesting that epigenetic networks are delicate and that their disruption probably initiates other events that cause cells to lose their function over time 8,9 .”

• cites [8] Zhang, W., Qu, J., Liu, G. H. & Belmonte, J. C. I. The ageing epigenome and its rejuvenation. Nat. Rev. Mol. Cell Biol. 21, 137–150 (2020). sourceNot checked — no full text of the source
• cites [9] Villeponteau, B. The heterochromatin loss model of aging. Exp. Gerontol. 32, 383–394 (1997). sourceNot checked — no full text of the source

The landmark paper used four transcription factors, OCT4, SOX2, KLF4 and MYC (OSKM), to erase cell identity and reset cell-type-specific epigenetic signatures.

“The landmark paper used four transcription factors, namely OCT4, SOX2, KLF4 and MYC (OSKM), to erase cell identity and reset cell-type-specific epigenetic signatures.”

• cites [18] Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006). sourceNot checked — no full text of the source

The observed frequency of tissue dysplasia and tumorigenesis implies that full cell reprogramming is not a viable anti-aging approach.

“Although this discovery holds great promise for regenerative medicine, the observed frequency of tissue dysplasia and tumorigenesis implies that full cell reprogramming is not a viable anti-aging approach.”

Early reports using cells derived from old donors to generate iPS cells demonstrated beneficial effects on senescence phenotypes and telomere length.

“Early reports using cells derived from old donors to generate iPS cells demonstrated beneficial effects on senescence phenotypes and telomere length [26][27][28] .”

• cites [26] Lapasset, L. et al. Rejuvenating senescent and centenarian human cells by reprogramming through the pluripotent state. Genes Dev. 25, 2248–2253 (2011). sourceNot checked yet
• cites [27] Marion, R. M. et al. Telomeres acquire embryonic stem cell characteristics in induced pluripotent stem cells. Cell Stem Cell 4, 141–154 (2009). sourceNot checked — no full text of the source
• cites [28] Suhr, S. T. et al. Telomere dynamics in human cells reprogrammed to pluripotency. PLoS ONE 4, e8124 (2009). sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

Epigenetic networks are delicate and their disruption probably initiates other events that cause cells to lose their function over time.

“suggesting that epigenetic networks are delicate and that their disruption probably initiates other events that cause cells to lose their function over time 8,9 .”

• cites [8] Zhang, W., Qu, J., Liu, G. H. & Belmonte, J. C. I. The ageing epigenome and its rejuvenation. Nat. Rev. Mol. Cell Biol. 21, 137–150 (2020). sourceNot checked — no full text of the source
• cites [9] Villeponteau, B. The heterochromatin loss model of aging. Exp. Gerontol. 32, 383–394 (1997). sourceNot checked — no full text of the source

Epigenetic reprogramming was first used by Shinya Yamanaka to revert differentiated cells in vitro back to a pluripotent state.

“One emerging strategy to intervene in the progression of biological aging is epigenetic reprogramming (Box 1), a technique first used by Shinya Yamanaka 18 to revert differentiated cells in vitro back to a pluripotent state.”

• cites [18] Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006). sourceNot checked — no full text of the source

Chromatin accessibility establishment occurs independently of DNA methylation.

“Moreover, enhancer chromatin opening generally follows OSK binding, which suggests that chromatin accessibility establishment occurs independently of DNA methylation.”

SOX2 engages the chromatin first and primes the target site for subsequent OCT4 binding.

“Singlemolecule imaging shows that SOX2 engages the chromatin first and primes the target site for subsequent OCT4 binding 47 .”

• cites [47] Chen, J. et al. Single-molecule dynamics of enhanceosome assembly in embryonic stem cells. Cell 156, 1274–1285 (2014). sourceNot checked — no full text of the source

The factors' expression levels and duration of treatment are sensitive components of the process.

“These initial findings identified the factors' expression levels and duration of treatment as sensitive components of the process and helped to establish parameters for in vivo rejuvenation applications.”

NANOG alone has a pivotal role in ameliorating senescence hallmarks and inducing rejuvenation in myogenic progenitor cells both in vitro and in vivo.

“Shahini et al. showed that NANOG alone has a pivotal role in ameliorating of senescence hallmarks and inducing rejuvenation in myogenic progenitor cells both in vitro and in vivo 61 .”

