Glaucoma Reversed by OSK Study

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Nature. Author manuscript; available in PMC 2021 June 02.
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Published in final edited form as:

Nature. 2020 December ; 588(7836): 124–129. doi:10.1038/s41586-020-2975-4.
Reprogramming to recover youthful epigenetic information and

restore vision
Yuancheng Lu1, Benedikt Brommer2,3,11, Xiao Tian1,11, Anitha Krishnan3,4,11, Margarita
Meer5,6,11, Chen Wang2,3, Daniel L. Vera1, Qiurui Zeng1, Doudou Yu1, Michael S.
Bonkowski1, Jae-Hyun Yang1, Songlin Zhou2,3, Emma M. Hoffmann3,4, Margarete M.
Karg3,4, Michael B. Schultz1, Alice E. Kane1, Noah Davidsohn7, Ekaterina Korobkina3,4,
Author Manuscript
Karolina Chwalek1, Luis A. Rajman1, George M. Church7, Konrad Hochedlinger8, Vadim N.

Gladyshev5, Steve Horvath9, Morgan E. Levine6, Meredith S. Gregory-Ksander3,4,*, Bruce R.
Ksander3,4,*, Zhigang He2,3,*, David A. Sinclair1,10,*,#
1.Departmentof Genetics, Blavatnik Institute, Paul F. Glenn Center for Biology of Aging Research,
Harvard Medical School, MA, USA;
2.Department of Neurology, Boston Children’s Hospital, Harvard Medical School, MA, USA;
3.Department of Ophthalmology, Harvard Medical School, Boston, MA, USA;
4.Schepens Eye Research Institute of Mass Eye & Ear, Harvard Medical School, MA, USA;
5.Division
of Genetics, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical
School, MA, USA;
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6.Department of Pathology, Yale School of Medicine, New Haven, CT, USA;

7.Department of Genetics, Wyss Institute for Biologically Inspired Engineering, Harvard University,
MA, USA;
Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research,
subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms
#
Correspondence: [email protected].
*Senior authors;
Contributions
Y.L. and D.A.S. conceived the project. Y.L., X.T., and D.A.S. wrote the manuscript with input from all co-authors. Y.L. was involved
in all experiments and analyses. M.S.B. and J.-H.Y. provided early training to Y.L.. B.B., C.W., Q.Z., D.Y., S.Z., and Z.H. contributed
to the optic nerve crush studies and imaging. A.K., D.Y., Q.Z., E.M.H., E.K., M.S.G.-K., and B.R.K. contributed to the glaucoma and
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ageing studies. M.M.K. and B.R.K. performed OCT imaging and analysis. M.M. and V.N.G. conducted ribosomal DNAm age analysis
for mouse RGCs. M.E.L. developed DNAm ageing signature. D.L.V. performed the RNA-seq and gene association analysis. X.T.
conducted human neuron experiments. S.H. conducted human methylation clock analysis. X.T., J.-H.Y., and K.H. helped with
transgenic mouse fibroblasts work. M.S.B., X.T., M.B.S., A.E.K., L.A.R., helped with systemic AAV9 experiments. N.D., and G.M.C.
helped with plasmid constructs and AAV9 production. K.C. helped with grant applications and project management. M.S.G.-K.,
B.R.K., Z.H., and D.A.S. jointly supervised this work.
Conflict of interest
D.A.S. is a consultant to, inventor of patents licensed to, board member of and equity owner of Iduna Therapeutics, a Life Biosciences
company developing epigenetic reprograming therapies. D.A.S. is an advisor to Zymo Research, an epigenetics tools company.
Additional disclosures are at https://genetics.med.harvard.edu/sinclair/people/sinclair-other.php. Y.L., L.A.R. and S.H. are equity
owners of Iduna Therapeutics, a Life Biosciences company. D.L.V. is an advisor to Liberty Biosecurity. M.S.B. is a shareholder in
MetroBiotech. K.C. is an equity owner in Life Biosciences and affiliates. N.D. and G.M.C. are co-founders of Rejuvenate Bio.
Disclosures for G.M.C. can be found at http://arep.med.harvard.edu/gmc/tech.html. M.E.L. is a bioinformatics advisor to Elysium
Health. Y.L., N.D. and D.A.S. are inventors on patents arising from this work (WO/2020/069373 and WO/2020/069339), filed by the
President and Fellows of Harvard College. The other authors declare no competing interests.
Lu et al. Page 2
8.Departmentof Molecular Biology, Cancer Center and Center for Regenerative Medicine,
Author Manuscript
Massachusetts General Hospital, MA, USA;

