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  1. Author Response:

    Reviewer #1 (Public Review):

    1. Figure 1A discusses Horvath et al. multi-tissue and skin and blood clocks but more clocks could be applied. It is recommended to add Hannum et al., Levine et al. PhenoAge, Lu et al. GrimAge, and Teschendorff epitoc2 clocks.

    We have applied other epigenetic clocks to our dataset and commented on their results in the first results section (see Supplementary Figure 1A). These clocks appeared to rejuvenate later in the reprogramming process, suggesting that the epigenome may be rejuvenated in stages.

    1. The study reports substantial differences in DNA methylation and chronological ages, which might be due to passage number. The passage number of these cells should be listed, if possible. Additionally, there seems to be a deviation when applying EPIC chip data to the Horvath et al. clock compared to its original platform. The authors may address in the Methods section whether this inconsistency has been addressed.

    We tried to use the lowest passage number available to reduce the effect of in vitro culture on epigenetic age. As such, cells were used at passage four after purchasing. The exact passage number at purchase is unfortunately not available from Thermo Fisher. These details have been added to the methods section. As for applying EPIC data to the Horvath clock, work by McEwen et al (2018) shows that DNA methylation age is highly correlated between 450K and EPIC array platforms. Nevertheless, we have applied multiple epigenetic clocks to our dataset including those trained for EPIC array data and stated this point in the methods section.

    1. It is discussed that fibroblast morphology is reversed. It would be good to quantify this morphological dynamics. For instance, whether cell size undergoes transition from mesenchymal to epithelial lineages and if any reversal is observed.

    This is an interesting point and we have quantified the morphological changes using confocal microscopy and measuring a ratio of roundness (the maximum length divided by the perpendicular width) of individual cells before, during and after maturation phase transient reprogramming (MPTR). Cells became temporarily rounder during MPTR (lower ratio) and then returned to an elongated state (higher ratio) which matched that of the starting fibroblasts (Figure 1D).

    1. The findings relate to cells >39 years old. Would the same significant effect be observed in younger cells?

    The effect of MPTR on cells of other ages (younger or older) is an interesting question (also raised by reviewer 3), but we feel it is beyond the scope of the current study. We have examined this idea in more detail in the discussion section.

    1. Citation 32 should be updated. Also, are there any common genes responsible for both rejuvenation and transient reprogramming?

    Citation 32 has been updated to the published article. We are not here able to distinguish between genes responsible for cellular rejuvenation relative to genes responsible for iPSC reprogramming. Indeed, the required genes for each process may be (partially) overlapping or unique. A number of transcriptional programs are activated by the Yamanaka factors, and it may be possible to disentangle these in the future to determine which genes are required for rejuvenation and which are required for reprogramming.

    1. Figure 1F (the PCA figure) has a disproportional percentage of PC1 and PC2. As PC1 represents the reprogramming trajectory, does PC2 have any obvious biological meaning?

    PC2 is composed of CpG sites that are temporarily hypermethylated or hypomethylated during reprogramming. These CpG sites are near genes that are involved in asymmetric protein localisation (according to gene ontology enrichment). We have included these observations in the results section.

    1. Of the cells that failed to reprogram, do they undergo cellular senescence? What are their predicted ages?

    Cells that fail to reprogram appear to remain fibroblast like (transcriptionally and epigenetically) and their epigenetic ages are unchanged (Figures 1A, 1D, 1F and 4B). The cell cultures continued to replicate for several weeks following the MPTR process with no obvious cellular senescence beyond what is usually observed in cultures of growing primary fibroblasts.

    1. It would be interesting to investigate multiple rounds of transient reprogramming, and the maximum number rounds that could be achieved.

    The effect of multiple rounds of MPTR is an interesting question as this may enhance the magnitude of rejuvenation. It is our view that this is beyond the scope of this manuscript, however, we have discussed this idea in the discussion section.

    1. Ohnishi et al. 2014 could be cited as a key article, which should not be missed when discussing the oncogenicity of these genes.

    The Ohnishi et al 2014 article has been cited in the discussion section.