• cites [61] Shahini, A. et al. Ameliorating the hallmarks of cellular senescence in skeletal muscle myogenic progenitors in vitro and in vivo. Sci. Adv. 7, eabe5671 (2021). sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

Active DNA methylation via TET and TDG enzymes is required for rejuvenation.

“and active DNA methylation via TET and TDG enzymes is required for rejuvenation 92 .”

• cites [92] Lu, Y. et al. Reprogramming to recover youthful epigenetic information and restore vision. Nature 588, 124–129 (2020). sourceNot checked yet

Specific small molecules such as 3-deazaneplanocin A, 5-azacytidine, sodium butyrate and RG108 have emerged as potential epigenetic modifiers.

“Notably, specific small molecules such as 3-deazaneplanocin A, 5-azacytidine, sodium butyrate and RG108 have emerged as potential epigenetic modifiers 72 .”

• cites [72] Hou, P. et al. Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds. Science 341, 651–654 (2013). sourceNot checked — no full text of the source

SOX2 alone can open the chromatin and bind target DNA sites before the arrival of OCT4 in vivo.

“This is also supported by in vivo studies, suggesting that SOX2 alone can open the chromatin and bind target DNA sites before the arrival of OCT4 (ref.51).”

• cites [51] Malik, V. et al. Pluripotency reprogramming by competent and incompetent POU factors uncovers temporal dependency for OCT4 and SOX2. Nat. Commun. 10, 3477 (2019). sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

The duration of OSKM overexpression has a pivotal role in the de-differentiation process.

“Ohnishi et al.21 provided evidence that the duration of OSKM overexpression has a pivotal role in the de-differentiation process.”

• cites [21] Ohnishi, K. et al. Premature termination of reprogramming in vivo leads to cancer development through altered epigenetic regulation. Cell 156, 663–677 (2014). sourceNot checked yet

The earliest phenotypic and transcriptional changes following OSKM expression begin with an immediate increase in cell proliferation induced by MYC.

“The earliest phenotypic and transcriptional changes following OSKM expression begin with an immediate increase in cell proliferation induced by MYC 58 .”

• cites [58] Smith, Z. D., Sindhu, C. & Meissner, A. Molecular features of cellular reprogramming and development. Nat. Rev. Mol. Cell Biol. 17, 139–154 (2016). sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

Optimal reprogramming requires threefold excess of OCT4 relative to the other factors.

“Supporting this notion, optimal reprogramming requires threefold excess of OCT4 relative to the other factors 36 .”

• cites [36] Papapetrou, E. P. et al. Stoichiometric and temporal requirements of OCT4, SOX2, KLF4, and c-MYC expression for efficient human iPSC induction and differentiation. Proc. Natl Acad. Sci. USA 106, 12759–12764 (2009). sourceNot checked — no full text of the source

Early loss of H3K27me3 is associated with acquiring a partially open or primed chromatin state.

“Consistent with this notion, early loss of H3K27me3 is associated with acquiring a partially open or primed chromatin state 87 .”

• cites [87] Hussein, S. M. I. et al. Genome-wide characterization of the routes to pluripotency. Nature 516, 198–206 (2014). sourceNot checked — no full text of the source

H3K9me3 inhibits full reprogramming of pre-iPS cells into stem cells, effectively functioning as a barrier to pluripotency.

“Using this system, Chen and colleagues found that H3K9me3 inhibits full reprogramming of pre-iPS cells into stem cells, effectively functioning as a barrier to pluripotency 88 .”

• cites [88] Chen, J. et al. H3K9 methylation is a barrier during somatic cell reprogramming into iPSCs. Nat. Genet. 45, 34–42 (2013). sourceNot checked — no full text of the source

This reveals a cooperative relationship between DNA methylation and histone modifications that was not previously appreciated.

“This reveals a cooperative relationship between DNA methylation and histone modifications that was not previously appreciated.”

Continuous expression of OSKM factors in vivo is limited by substantial safety concerns including severe weight loss, the formation of circulating totipotent stem cells and teratomas in several organs.