9.Department of Human Genetics, David Geffen School of Medicine, University of California Los
Angeles, CA, USA;
10.Laboratory for Aging Research, Department of Pharmacology, School of Medical Sciences, The
University of New South Wales, Sydney, Australia;
11.These authors contributed equally: B. B., X. T., A. K., M. M.;
Abstract
Ageing is a degenerative process that leads to tissue dysfunction and death. A proposed cause of
ageing is the accumulation of epigenetic noise that disrupts gene expression patterns, leading to
decreases in tissue function and regenerative capacity1–3. Changes to DNA methylation patterns
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over time form the basis of ageing clocks4, but whether older individuals retain the information
needed to restore these patterns—and, if so, whether this could improve tissue function—is not
known. Over time, the central nervous system (CNS) loses function and regenerative capacity5–7.
Using the eye as a model CNS tissue, here we show that ectopic expression of Oct4 (also known
as Pou5f1), Sox2 and Klf4 genes (OSK) in mouse retinal ganglion cells restores youthful DNA
methylation patterns and transcriptomes, promotes axon regeneration after injury, and reverses
vision loss in a mouse model of glaucoma and in aged mice. The beneficial effects of OSK-
induced reprogramming in axon regeneration and vision require the DNA demethylases TET1 and
TET2. These data indicate that mammalian tissues retain a record of youthful epigenetic
information—encoded in part by DNA methylation—that can be accessed to improve tissue
function and promote regeneration in vivo.
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The metaphor of the epigenetic landscape, first invoked by Waddington to explain

embryonic development8, is increasingly seen as relevant to the other end of life9. Evidence
from yeast and mammals supports an Information Theory of Ageing10,11, in which the loss
of epigenetic information disrupts youthful gene expression patterns1–3, leading to cellular
dysfunction and senescence12,13.
DNA methylation patterns are laid down during embryonic development to establish cell
type and function. During ageing, for reasons that are unclear, these patterns change in ways
that can be used to calculate DNA methylation (DNAm) age, a representation of biological
age that can predict future health and lifespan4. In cell culture, the ectopic expression of the
four Yamanaka transcription factors Oct4, Sox2, Klf4, and c-Myc (OSKM) can reprogram
cultured somatic cells to become pluripotent stem cells14, a process that erases cellular
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identity and resets DNAm age4,15. In a premature ageing mouse model of Hutchinson-
Gilford progeria syndrome (HGPS), cyclic transgene-mediated expression of these four
genes alleviates symptoms and extends lifespan, raising the possibility that OSKM might
counteract normal ageing, too16. Continuous expression of all four factors in normal mice,
however, often induces teratomas17–19 or is fatal within days16, ostensibly due to tissue
dysplasia18.

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Ageing is generally considered a unidirectional process akin to an increase in entropy.

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Living systems, however, are open not closed, and in some cases can fully reset biological
age, examples being “immortal” jellyfish and the cloning of animals by nuclear transfer.
Having previously found evidence for epigenetic noise as an underlying cause of ageing3,13,
we wondered whether mammalian cells might retain a faithful copy of epigenetic
information from earlier in life that could serve as instructions to reverse ageing10.
Reset of ageing signatures, not identity

Our first goal was to find a way to reset the epigenome without erasing cell identity. Among
the Yamanaka factors, c-Myc is an oncogene that reduces mouse lifespan20 and is not
required for the initiation of cellular reprogramming21. Therefore, we excluded c-Myc from
our experiments, expressing only Oct4, Sox2, and Klf4 (OSK) in fibroblasts from old mice
and monitoring the effect on genes known to be altered with age, including H2A, H2B,
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LaminB1, and Chaf1b13. Even without c-Myc, expression of OSK for five days promoted
youthful mRNA profile, with no apparent loss of cellular identity or the induction of Nanog,
a transcription factor indicative of pluripotency and oncogenicity (Extended Data Fig. 1a–c).
Next we tested the long-term safety of ectopically expressing OSK in vivo. To deliver and
control OSK expression in mice, we engineered a dual adeno-associated virus (AAV) system
under the tight control of a tetracycline response element (TRE) promoter (Fig. 1a). The
TRE promoter can be activated either by reverse tetracycline-controlled transactivator (rtTA)
in the presence of the tetracycline derivative doxycycline (DOX) (‘Tet-On’) or by
tetracycline-controlled transactivator (tTA) in the absence of DOX (‘Tet-Off’). Extraneous
AAV sequences were removed from the vector to accommodate OSK as a poly-cistron. To
test if AAV-mediated OSK expression was toxic in vivo, we co-infected young (5-month-
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old) and aged (20-month-old) mice with AAV9-rtTA;TRE-OSK via intravenous delivery.
OSK expression was then induced by providing Dox in the drinking water, the rationale
being that AAV9’s weak tropism for the digestive system might avoid the dysplasia and
weight loss seen in transgenic mouse models18 (Extended Data Fig. 1d–i). After 10–18
months of continuous OSK induction, no increase in tumor incidence or negative effects on
overall health were observed, indicating that cellular identity was maintained in OSK-
expressing cells (Extended Data Fig.1 j–n).
OSK stimulates axon regeneration

During ageing, almost all species experience a decline in regenerative potential. In
mammals, one of the first systems to lose regenerative potential is the CNS5–7. Retinal
ganglion cells (RGCs) of the CNS project axons away from the retina to form the optic
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nerve. If damaged, RGCs of embryos and neonates can regenerate axons, but this capacity is
lost within days after birth, likely due to a molecular program intrinsic to the RGCs6,22.
Attempts to reverse optic nerve damage have been only moderately successful, with no
treatments able to restore eyesight either in late-stage glaucoma or in old age23.
To test whether it is possible to provide adult RGCs with the regenerative capacity of
younger RGCs, we induced OSK in an optic nerve crush injury model. For retinal gene