    1. There are only 6 rejuvenation genes overlapping in figure 4f. Would DNAm age be reversed if all 6 genes are overexpressed? Would it erase cell identity?

    The genes in figure 4F have both rejuvenated levels of expression and DNA methylation, however, these genes are likely to be downstream of rejuvenation pathways as they are not known to possess transcription factor activity. As a result, upregulating and downregulating these genes as appropriate may not recapitulate the effects of reprogramming induced rejuvenation. In addition, there may be further overlaps that are not captured due to the limited CpGs covered by the DNA methylation array.

    Reviewer #3 (Public Review):

    In this manuscript, the authors investigate the potential of cellular reprogramming for the rejuvenation of age-associated phenotypes in human fibroblasts. Importantly, compared to previous studies in this field, the authors go one step further in the degree of induction of cellular reprogramming by expressing the reprogramming factors for a longer time until the maturation phase (MPTRs). Using this approach, they demonstrate not only the rejuvenation of fibroblasts at the transcriptional and epigenetic level but also the restoration of cellular identity following partial dedifferentiation. First, the authors showed that DNA methylation age is progressively decreased following the expression of the Yamanaka factors using a doxycycline inducible lentiviral system. In addition, they demonstrate that although fibroblasts transiently lose their identity during reprogramming to the maturation phase (transient reprogramming intermediate), this identity is recovered after the expression of the factors has been terminated following doxycycline withdrawal. Subsequently, analysis of DNA methylation at promoters and enhancers associated with fibroblast identity, reveals what they authors refer to as epigenetic memory, which might be responsible for the restoration of fibroblast identity following termination of transient reprogramming. Lastly, the authors elegantly demonstrate the rejuvenation of human fibroblasts following reprogramming at the transcriptional and epigenetic level using clocks based on these analyses, as well as the restoration of epigenetic marks and expression of dysregulated genes associated with fibroblast function.

    This is very interesting manuscript that follows up on previous studies demonstrating the amelioration of age-associated phenotypes by cellular reprogramming. Although the concept presented in the manuscript is not completely novel, the authors try to go one step further by investigating the rejuvenation of aging phenotypes and recovery of cellular identity following a more extensive and longer reprogramming protocol. This manuscript elegantly reinforces the potential of cellular reprogramming for the rejuvenation of aging and proposes a hypothesis for the restoration of cellular identity after cellular reprogramming based on the existence of a certain degree of epigenetic memory in the cells. This manuscript will be of interest to scientist working in cellular reprogramming, aging and epigenetics. Nevertheless, before publication the authors would need to address the points stated below:

    Major points:

    • The timeline of induction and termination of cellular reprogramming by addition and withdrawal of doxycycline is not very clear across the manuscript. This is a critical aspect of the manuscript that should be better explained at the beginning of the results as well as in the methods section. Specifically, the authors only refer to the time of analysis following withdrawal of doxycycline in the legend of Figure 1B. Even there, the authors mentioned that "Sorted cells were also further cultured and grown in the absence of doxycycline for at least four weeks". The precise timeline of induction and termination of reprogramming by addition and withdrawal of doxycycline is critical for the whole message of the manuscript and therefore should be explained in much more detail. How long after doxycycline withdrawal were the transient reprogrammed fibroblasts analyzed?

    The amount of time that doxycycline was withdrawn has been further clarified in results and methods sections. This was 4 weeks in the first experiment and 5 weeks in the second experiment. Cells had returned to fibroblast morphology by four weeks in the second experiment, however, they needed to be further expanded to generate enough material for downstream analyses.

    • In the same line, the authors described in the methods section that transient reprogrammed sorted cells were replated on irradiated mouse embryonic fibroblasts (iMEFs) in fibroblast medium without doxycycline. Were the negative control cells processed in the same way and plated on top of iMEFs? Was there any effect observed due to the growth of fibroblasts on top of iMEFs? How long were the cells cultured on top of iMEFs before culturing under normal conditions? Could the authors explain the rationale for the use of iMEFs during this protocol?

    All cells (including the negative controls) were initially replated onto iMEFs after flow sorting to replicate the culture conditions before the flow sort and aid in the reattachment of cells to the culture surface. At subsequent passages, cells were replated without iMEFs. Culturing on iMEFs had no obvious effect on negative control cells, which were similar (based on transcriptome and morphology) to cells that had never been grown with iMEFs. These points have been added to the methods section.