“Although developments in cellular reprogramming have proposed several new and promising medical avenues [19][20][21] , continuous expression of OSKM factors in vivo is limited by substantial safety concerns including severe weight loss, the formation of circulating totipotent stem cells and teratomas in several organs 19 .”

• cites [19] Abad, M. et al. Reprogramming in vivo produces teratomas and iPS cells with totipotency features. Nature 502, 340–345 (2013). sourceNot checked — no full text of the source

Cell proliferation does not seem to be always required to reach pluripotency.

“Nonetheless, cell proliferation does not seem to be always required to reach pluripotency.”

• AssumptionRests on an assumption: Overexpression of LIN28 increases cell proliferation and accelerates iPS cell formation, but overexpression of NANOG achieves the same goal independent of cell proliferation.

MYC is not required to initiate reprogramming.

“Although recent studies suggest that MYC is not required to initiate reprogramming 56,57 , it is considered one of the most potent amplifiers of reprogramming 58 .”

• cites [56] Nakagawa, M. et al. Generation of induced pluripotent stem cells without MYC from mouse and human fibroblasts. Nat. Biotechnol. 26, 101–106 (2008). sourceNot checked — no full text of the source
• cites [57] Rand, T. A. et al. MYC Releases early reprogrammed human cells from proliferation pause via retinoblastoma protein inhibition. Cell Rep. 23, 361–375 (2018). sourceNot checked yet

Full reprogramming is relatively inefficient, with ~3% of cells reaching full reprogramming in the first round.

“It is worth noting, however, that full reprogramming is relatively inefficient, with ~3% of cells reaching full reprogramming in the first round 83 .”

• cites [83] Polo, J. M. et al. A molecular roadmap of reprogramming somatic cells into iPS cells. Cell 151, 1617–1632 (2012). sourceNot checked yet

Reducing H3K9me3 and H3K36me3 through inducible expression of a demethylase (KDM4B) improves iPS cell reprogramming efficiency.

“Similarly, reducing H3K9me3 and H3K36me3 through inducible expression of a demethylase (KDM4B) improves iPS cell reprogramming efficiency 89 .”

• cites [89] Wei, J. et al. KDM4B-mediated reduction of H3K9me3 and H3K36me3 levels improves somatic cell reprogramming into pluripotency. Sci. Rep. 7, 7514 (2017). sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

Changes to methylation occur in parallel with the observed alteration in chromatin contacts.

“There is also evidence that changes to methylation occur in parallel with the observed alteration in chromatin contacts, and active DNA methylation via TET and TDG enzymes is required for rejuvenation 92 .”

• cites [92] Lu, Y. et al. Reprogramming to recover youthful epigenetic information and restore vision. Nature 588, 124–129 (2020). sourceNot checked yet

Cells transition from the mesenchymal-to-epithelial state that is actively induced during the early phase and is required to progress through the reprogramming process.

“At this point, cells also transition from the mesenchymal-to-epithelial state that is actively induced during this early phase and are required to progress through the reprogramming process [94][95][96] .”

• cites [94] Li, R. et al. A mesenchymal-to-epithelial transition initiates and is required for the nuclear reprogramming of mouse fibroblasts. Cell Stem Cell 7, 51–63 (2010). sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

• cites [95] Liu, X. et al. Sequential introduction of reprogramming factors reveals a time-sensitive requirement for individual factors and a sequential EMT-MET mechanism for optimal reprogramming. Nat. Cell Biol. 15, 829–838 (2013). sourceNot checked — no full text of the source
• cites [96] Samavarchi-Tehrani, P. et al. Functional genomics reveals a BMP-driven mesenchymal-to-epithelial transition in the initiation of somatic cell reprogramming. Cell Stem Cell 7, 64–77 (2010). sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

Ocampo and colleagues established the potential for the technique to modulate phenotypes of aging.

“Through careful fine-tuning of in vivo reprogramming protocols, subsequent research from Ocampo and colleagues later established the potential for the technique to modulate phenotypes of aging.”

Such changes were highly correlated with altered cell division, DNA replication, chromatin modification and DNA damage response protein levels.

“Such changes were highly correlated with altered cell division, DNA replication, chromatin modification and the DNA damage response protein levels 84 .”