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delivery, Tet-Off AAV2s carrying OSK as a single polycistron were injected into the vitreous
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body, resulting in Dox-responsive gene expression in around 40% of RGCs, as estimated by

immunostaining (Fig. 1b, Extended Data Fig. 2a, b). Even after 15 months of continuous
induction, OSK expression did not induce any tumors or structural changes in the retina
(Extended Data Fig. 2c–i, Supplementary Videos).
In a separate cohort, RGCs were infected with Tet-Off AAV2 and optic nerve crush was
performed two weeks later (Fig. 1c). After another two weeks, Alexa Fluor 555-conjugated
cholera toxin subunit B (CTB), an anterograde axonal tracer, was delivered by intravitreal
injection to quantify axon regeneration. Independent of RGC proliferation (Extended Data
Fig. 3a, b), the greatest extent of axon regeneration and RGC survival occurred when all
three genes were delivered and expressed as a polycistron within the same AAV particle
(Fig. 1d, e). Strikingly, when polycistronic OSK was induced for 12–16 weeks, regenerating
and sprouting RGC axon fibers extended over 5 mm into the chiasm, where the optic nerve
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connects to the brain (Extended Data Fig. 3c, d). Suppression of polycistronic OSK by Dox
treatment prevented both regenerative and survival effects (Fig. 1d, e, Extended Data Fig.
3e).
When Oct4, Sox2, and Klf4 were co-delivered via separate monocistronic AAVs, no effect
on axon regeneration was observed, ostensibly due to the lower frequency of co-infection or
inconsistent stoichiometry (Fig. 1d, Extended Data Fig. 3f–i). When delivered singly, Oct4
or Sox2 alone increased RGC survival slightly (Extended Data Fig. 3e), but none of the
single factors alone had any effect on regenerative capacity (Fig. 1d). Because Klf4 can
repress rather than promote axonal growth24, we tested a dual-cistron of only Oct4 and Sox2
and observed no regenerative effect.
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Many regenerative approaches that work well in young individuals often fail in older
ones25,26. In 12-month-old mice, OSK treatment doubled RGC survival, similar to what was
observed in 1- and 3-month-old mice (Extended Data Fig. 4a). Though axon regeneration
was slightly weaker in the older mice (Extended Data Fig. 4b), when treatment was extended
for three more weeks (5 weeks post crush; wpc), robust axon regeneration still occurred,
with no increase in RGC number (Fig. 1f, Extended Data Fig. 4c, d). These data indicate that
ageing does not greatly diminish the ability of OSK to induce axon regeneration.
To test whether OSK was acting directly on RGCs or via amacrine cells27, the other type of
retinal cells that AAV2 can infect following intravitreal injection, we introduced tTA in
double-floxed inverse orientation (AAV2-FLEx-tTA) with AAV2-TRE-OSK into two Cre
transgenic mouse lines, so that OSK selectively expressed either in RGCs (Vglut2-CRE) or
amacrine cells (Vgat-CRE) (Extended Data Fig. 4e, f). OSK expression in RGCs alone
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promoted axon regeneration, whereas expression in amacrine cells did not (Fig. 1g,
Extended Data Fig. 4g, h). The survival frequency of OSK-positive RGCs was roughly three
times that of nearby OSK-negative cells or that of RGCs in GFP-expressing retinas, resulting
in a greater relative abundance post-injury (Extended Data Fig. 4i–k). Thus, the ability of
OSK to increase survival appears to be mediated within the RGC and is largely cell-
autonomous. The mRNA levels of Stat3, a pro-regeneration gene28, robustly increased in
response to OSK and was maintained post-injury (Extended Data Fig. 5a, Supplementary

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Table 1). Stat3 upregulation, however, does not appear to be mediated by PTEN-mTOR-S6K
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and SOCS3, two canonical Stat3 regulatory pathways that affect RGC axon
regeneration29,30 (Extended Data Fig. 5b, c, Supplementary Table 1).
OSK counters injury-induced DNAm ageing

Currently, there is no known treatment that can induce robust RGC axon regeneration when
started after crush injury. Using our Tet-On AAV system, which rapidly induces OSK
expression (Fig. 2a, Extended Data Fig. 2b and 5d, e), we observed significant improvement
in axon regeneration and RGC survival, even when OSK was induced post-injury. The
longer the duration of the OSK induction, the greater the distance the axons extended, with
no sign of RGC proliferation (Fig. 2b, c, Extended Data Fig. 5f).
Given the effectiveness of OSK post-injury and the ability of Yamanaka factors to reverse
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DNAm age in vitro4,15,31,32, we speculated that OSK might have been able to regenerate
axons by counteracting the effect of injury on DNA methylation. The DNAm age of RGCs
was calculated based on a ribosomal DNA methylation clock33, which provided the best site
coverage (67/72 CpG sites) and was highly correlated with chronological age (Extended
Data Fig. 5g). Day 4-injured RGCs experienced an acceleration of DNAm age, while OSK
expression counteracted this effect (Fig. 2d). Patterns of global DNA methylation were also
altered by injury in a way similar to ageing, without affecting average DNA methylation
levels (Extended Data Fig. 5h, i). Strikingly, OSK reversed the global DNA methylation
changes caused by injury, with enrichment at genes associated with light detection and
synaptic transmission (Fig. 2e, Extended Data Fig. 5j, k).
Regeneration requires DNA demethylation