    • There is a certain degree of contradiction between the results shown in some sections of the manuscript as well as the discussion of the results. In one hand, the authors claim that reprogramming can rejuvenate fibroblasts not only at the transcriptional but also at the epigenetic level, more specifically at the level of DNA methylation, and that this rejuvenation is maintained even weeks after the induction of reprogramming has stopped. On the other hand, the authors propose that epigenetic memory based on the absence of changes in DNA methylation in promoters and enhancer of fibroblasts-related genes allow the restoration of cellular identity following reprogramming. Why epigenetic age does not display this memory? Why some regions will maintain DNA methylation memory but the site used for the analysis of epigenetic age by DNA methylation show changes in methylation status and therefore rejuvenation? The authors should discuss in more detail this important aspect of their data.

    This is a very interesting point. Cell identity CpG sites (at fibroblast specific enhancers) and age-associated CpG sites (used to calculate epigenetic age) are distinct. In addition, DNA methylation has different dynamics at different times during reprogramming, with cell identity CpG sites changing methylation during the stabilisation phase, and age-associated CpG sites changing methylation during the maturation phase. This suggests these sites may be regulated by different mechanisms. In addition, methylation changes at age-associated CpG sites include both gain and loss of methylation, implying these are targeted methylation changes rather than global changes. These points have been highlighted in the discussion section.

    • In Figure 3A the changes induced by reprogramming in the PC2 direction look orthogonal to the aging changes observed in PC1. Could the authors maybe use a more stringent criteria for the selection of age-related genes. How do they explain the direction of these changes? In the same line, have the authors tested the effect of MPTRs in young fibroblasts? Could young fibroblasts be included in Figure 3C?

    Our method for identifying age-associated genes is already quite stringent as we have applied Bonferroni multiple testing correction to the p values generated by the Pearson correlation analysis.

    We have not yet attempted MPTR on young fibroblasts, this is certainly an interesting future direction but we feel it is beyond the scope of the current study. Instead, we have discussed this idea in the discussion section.

    Unfortunately, we cannot include young fibroblasts in figure 3C as they have been included in the training dataset for the transcription clock.

    • Based on the standard protocols used for the culture of fibroblasts using culture medium containing fetal bovine serum (FBS), I hypothesis that the recovery of cellular identity following reprogramming is mainly due to differentiation signals coming from factors present in the medium. For these reasons, knockout serum (KSR) is used at later stages (day 8) of reprogramming to allow generation of iPSCs. The authors should rule out the possibility that recovery of fibroblast identity is due to the culture of reprogramed fibroblasts in FBS containing medium. For this purpose, the authors should test whether the fibroblast identity can be recovered following doxycycline withdrawal by culturing the cell in KSR or 1% FBS containing medium instead of 10% FBS. This is a very important concept for the message that the manuscript tries to communicate regarding an epigenetic memory responsible for the recovery of fibroblast identity.

    The effect of FBS in promoting the return to fibroblast identity is an interesting possibility. As mentioned above in response to the essential revisions, we attempted to investigate this by growing cells in fibroblast medium containing 10% KSR instead of 10% FBS after withdrawal of doxycycline, however, KSR containing medium was unable to support long term culture of fibroblasts. In addition, we examined the effect of 1% FBS on fibroblasts and found that their growth rate was substantially impeded, which would be major confounder.

    Minor points:

    • The authors claim that their maturation phase transient reprogramming protocol (MPTRs) induces further rejuvenation compared to previous studies because of the use of a longer induction timeline. Nevertheless, the authors mentioned that cells took around 50 days to reach a fully pluripotent state. This is a very long timeline to reach pluripotency compared to previous studies inducing reprogramming in mouse or human fibroblasts where typically iPSCs can be generated after 2 or 3 weeks following expression of the reprogramming factors. In the same line, secondary systems based on the use of cells isolated from transgenic mice carrying a cassette for the expression of Yamanaka factors have been shown to be very rapid and efficient in the generation of iPSCs. For these reasons, the authors should not use during their discussion a direct comparison with previous studies based just on time of induction since it is possible than systems with a more efficient or higher expression of the reprogramming factors and therefore a much more rapid alteration of cell fate have been used in previous studies.