• cites [84] Hansson, J. et al. Highly coordinated proteome dynamics during reprogramming of somatic cells to pluripotency. Cell Rep. 2, 1579–1592 (2012). sourceNot checked yet

Cells switch from oxidative phosphorylation to glycolysis during the early reprogramming phase.

“Interestingly, cells switch from oxidative phosphorylation to glycolysis during this early reprogramming phase 58 .”

• cites [58] Smith, Z. D., Sindhu, C. & Meissner, A. Molecular features of cellular reprogramming and development. Nat. Rev. Mol. Cell Biol. 17, 139–154 (2016). sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

The presence of MYC increases OSK binding by twofold.

“The presence of MYC increases OSK binding by twofold 53 , and MYC binding increases by 40-fold with OSK, supporting the notion that the modulatory activities of OSK and MYC on each other are bidirectional.”

• cites [53] Soufi, A., Donahue, G. & Zaret, K. S. Facilitators and impediments of the pluripotency reprogramming factors’ initial engagement with the genome. Cell 151, 994–1004 (2012). sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

The Ocampo reprogramming protocol improved recovery from diabetes and muscle injury in old wild-type mice.

“In addition, the Ocampo reprogramming protocol improved recovery from diabetes and muscle injury in old wild-type mice, confirming their results in the context of normal physiological aging.”

• cites [15] Ocampo, A. et al. In vivo amelioration of age-associated hallmarks by partial reprogramming. Cell 167, 1719–1733.e1712 (2016). sourceNot checked yet

These findings are consistent with the proposed dual role of KLF4 in both transcriptional activation and repression.

“These findings are consistent with the proposed dual role of KLF4 in both transcriptional activation and repression.”

OCT4 and SOX2 are mainly responsible for increasing chromatin accessibility during full iPS cell reprogramming.

“OCT4 and SOX2 are mainly responsible for increasing chromatin accessibility during full iPS cell reprogramming, and KLF4 (as well as MYC, described in detail below) is believed to drive the first wave of transcriptional activation 42 .”

• cites [42] Jerabek, S. et al. Changing POU dimerization preferences converts OCT6 into a pluripotency inducer. EMBO Rep. 18, 319–333 (2017). sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

Continued proliferation and OSKM expression eventually convert all cells to iPS cells.

“However, there is evidence that continued proliferation and OSKM expression eventually convert all cells to iPS cells, although such treatment can make cells vulnerable to apoptosis and cellular senescence 85,93 .”

• cites [85] Schwarz, B. A. et al. Prospective isolation of poised iPSC intermediates reveals principles of cellular reprogramming. Cell Stem Cell 23, 289–305 (2018). sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

• cites [93] Hanna, J. et al. Direct cell reprogramming is a stochastic process amenable to acceleration. Nature 462, 595–601 (2009). sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

KLF4 contains both activator and repressor domains, conferring a dual function during cell differentiation.

“KLF4 is a transcription factor containing both activator and repressor domains, conferring a dual function during cell differentiation 52 (Fig. 1c).”

• cites [52] Schuetz, A. et al. The structure of the KLF4 DNA-binding domain links to self-renewal and macrophage differentiation. Cell Mol. Life Sci. 68, 3121–3131 (2011). sourceNot checked — no full text of the source

Partial de-differentiation results in cardiomyocytes correlated with the cardiomyocyte developmental stage.

“partial de-differentiation results correlated with the cardiomyocyte developmental stage”

• cites [112] Chen, Y. et al. Reversible reprogramming of cardiomyocytes to a fetal state drives heart regeneration in mice. Science 373, 1537–1540 (2021). sourceNot checked — no full text of the source

There is a lack of suitable and universal molecular biomarkers for measuring age reversal.

“Another limiting factor in advancing the field is the lack of suitable and universal molecular biomarkers for measuring age reversal.”

The rejuvenation effect of single-pulse OSKM in naturally aged mice was more prominent in the pancreas than in the spleen, liver and blood.

“The study demonstrated that the rejuvenation effect was more prominent in the pancreas than the spleen, liver and blood 106 .”

• cites [106] Chondronasiou, D. et al. Multi-omic rejuvenation of naturally aged tissues by a single cycle of transient reprogramming. Aging Cell 21, e13578 (2022). sourceNot checked yet

A single pulse of continuous OSKM induction for 7 days (0.2 mg/ml DOX) in naturally aged mice can trigger epigenetic, transcriptomic and metabolomic rejuvenation patterns.