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Next, we asked whether the DNA methylation changes induced by OSK are necessary for
RGC survival and axon regrowth. DNA demethylation is catalyzed by ten-eleven-
translocation methylcytosine dioxygenases (TET1–3), either passively through DNA
replication or actively, even in non-replicating cells, by reversion to cytosine via thymine
DNA glycosylase (TDG)34. Because Tet1 and Tet2, but not Tet3, were upregulated by
OSK35,36 (Extended Data Fig. 6a), we tested the effect of knocking down Tet1 or Tet2 (sh-
Tet1 or sh-Tet2) using AAV37 in the context of damaged, OSK-treated RGCs (Extended
Data Fig. 6b–d).
The knockdown of either Tet1 or Tet2 blocked the ability of OSK to increase Stat3 mRNA
levels and promote RGC survival and axon regeneration (Extended Data Fig. 6e–g).
Knocking out Tet2 in RGCs by delivering AAV2-Cre30 intravitreally to Tet2flox/flox mice
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also abrogated the increase in regeneration and survival (Fig. 2f, Extended Data Fig. 6h–j).
While OSK-expressing RGCs showed no evidence of DNA replication by BrdU (Extended
Data Fig. 3a, b) and knockdown of TDG38 completely abolished the beneficial effects of
OSK (Extended Data Fig. 7a–d), a global reduction of DNA methylation mediated by
overexpression of the Tet1 catalytic domain39 had no protective or regenerative effect
(Extended Data Fig. 7e–h). These data indicate that active DNA demethylation is necessary

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for OSK, but not sufficient by itself, to protect RGCs and regenerate axons (Extended Data
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Fig. 7i).
Axon regeneration in human neurons

Paralleling the mouse RGC data, OSK expression in differentiated human neurons
effectively counteracted axonal loss and the advancement of DNAm age induced by
vincristine, a chemotherapeutic drug that induces axon injury, in the absence of cell
proliferation or global DNA demethylation (Extended Data Fig. 8a–g). Nine days after
damage, the neurite area was 15-fold greater in the rejuvenated OSK-transduced cells
compared to controls (Extended Data Fig. 8h, i).
OSK-mediated recovery of neurite area, as well as both the number and length of axons,
were blocked by Tet2 knockdown, but not by mTOR-S6K inhibition (Extended Data Fig. 8j–
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s). Again, overexpression of the Tet1 catalytic domain on its own failed to promote axon
regeneration (Extended Data Fig. 8t). Thus, the ability of OSK to reprogram neurons and
promote axon growth in a Tet2-dependent manner appears to be conserved in both mice and
humans.
Recovery of vision in mice with glaucoma

We then tested whether OSK induction could restore the function of RGCs in a disease
setting. Glaucoma is a leading cause of age-related blindness worldwide, characterized by a
progressive loss of RGCs and their axons and often coinciding with increased intraocular
pressure (IOP). Elevated IOP was induced unilaterally for 21 days by injection of
microbeads into the anterior chamber (Fig. 3a, b)40, with saline injections serving as non-
glaucomatous controls. At the 4-week time point, after a significant decrease in axonal
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density and the number of RGCs (baseline, Extended Data Fig. 9a, b), AAVs were injected
intravitreally and OSK expression was induced for another 4 weeks (Fig. 3a). Compared to
glaucomatous eyes that received either PBS or AAVs with no OSK induction (−OSK), the
OSK-treated glaucomatous eyes (+OSK) presented with a restoration in axon density
equivalent to that in the non-glaucomatous eyes, with no evidence of RGC proliferation
(Extended Data Fig. 9c, d).
The OSK-treated eyes also experienced a significant increase in visual acuity relative to the
pre-treatment function baseline, restoring about half of visual acuity in the optomotor
response (OMR) assay (Fig. 3c, d). A similar improvement associated with OSK expression
was also seen in pattern electroretinogram (PERG) analysis (N1+P1), a readout of electrical
waves generated specifically by RGCs (Fig. 3e, f). To our knowledge, this is the first
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example of vision loss reversal after glaucomatous injury has occurred, with previous
attempts focusing on neuroprotection delivered at an early stage to prevent further disease
progression41,42 (Supplementary discussion).
Restoration of vision in old mice

The finding that OSK induction could effectively restore the DNAm age of RGCs after
injury indicated that vision loss caused by natural ageing might be reversible too. To test