    We could isolate iPSCs that could be cultured without doxycycline earlier than this at 30 days of reprogramming. However, we analysed iPSCs after 50 days of reprogramming to ensure the stabilisation phase had been completed and donor memory was erased. We agree that other reprogramming systems can achieve pluripotency faster than lentiviral based systems so we have acknowledged this in the discussion section.

    • In Figure 1D, principal component analysis shows "Not reprogramming" fibroblasts represented by a cross that cluster between young, old, and rejuvenated fibroblasts. What cells are the authors referring to? Are these cells represented in the diagram included in Figure 1B? Why do they cluster differently compared to the other fibroblasts?

    The ‘not reprogramming cells’ in figure 1E are reference data samples from our Sendai virus reprogramming experiment that were annotated as ‘failing to reprogram’ based on surface markers. Therefore, these samples are not in figure 1B. Like the ‘failing to reprogram intermediates’, they may be expressing the Yamanaka factors, and have therefore moved along PC1 relative to fibroblasts, but they have failed to upregulate SSEA4. We have clarified this in the figure legend and methods section.

    • The authors propose in the discussion that the use of transient reprogramming protocols like the one presented in the manuscript could be used in vivo to safely induce the rejuvenation of tissues and organs. Although previous studies have shown that this in fact the case, the authors should be aware that the recovery of cellular identity might be easier to achieve in vitro where differentiation signals like the ones in FBS are present during in vitro culture but not present anymore in an adult fully differentiated animal. For these reasons, the authors should be cautious when discussing the potential of in vivo application of these approaches and their safety.

    This is an interesting point and we have clarified that our method may be suitable for ex vivo approaches where our method is performed on cells in vitro.

    • The figure legend of Supplementary Figure 2B shows iPSC but this population is not present in the figure.

    The figure legend for supplementary figure 2B has been corrected to remove the iPSC group, which the reviewer correctly noticed is not present in the heatmap.

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  2. Evaluation Summary:

    This study describes a novel "maturation phase transient reprogramming" (MPTR) method to restore the epigenome of cells to a more youthful state. The authors demonstrate the effectiveness of the method to reverse several age-related changes including remodeling of the transcriptome. The method appears to peform favorably compared to other transient reprogramming protocols, and the study will be of interest to developmental biologists as well as researchers that study ageing.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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  3. Reviewer #1 (Public Review):

    Gill et al. investigated organismal rejuvenation, an emerging topic in longevity and aging research. They explored the timing of Yamanaka factor expression and developed a transient reprogramming-withdrawal protocol that supports restoration of cellular identity. They also used omics methods to prove that the transcription profile and epigenetic memory of fibroblast identity are preserved, while the age profile is reversed. They marked an outline of the genes specifically related to the rejuvenation of the epigenome and transcriptome.

    This study fills an important gap in our knowledge and presents a fascinating finding that human cells may achieve age reversal without going to the full pluripotency state, which further offers an intriguing insight that the reversal of biological age can be somehow separated from the reversal of cell differentiation status. This manuscript should be of great interest to scientists working on aging, epigenetics and development.