“Chondronasiou and colleagues demonstrated that a single pulse of continuous OSKM induction for 7 days (using 0.2 mg ml -1 of DOX) in naturally aged mice can trigger epigenetic, transcriptomic and metabolomic rejuvenation patterns (Fig. 3d)”

• cites [106] Chondronasiou, D. et al. Multi-omic rejuvenation of naturally aged tissues by a single cycle of transient reprogramming. Aging Cell 21, e13578 (2022). sourceNot checked yet

Rejuvenation was retained 6 days after interrupting OSKMLN treatment.

“rejuvenation was retained 6 days after interrupting the treatment.”

• cites [17] Sarkar, T. J. et al. Transient non-integrative expression of nuclear reprogramming factors promotes multifaceted amelioration of aging in human cells. Nat. Commun. 11, 1545 (2020). sourceNot checked yet

Extended partial reprogramming treatment leads to more pronounced rejuvenation effects than short-term treatment when examining epigenetic clocks, transcriptomics and metabolomics in several organs and tissues, particularly skin and kidney.

“they found that an extended treatment regimen leads to more pronounced rejuvenation effects as compared with short term when examining epigenetic clocks, transcriptomics and metabolomics in several different organs and tissues (skin and kidney in particular).”

• cites [105] Browder, K. C. et al. In vivo partial reprogramming alters age-associated molecular changes during physiological aging in mice. Nat. Aging 2, 243–253 (2022). sourceNot checked — no full text of the source

When the same protocol was applied to nonprogeric mice, the effects were markedly less pronounced, with lifespan only being extended by 18 weeks.

“Interestingly, when the same protocol was applied to nonprogerice mice, the effects were markedly less pronounced, with lifespan only being extended by 18 weeks.”

• cites [104] Alle, Q. et al. A single short reprogramming early in life initiates and propagates an epigenetically related mechanism improving fitness and promoting an increased healthy lifespan. Aging Cell 21, e13714 (2022). sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

Similar results to OSK-induced axon regeneration were obtained in human neurons.

“Similar results were also obtained in human neurons”

• cites [92] Lu, Y. et al. Reprogramming to recover youthful epigenetic information and restore vision. Nature 588, 124–129 (2020). sourceNot checked yet

Expressing OSKM for 3 weeks in fully differentiated bronchiole secretory cells from 4F mice generated induced progenitor-like cells in vitro.

“The authors generated 'induced progenitor-like cells' in vitro by expressing OSKM for 3 weeks in fully differentiated bronchiole secretory cells from 4F mice (Fig. 4a).”

• cites [75] Guo, L. et al. Generation of induced progenitor-like cells from mature epithelial cells using interrupted reprogramming. Stem Cell Rep. 9, 1780–1795 (2017). sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

Specific overexpression of OSKM factors in MuSCs did not improve muscle regeneration.

“the specific overexpression of the factors in MuSCs did not improve muscle regeneration”

• cites [111] Wang, C. et al. In vivo partial reprogramming of myofibers promotes muscle regeneration by remodeling the stem cell niche. Nat. Commun. 12, 3094 (2021). sourceNot checked yet

The DNA methylation clock can be reversed in vivo by reprogramming.

“The demonstration that the DNA methylation clock can be reversed in vivo by reprogramming”

• cites [92] Lu, Y. et al. Reprogramming to recover youthful epigenetic information and restore vision. Nature 588, 124–129 (2020). sourceNot checked yet

In nonprogeric mice, partial reprogramming effects were markedly less pronounced, extending lifespan by only 18 weeks.

“applied to nonprogerice mice, the effects were markedly less pronounced, with lifespan only being extended by 18 weeks.”

Roux and colleagues obtained a similar partial de-differentiation event in adipogenic and mesenchymal stem cells from old and young mice.

“Roux and colleagues obtained a similar partial de-differentiation event in adipogenic and mesenchymal stem cells from old and young mice 113”

• cites [113] Roux, A. E. et al. Diverse partial reprogramming strategies restore youthful gene expression and transiently suppress cell identity. Cell Syst 13, 574–587 (2022). sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

Collectively, the works described suggest that timing and the identity of each factor and their relative stoichiometry require fine-tuning in different cell types to achieve a rejuvenated phenotype.