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this, we treated 3- and 11-month-old mice with −OSK or +OSK AAVs and induced OSK for
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4 weeks (Fig. 4a). OMR and PERG measurements were then carried out. In the absence of
OSK induction, there was a significant reduction in visual acuity and RGC electrical activity
by one year of age, a loss that was restored by OSK induction (Fig. 4b, Extended Data Fig.
9e). Restoration of visual acuity was not seen in 18-month-old mice (Extended Data Fig. 9e,
f), likely because an age-dependent increase in corneal opacity43 interfered with assessment.
Because there was no obvious increase in the number of RGC or axon density in the 12-
month-old mice treated with +OSK AAV (Extended Data Fig. 9g, h), we hypothesized that
the visual improvements were due to transcriptomic changes. RGCs from 12-month-old
mice, either untreated or treated with OSK, were FACS-purified and analyzed by RNA-seq.
Compared to RGCs from 5-month-old young mice, the mRNA levels of 464 genes were
altered during ageing. Remarkably, ~90% of them were restored to youthful levels by OSK
treatment (Fig. 4c, d, Extended Data Fig. 9i, Supplementary Table 2). Of the 268 age-
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downregulated genes, 44 are linked to sensory perception and axon targeting44, raising the
possibility that they contribute to the decline in vision caused by ageing (Fig. 4d, Extended
Data Fig. 9j and 10a). Another 196 genes that were up-regulated during ageing are involved
in ion transport or neuronal projection development, including Efemp1 (Extended Data Fig.
10b–d), a Tet1- and Tet2-regulated gene associated with glaucoma45 and age-related macular
degeneration (AMD)46.
To gain insights into how DNA methylation changes might be involved in the effects of
OSK, we used machine learning to identify a methylation ageing signature based on 1,226
sites from genome-wide DNAm datasets, derived from the first principal component (PC1)
in multi-age training samples (Supplementary Table 3, Extended Data Fig 10e). In an
independent set of test samples, OSK reversed the DNAm ageing signature in the context of
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both injury and ageing (Fig. 4e and Extended Data Fig 10f). Interestingly, the signature CpG
sites were associated with genes involved in synaptic and neuronal processes and enriched
for the binding of polycomb repressive complex 2 (PRC2) and its histone methyltransferase
product H3K27me3, both of which are known to recruit Tet enzymes47,48 (Extended Data
Fig 10g–i). Knockdown of either Tet1 or Tet2 altered the methylation status of the signature
CpG sites in a similar way and counteracted the effect of OSK on DNAm age (Extended
Data Fig. 10j–l). Both Tet1 and Tet2 were also required to improve RGC function and vision
in 12-month-old mice (Fig. 4f, Extended Data Fig. 10m), indicating that changes in DNA
methylation may play a functional role in the restoration of vision in old mice by OSK.
Discussion
Here, we show that it is possible to safely reverse the age of a complex tissue and restore its
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biological function in vivo. Using the eye as a model system, we present evidence that
ectopically expressed OSK safely induces in vivo epigenetic restoration of aged CNS
neurons, without causing a loss of cell identity or pluripotency. Instead, OSK promotes a
youthful epigenetic signature and gene expression pattern that causes the neurons to function
as though they were young again. The requirement for active demethylation in this process
supports the idea that changes in DNA methylation patterns are involved in the ageing
process and its functional reversal (Extended Data Fig. 10n). That said, we do not wish to

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imply that DNA methylation is the only epigenetic mark involved in this process. It likely
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involves other transcription factors and epigenetic modifications, such as the PRC2 complex
and H3K27me3, both of which are involved in maintaining stem cell plasticity and are
associated with DNAm clock sites4,49,50.
Perhaps the most interesting question raised by these data is: How do cells encode and store
youthful epigenetic information? Possibilities for information storage include covalent DNA
modifications, DNA binding proteins, RNA-guided chromatin modifying factors, and RNA-
DNA hybrids that are established early in life. The role of these youth marks would be akin
to Shannon’s “Observer” in Information Theory, which preserves an original backup copy of
information in case it is lost or obscured by noise11. We envision that epigenetic
reprogramming, either by gene therapy or other means, could promote tissue repair and the
reversal of age-related decline in humans.
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Methods
Mouse lines
For optic nerve crush and glaucoma model experiments, C57BL6/J wild type mice were
purchased from the Jackson Laboratory (000664). Young and old females from the NIA
Aged Rodent Colonies were used for ageing experiments. Rosa26-M2rtTA/Col1a1-tetOP-
OKS-mCherry alleles were from the Hochedlinger lab (Massachusetts General Hospital)51.
Rosa-CAG-lox-STOP-lox-Tomato mice were provided by Fan Wang (McGovern Institute).
Vglut2-IRES-Cre (016963), Vgat-IRES-Cre (016962), Tet2flox/flox (017573)52 and Rosa26-
M2rtTA/Col1a1-tetOP-OSKM (011011) were purchased from the Jackson Laboratory. All
animal work was approved by the Institutional Animal Care and Use Committees (IACUCs)
at Harvard Medical School, Boston Children’s Hospital, and the Mass Eye and Ear
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Institution. Animals were housed under 12-hour light/dark cycles 6am-6pm-6am, 70–72°F
ambient temperature, and 40–50% humidity.
Surgery
Mice were anesthetized by intraperitoneal injection of a mixture of ketamine (100 mg/kg;
Ketaset; Fort Dodge Animal Health, Fort Dodge, IA) and xylazine (10 mg/kg; TranquiVed;
Vedco, Inc., St. Joseph, MO) supplemented by topical application of proparacaine to the
ocular surface (0.5%; Bausch & Lomb, Tampa, FL). All animal procedures were approved
by the IACUC of the respective institutions and according to appropriate animal welfare
regulations.
Production of adeno associated viruses (AAVs)