    1. Figure 1A discusses Horvath et al. multi-tissue and skin and blood clocks but more clocks could be applied. It is recommended to add Hannum et al., Levine et al. PhenoAge, Lu et al. GrimAge, and Teschendorff epitoc2 clocks.
    2. The study reports substantial differences in DNA methylation and chronological ages, which might be due to passage number. The passage number of these cells should be listed, if possible. Additionally, there seems to be a deviation when applying EPIC chip data to the Horvath et al. clock compared to its original platform. The authors may address in the Methods section whether this inconsistency has been addressed.
    3. It is discussed that fibroblast morphology is reversed. It would be good to quantify this morphological dynamics. For instance, whether cell size undergoes transition from mesenchymal to epithelial lineages and if any reversal is observed.
    4. The findings relate to cells >39 years old. Would the same significant effect be observed in younger cells?
    5. Citation 32 should be updated. Also, are there any common genes responsible for both rejuvenation and transient reprogramming?
    6. Figure 1F (the PCA figure) has a disproportional percentage of PC1 and PC2. As PC1 represents the reprogramming trajectory, does PC2 have any obvious biological meaning?
    7. Of the cells that failed to reprogram, do they undergo cellular senescence? What are their predicted ages?
    8. It would be interesting to investigate multiple rounds of transient reprogramming, and the maximum number rounds that could be achieved.
    9. Ohnishi et al. 2014 could be cited as a key article, which should not be missed when discussing the oncogenicity of these genes.
    10. There are only 6 rejuvenation genes overlapping in figure 4f. Would DNAm age be reversed if all 6 genes are overexpressed? Would it erase cell identity?

    Was this evaluation helpful?
  4. Reviewer #2 (Public Review):

    In their manuscript, Gill et al describe a novel reprogramming approach by transient expression of Yamanaka factors in fibroblasts up to that maturation phase of reprogramming to test for rejuvenation of fibroblast. Guided initially by epigenetic clocks, it was determined that transient expression of Yamanaka factors in the range of 10-17 days resulted in a likely optimal level of rejuvenation while still maintaining their fibroblastic nature. The data supports this conclusion very well. There are some shortcomings with respect to characterization of the extent of the functional rejuvenation of fibroblasts and identification of limits of the protocol.

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  5. Reviewer #3 (Public Review):

    In this manuscript, the authors investigate the potential of cellular reprogramming for the rejuvenation of age-associated phenotypes in human fibroblasts. Importantly, compared to previous studies in this field, the authors go one step further in the degree of induction of cellular reprogramming by expressing the reprogramming factors for a longer time until the maturation phase (MPTRs). Using this approach, they demonstrate not only the rejuvenation of fibroblasts at the transcriptional and epigenetic level but also the restoration of cellular identity following partial dedifferentiation. First, the authors showed that DNA methylation age is progressively decreased following the expression of the Yamanaka factors using a doxycycline inducible lentiviral system. In addition, they demonstrate that although fibroblasts transiently lose their identity during reprogramming to the maturation phase (transient reprogramming intermediate), this identity is recovered after the expression of the factors has been terminated following doxycycline withdrawal. Subsequently, analysis of DNA methylation at promoters and enhancers associated with fibroblast identity, reveals what they authors refer to as epigenetic memory, which might be responsible for the restoration of fibroblast identity following termination of transient reprogramming. Lastly, the authors elegantly demonstrate the rejuvenation of human fibroblasts following reprogramming at the transcriptional and epigenetic level using clocks based on these analyses, as well as the restoration of epigenetic marks and expression of dysregulated genes associated with fibroblast function.

    This is very interesting manuscript that follows up on previous studies demonstrating the amelioration of age-associated phenotypes by cellular reprogramming. Although the concept presented in the manuscript is not completely novel, the authors try to go one step further by investigating the rejuvenation of aging phenotypes and recovery of cellular identity following a more extensive and longer reprogramming protocol. This manuscript elegantly reinforces the potential of cellular reprogramming for the rejuvenation of aging and proposes a hypothesis for the restoration of cellular identity after cellular reprogramming based on the existence of a certain degree of epigenetic memory in the cells. This manuscript will be of interest to scientist working in cellular reprogramming, aging and epigenetics. Nevertheless, before publication the authors would need to address the points stated below:

    Major points:

    - The timeline of induction and termination of cellular reprogramming by addition and withdrawal of doxycycline is not very clear across the manuscript. This is a critical aspect of the manuscript that should be better explained at the beginning of the results as well as in the methods section. Specifically, the authors only refer to the time of analysis following withdrawal of doxycycline in the legend of Figure 1B. Even there, the authors mentioned that "Sorted cells were also further cultured and grown in the absence of doxycycline for at least four weeks". The precise timeline of induction and termination of reprogramming by addition and withdrawal of doxycycline is critical for the whole message of the manuscript and therefore should be explained in much more detail. How long after doxycycline withdrawal were the transient reprogrammed fibroblasts analyzed?