“Collectively, the works described suggest that timing and the identity of each factor and their relative stoichiometry require fine-tuning in different cell types to achieve a rejuvenated phenotype.”

OSK counteracts cellular damage-induced epigenetic aging to allow cells to regain youthful gene expression patterns and functions.

“a change that OSK counteracts to allow cells to regain youthful gene expression patterns and functions.”

• cites [92] Lu, Y. et al. Reprogramming to recover youthful epigenetic information and restore vision. Nature 588, 124–129 (2020). sourceNot checked yet

The different effects of OSKM overexpression on cell types within the same organ are probably due to different plasticity or susceptibility of the cell types to reprogramming.

“This is probably due to the different plasticity or susceptibility of the different cell types to reprogramming”

• cites [107] Chondronasiou, D. et al. Deciphering the roadmap of in vivo reprogramming toward pluripotency. Stem Cell Rep 17, 2501–2517 (2022). sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

Dynamic DNA methylation has a critical role during partial reprogramming.

“indicating a critical role for dynamic DNA methylation during partial reprogramming.”

• cites [92] Lu, Y. et al. Reprogramming to recover youthful epigenetic information and restore vision. Nature 588, 124–129 (2020). sourceNot checked yet

Long-term partial reprogramming with cycling DOX in 4F/+ mice did not produce teratoma formation.

“They did not observe teratoma formation during the long-term treatment.”

• cites [105] Browder, K. C. et al. In vivo partial reprogramming alters age-associated molecular changes during physiological aging in mice. Nat. Aging 2, 243–253 (2022). sourceNot checked — no full text of the source

Safety of partial reprogramming remains unresolved.

“this issue remains unresolved.”

When OSK were co-delivered as monocistronic vectors, no axon regeneration was observed.

“when OSK were co-delivered as monocistronic vectors, no axon regeneration was observed”

• cites [92] Lu, Y. et al. Reprogramming to recover youthful epigenetic information and restore vision. Nature 588, 124–129 (2020). sourceNot checked yet

Effects induced by partial reprogramming of cardiomyocytes were almost wholly reversed after 6 days of DOX withdrawal.

“the effects induced were almost wholly reversed after 6 days of DOX withdrawal 112”

• cites [112] Chen, Y. et al. Reversible reprogramming of cardiomyocytes to a fetal state drives heart regeneration in mice. Science 373, 1537–1540 (2021). sourceNot checked — no full text of the source

Adult mouse cardiomyocytes can be partially reprogrammed in vivo from an adult to a fetal stage where they proliferate without losing identity.

“The authors were able to partially reprogram in vivo adult mouse cardiomyocytes, bringing them from an adult to a fetal stage where the cells could proliferate without losing their identity.”

• cites [112] Chen, Y. et al. Reversible reprogramming of cardiomyocytes to a fetal state drives heart regeneration in mice. Science 373, 1537–1540 (2021). sourceNot checked — no full text of the source

Day 13 is a critical time point in the rejuvenation process.

“suggesting that day 13 is a critical time point in the rejuvenation process”

• cites [74] Gill, D. et al. Multi-omic rejuvenation of human cells by maturation phase transient reprogramming. Elife 11, e71624 (2022). sourceNot checked yet

Reprogramming is dependent on chromatin reshaping.

“suggesting that reprogramming is dependent on chromatin reshaping.”

• cites [76] Hishida, T. et al. In vivo partial cellular reprogramming enhances liver plasticity and regeneration. Cell Rep. 39, 110730 (2022). sourceNot checked yet

Reprogramming was later shown to exert mosaic-like expression patterns in tissues.

“which (as previously discussed) were later shown to exert mosaiclike expression patterns in tissues 113 .”

• cites [113] Roux, A. E. et al. Diverse partial reprogramming strategies restore youthful gene expression and transiently suppress cell identity. Cell Syst 13, 574–587 (2022). sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

Manipulation of specific pathways may be critical to achieving rejuvenation.