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Vectors of AAV-TRE-OSK were made by cloning mouse Oct4, Sox2 and Klf4 cDNA into an
AAV plasmid consisting of the Tetracycline Response Element (TRE3G promoter) and the
SV40 element. The other homemade vectors were made using a similar strategy or directly
chemically synthesized. All pAAVs, as listed (Supplementary Table 4), were then packaged
into AAVs of serotype 2/2, 2/DJ or 2/9 (titers: > 5×1012 genome copies/ml). AAVs were
produced by the Boston Children’s Hospital Viral Core.

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Systemic delivery of AAV9

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Expression in internal organs was achieved through retro-orbital injection of AAV9: 7×1011
gc of UBC-rtTA and 3×1011 gc of TRE-OSK for 5-month-old mice; 7×1011 gc of UBC-rtTA
and 5×1011 gc of TRE-OSK (or TRE-GFP) for 20-month-old mice. To induce OSK
expression, doxycycline (1 mg/ml; MP Biochemicals) was given in drinking water
continuously until the end of the experiment, 3 weeks post-AAV injection.
Cell culture of ear fibroblasts

Ear fibroblasts were isolated from Reprogramming 4F (Jackson Laboratory 011011) or 3F
(Hochedlinger lab, Massachusetts General Hospital) mice and cultured at 37ºC in DMEM
(Invitrogen) containing non-essential amino acids, 10% tetracycline-free fetal bovine serum
(TaKaRa Bio, 631106) and 1% penicillin/streptomycin (ThermoFisher Scientific,
15140122). EFs of transgenic OSKM and OSK mice were passaged to P3 and treated with
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doxycycline (2 µg/ml) for the indicated time periods in the culture medium. All cell lines
used were mycoplasma negative.
AAV2 intravitreal injection

Adult animals were anesthetized with ketamine/xylazine (100/10 mg/kg), and then AAV (1–
2 µl) was injected intravitreally, just posterior to the limbus with a fine glass pipette attached
to a Hamilton syringe using plastic tubing. In the elevated IOP model, mice received a 1 µl
intravitreal injection between 3–4 weeks following microbead injection. The injected
volume of AAV-sh-RNA is 1/5th the volume of other AAVs.
Optical Coherence Tomography (OCT)

OCT images were taken with a Bioptigen Envisu R-Class OCT (Leica Microsystems). The
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animals were anesthetized with a ketamine/xylazine (100–200/20 mg/kg) cocktail and eyes
were treated with a drop of 1% tropicamide solution to dilate the pupils and a drop of
GenTeal gel to keep the lens hydrated. Full retinal OCT scans were obtained for all eyes
(1000× 100× 10). 100 B-scans were converted into 7 fps videos with ImageJ. Representative
OCT images of the retina were taken near the optic nerve head, and the imaging location
was marked on the volume intensity projection image with a white line. The retinal
thickness in each eye was measured using 4 B-scans at a distance of 50 – 600 µm on both
sides of the optic nerve head and averaged using ImageJ.
Creation of retinoblastoma tumors

In order to create the space to inject the retinoblastoma tumor cells into the subretinal space,
a transient retinal detachment was created. Intraocular pressure was decreased by first
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making a corneal incision, followed by a subretinal injection of 10,000 retinoblastoma tumor

cells (Rb116) via a 30-gauge needle in a total volume of 10 μl. Two weeks post-injection the
mice were observed via OCT.
Optic nerve crush

Two weeks after intravitreal AAV injection, the optic nerve was accessed intraorbitally and
crushed in anesthetized animals using a pair of Dumont #5 forceps (FST). Alexa-conjugated

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cholera toxin beta subunit (CTB-555, 1 mg/ml; 1–2 µl) injection was performed 2–3 days
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before euthanasia to trace regenerating RGC axons. More detailed surgical methods were
described previously29.
In vivo doxycycline induction or suppression

Induction of the Tet-On or suppression of the Tet-Off AAV2 systems in the retina was
performed by administration of doxycycline (2 mg/ml) (Sigma) in the drinking water.
Induction of Tet-On AAV9 system systemically was performed by administration of
doxycycline (1 mg/ml) (USP grade, MP Biomedicals 0219895505) in the drinking water.
Quantification of axon regeneration for the optic nerve crush model