    - In the same line, the authors described in the methods section that transient reprogrammed sorted cells were replated on irradiated mouse embryonic fibroblasts (iMEFs) in fibroblast medium without doxycycline. Were the negative control cells processed in the same way and plated on top of iMEFs? Was there any effect observed due to the growth of fibroblasts on top of iMEFs? How long were the cells cultured on top of iMEFs before culturing under normal conditions? Could the authors explain the rationale for the use of iMEFs during this protocol?

    - There is a certain degree of contradiction between the results shown in some sections of the manuscript as well as the discussion of the results. In one hand, the authors claim that reprogramming can rejuvenate fibroblasts not only at the transcriptional but also at the epigenetic level, more specifically at the level of DNA methylation, and that this rejuvenation is maintained even weeks after the induction of reprogramming has stopped. On the other hand, the authors propose that epigenetic memory based on the absence of changes in DNA methylation in promoters and enhancer of fibroblasts-related genes allow the restoration of cellular identity following reprogramming. Why epigenetic age does not display this memory? Why some regions will maintain DNA methylation memory but the site used for the analysis of epigenetic age by DNA methylation show changes in methylation status and therefore rejuvenation? The authors should discuss in more detail this important aspect of their data.

    - In Figure 3A the changes induced by reprogramming in the PC2 direction look orthogonal to the aging changes observed in PC1. Could the authors maybe use a more stringent criteria for the selection of age-related genes. How do they explain the direction of these changes? In the same line, have the authors tested the effect of MPTRs in young fibroblasts? Could young fibroblasts be included in Figure 3C?

    - Based on the standard protocols used for the culture of fibroblasts using culture medium containing fetal bovine serum (FBS), I hypothesis that the recovery of cellular identity following reprogramming is mainly due to differentiation signals coming from factors present in the medium. For these reasons, knockout serum (KSR) is used at later stages (day 8) of reprogramming to allow generation of iPSCs. The authors should rule out the possibility that recovery of fibroblast identity is due to the culture of reprogramed fibroblasts in FBS containing medium. For this purpose, the authors should test whether the fibroblast identity can be recovered following doxycycline withdrawal by culturing the cell in KSR or 1% FBS containing medium instead of 10% FBS. This is a very important concept for the message that the manuscript tries to communicate regarding an epigenetic memory responsible for the recovery of fibroblast identity.

    Minor points:
    - The authors claim that their maturation phase transient reprogramming protocol (MPTRs) induces further rejuvenation compared to previous studies because of the use of a longer induction timeline. Nevertheless, the authors mentioned that cells took around 50 days to reach a fully pluripotent state. This is a very long timeline to reach pluripotency compared to previous studies inducing reprogramming in mouse or human fibroblasts where typically iPSCs can be generated after 2 or 3 weeks following expression of the reprogramming factors. In the same line, secondary systems based on the use of cells isolated from transgenic mice carrying a cassette for the expression of Yamanaka factors have been shown to be very rapid and efficient in the generation of iPSCs. For these reasons, the authors should not use during their discussion a direct comparison with previous studies based just on time of induction since it is possible than systems with a more efficient or higher expression of the reprogramming factors and therefore a much more rapid alteration of cell fate have been used in previous studies.

    - In Figure 1D, principal component analysis shows "Not reprogramming" fibroblasts represented by a cross that cluster between young, old, and rejuvenated fibroblasts. What cells are the authors referring to? Are these cells represented in the diagram included in Figure 1B? Why do they cluster differently compared to the other fibroblasts?

    - The authors propose in the discussion that the use of transient reprogramming protocols like the one presented in the manuscript could be used in vivo to safely induce the rejuvenation of tissues and organs. Although previous studies have shown that this in fact the case, the authors should be aware that the recovery of cellular identity might be easier to achieve in vitro where differentiation signals like the ones in FBS are present during in vitro culture but not present anymore in an adult fully differentiated animal. For these reasons, the authors should be cautious when discussing the potential of in vivo application of these approaches and their safety.

    - The figure legend of Supplementary Figure 2B shows iPSC but this population is not present in the figure.

    Was this evaluation helpful?