“suggesting that manipulation of specific pathways may be critical to achieving rejuvenation 111”

• cites [111] Wang, C. et al. In vivo partial reprogramming of myofibers promotes muscle regeneration by remodeling the stem cell niche. Nat. Commun. 12, 3094 (2021). sourceNot checked yet

Partial reprogramming of a specific cell type can revert aging by promoting cell regeneration.

“similar work showing that partial reprogramming of a specific cell type can revert aging by promoting cell regeneration came from Chen and colleagues 112”

• cites [112] Chen, Y. et al. Reversible reprogramming of cardiomyocytes to a fetal state drives heart regeneration in mice. Science 373, 1537–1540 (2021). sourceNot checked — no full text of the source

The phenotype observed in partially reprogrammed cardiomyocytes depends on the epigenetic state of the cells.

“confirming the dependence of the phenotype observed on the epigenetic state of the cells.”

• cites [112] Chen, Y. et al. Reversible reprogramming of cardiomyocytes to a fetal state drives heart regeneration in mice. Science 373, 1537–1540 (2021). sourceNot checked — no full text of the source

Single-cell transcriptome analysis showed suppression of somatic cell identity genes and induction of hallmark pluripotency programs in adipogenic and mesenchymal stem cells after 3 days of OKSM DOX exposure and 3 days of DOX withdrawal.

“the transcriptome of each cell population revealed suppression of somatic cell identity genes in both cell types while inducing hallmark pluripotency programs following 3 days of OKSM DOX exposure and 3 days of DOX withdrawal”

• cites [113] Roux, A. E. et al. Diverse partial reprogramming strategies restore youthful gene expression and transiently suppress cell identity. Cell Syst 13, 574–587 (2022). sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

OSKM overexpression has drastically different effects on different cell types within the same organ.

“This study also highlights how OSKM overexpression has drastically different effects on different cell types within the same organ.”

• cites [107] Chondronasiou, D. et al. Deciphering the roadmap of in vivo reprogramming toward pluripotency. Stem Cell Rep 17, 2501–2517 (2022). sourceNot checked yet

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Adeno-associated viruses encoding an inducible OSK system extended the median remaining lifespan of extremely old mice by 109% compared with untreated mice.

“Macip and colleagues who reported in 2023 that adeno-associated viruses encoding an inducible OSK system extended the median remaining lifespan of extremely old mice by 109% compared with untreated mice”

• cites [108] Macip, C. C. et al. Gene therapy mediated partial reprogramming extends lifespan and reverses age-related changes in aged mice. Preprint at bioRxiv, https://doi.org/10.1101/2023.01.04.522507 (2023). sourceNot checked yet

Continuous ectopic overexpression of OCT4 resulted in dysplastic growth in epithelial tissues.

“continuous ectopic overexpression of OCT4 also resulted in dysplastic growth in epithelial tissues 110”

• cites [110] Hochedlinger, K., Yamada, Y., Beard, C. & Jaenisch, R. Ectopic expression of OCT4 blocks progenitor-cell differentiation and causes dysplasia in epithelial tissues. Cell 121, 465–477 (2005). sourceNot checked yet

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Other transcription factors might represent a safer alternative to induce epigenetic rejuvenation.

“further supporting the idea that other transcription factors might represent a safer alternative to induce epigenetic rejuvenation as also reported by other groups 61,118 .”

• cites [61] Shahini, A. et al. Ameliorating the hallmarks of cellular senescence in skeletal muscle myogenic progenitors in vitro and in vivo. Sci. Adv. 7, eabe5671 (2021). sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

• cites [118] Ribeiro, R. et al. In vivo cyclic induction of the FOXM1 transcription factor delays natural and progeroid aging phenotypes and extends healthspan. Nat. Aging 2, 397–411 (2022). sourceNot checked — no full text of the source

Alle and colleagues demonstrated that rejuvenation outcomes might be more consistent by modifying OSKM methods.

“A recent paper published by Alle and colleagues demonstrated that rejuvenation outcomes might be more consistent by modifying OSKM methods (Fig. 3c) 104 .”

• cites [104] Alle, Q. et al. A single short reprogramming early in life initiates and propagates an epigenetically related mechanism improving fitness and promoting an increased healthy lifespan. Aging Cell 21, e13714 (2022). sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.