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

Optic nerves and the connecting chiasm were dehydrated in methanol for 5 min, then
incubated overnight with Visikol® HISTO-1™. The next day, nerves were transferred to
Visikol® HISTO-2™ and then incubated for 3 hrs. Finally, optic nerves and connecting
chiasm were mounted with Visikol® HISTO-2™.
Immunofluorescence
Wholemount retinas were blocked with horse serum 4°C overnight then incubated at 4°C for
3 days with primary antibodies diluted in PBS, BSA (3%) Triton X-100 (0.5%). Then,
tissues were incubated at 4°C overnight with appropriate Alexa Fluor-conjugated secondary
antibodies (Alexa 405, 488, 567, 674; Invitrogen) diluted with the same blocking solution as
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the primary antibodies, generally used at 1:400 final dilution. Frozen sections were stained
overnight with primary antibodies at 4°C and then secondary antibodies at room temperature
for 2 hrs. Between changes of solutions, all wholemounts or slices were washed 3 × 5 min
each time. Sections or wholemount retinas were mounted with Vectashield Antifade
Mounting Medium. Antibodies used: Mouse anti-Oct4 (1:100, BD bioscience 611203),
Rabbit anti-Sox2 (1:100, Cell signaling 14962), Goat anti-Klf4 (1:100, R&D system
AF3158), Rabbit anti-phosphorylated S6 Ser235/236 (1:100, Cell Signaling 4857), Mouse
anti-Brn3a (1:200, EMD Millipore MAB1585), Rabbit anti-Ki67(1:100, Abcam ab15580),
Mouse anti-AP2 alpha (1:100, Developmental Studies Hybridoma Bank 3B5), Rabbit anti-
pStat3 (Tyr705) (1:100, Cell signaling 9145S), Rat anti-HA (1:400, Roche 11867423001),
Rabbit anti-5mC (1:100, Cell signaling 28692S), Rabbit anti-5hmC (1:100, Active Motif
39769), Rat anti-BrdU (1:200, Abcam ab6326), Rabbit anti-Olig2 (1:100, Novusbio NBP1–
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28667) and Chicken anti-GFP (1:10,000, Aves Labs GFP-1020), and Guinea pig anti-
RBPMS (1:400, Raygene custom order A008712 to peptide
GGKAEKENTPSEANLQEEEVRC). Note that successful staining for anti-5mC,
anti-5hmC, anti-Ki67, and anti-BrdU requires pre-treatment of 2 N HCl for 30min at room
temperature, followed by 3 washes of 0.1 M Sodium Borate pH 8.3 and PBST each before
the serum blocking.

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5-Bromo-2′-deoxyuridine (BrdU) labelling

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Post crush injury, mice were injected intraperitoneally with BrdU (Sigma, B5002) at a dose
of 100 mg/kg daily the week before sacrifice. Optic nerves and retinas were collected either
1 or 2 wpc. Optic nerve sections and retina wholemounts were then performed with the same
procedure described for immunofluorescence (including HCl pre-treatment) to complete the
staining.
Western blot
SDS-PAGE and Western blot analysis was performed according to standard procedures and
detected with an ECL detection kit. Antibodies used: Rabbit anti-Sox2 (1:100, EMD
Millipore, AB5603), Mouse anti-Klf4 (1:1,000, ReproCell, 09–0021), Rabbit anti-p-S6
(S240/244) (1:1,000, CST, 2215), Mouse anti-S6 (1:1,000, CST, 2317), Mouse anti-β-
Tubulin (1:1,000, Sigma-Aldrich, 05–661), Mouse anti-β-Actin−Peroxidase antibody
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(1:20,000, Sigma-Aldrich, A3854).
RGC survival and phospho-S6 signal

RBPMS-positive cells in the ganglion layer were stained with an anti-RBPMS antibody
(1:400, Raygene custom order A008712 to peptide GGKAEKENTPSEANLQEEEVRC),
and a total of four 10× fields per retina, one in each quadrant, were enumerated. The average
number of viable RGCs per field was determined. Phospho-S6 (1:100, Cell Signaling 4857)
staining was performed under the same conditions and the percentages of phopsho-S6-
positive RGCs were obtained by comparing the value to the number of viable RGCs in the
same field.
Human neuron differentiation and regeneration assay

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We chose to use differentiated human neurons that would maintain ageing signatures53. SH-
SY5Y neuroblastoma cells were obtained from the American Tissue Culture Collection
(ATCC, CRL-2266). Cells were confirmed as negative for mycoplasma, then cultured in a
1:1 mixture of EMEM (ATCC, 30–2003) and F12 medium (ThermoFisher Scientific,
11765054) supplemented with 10% FBS (Sigma, F0926) and 1% penicillin/streptomycin
(ThermoFisher Scientific, 15140122). Cells were cultured at 37°C with 5% CO2 and 3% O2.
Cells were passaged at ~80% confluency. SH-SY5Y cells were differentiated into neurons as
previously described54 with modifications. Briefly, 1 day after plating, cell differentiation
was induced for 3 days using EMEM/F12 medium (1:1) containing 2.5% FBS, 1× penicillin/
streptomycin, and 10 µM all-trans retinoic acid (ATRA, Stemcell Technologies, 72264)
(Differentiation Medium 1), followed by a 3 day incubation in EMEM/F12 (1:1) containing
1% FBS, 1 × penicillin/streptomycin, and 10 µM ATRA (Differentiation Medium 2). Cells
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were then split into 35 mm cell culture plates coated with poly-D-lysine (ThermoFisher
Scientific, A3890401). A day after splitting, neurons were matured in serum-free
neurobasal/B27 plus culture medium (ThermoFisher Scientific, A3653401) containing 1 ×
Glutamax (ThermoFisher Scientific, 35050061), 1 × penicillin/streptomycin, and 50 ng/ml
BDNF (Alomone labs) (Differentiation Medium 3) for at least 5 days. Differentiated SH-
SY5Y cells were transduced with AAV.DJ vectors at 106 genome copies/cell. Five days after
transduction, vincristine (100 nM; Sigma, V8879) was added for 24 hrs to induce neurite

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degeneration. Neurons were then washed once with PBS and fresh Differentiation Medium 3
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was added back to the plates. Neurite outgrowth was monitored for 2–3 weeks by taking
phase-contrast images at 100x magnification every 3–4 days. Neurite area, axon number and
length of each cluster of neurons were quantified using Image J.
Cell cycle analysis

Cells were harvested and fixed with 70% cold ethanol for 16 hrs at 4°C. After fixation, cells
were washed twice with PBS and incubated with PBS containing 50 μg/mL propidium
iodide (Biotium, 40017) and 100 μg/mL RNase A (Omega) for 1 hr at room temperature. PI-
stained samples were analyzed on a BD LSR II analyzer and only single cells were gated for
analysis. Cell cycle profiles were analyzed using FCS Express 6 (De Novo Software).
Human neuron DNA methylation analyses

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DNA was extracted using the Zymo Quick DNA mini-prep plus kit (D4069) and DNA
methylation levels were measured on Illumina 850 EPIC arrays. The Illumina BeadChip
(EPIC) measured bisulfite-conversion-based, single-CpG resolution DNA methylation levels
at different CpG sites in the human genome. Data were generated via the standard protocol
of Illumina methylation assays (GSE147436), which quantifies methylation levels by the β
value using the ratio of intensities between methylated and unmethylated alleles.
Specifically, the β value was calculated from the intensity of the methylated (M
corresponding to signal A) and unmethylated (U corresponding to signal B) alleles, as the
ratio of fluorescent signals β = Max(M,0)/(Max(M,0)+ Max(U,0)+100). Thus, β values
ranged from 0 (completely unmethylated) to 1 (completely methylated). “Noob”
normalization was implemented using the “minfi” R package55,56. The mathematical
algorithm and available software underlying the skin & blood clock for in vitro studies
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(based on 391 CpGs) was previously published57.
Microbead-induced elevated intraocular pressure (IOP)

Elevation of IOP was induced unilaterally by injection of polystyrene microbeads
(FluoSpheres; Invitrogen, Carlsbad, CA; 15-μm diameter) to the anterior chamber of the
right eye of each animal under a surgical microscope, as previously reported40. Briefly,
microbeads were prepared at a concentration of 5.0 × 106 beads/mL in sterile physiologic
saline. A 2 μL volume was injected into the anterior chamber through a trans-corneal
incision using a sharp glass micropipette connected to a Hamilton syringe (World Precision
Instruments Inc., Sarasota, FL) followed by an air bubble to prevent leakage. Any mice that
developed signs of inflammation (clouding or an edematous cornea) were excluded.
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IOP measurements
IOPs were measured with a rebound TonoLab tonometer (Colonial Medical Supply, Espoo,
Finland), as previously described40. Mice were anesthetized by 3% isoflurane in 100%
oxygen (induction) followed by 1.5% isoflurane in 100% oxygen (maintenance) delivered
with a precision vaporizer. IOP measurement was initiated within 2–3 min after the loss of a
toe or tail pinch reflex. Anesthetized mice were placed on a platform, and the tip of the
pressure sensor was placed approximately 1/8 inch from the central cornea. Average IOP

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was displayed automatically after 6 measurements after elimination of the highest and
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lowest values. The machine-generated mean was considered as one reading, and six readings
were obtained for each eye. All IOPs were taken at the same time of day (between 10:00 and
12:00 hrs) due to the variation of IOP throughout the day.
Optomotor response
Visual acuity of mice was measured using an optomotor reflex-based spatial frequency
threshold test58. Mice were able to freely move and were placed on a pedestal located in the
center of an area formed by four computer monitors arranged in a quadrangle. The monitors
displayed a moving vertical black and white sinusoidal grating pattern. A blinded observer,
unable to see the direction of the moving bars, monitored the tracking behavior of the
mouse. Tracking was considered positive when there was a movement of the head (motor
response) to the direction of the bars or rotation of the body in the direction concordant with
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the stimulus. Each eye was tested separately depending on the direction of rotation of the
grating. The staircase method was used to determine the spatial frequency start from 0.15 to
0.40 cycles/deg, with intervals of 0.05 cycles/deg. Rotation speed (12°/s) and contrast
(100%) were kept constant. Responses were measured before and after treatment by
individuals blinded to the group of the animal and the treatment. Mice that had intravitreal
bleeding or developed signs of inflammation (clouding or an edematous cornea) during or
post intravitreal injection were excluded from OMR and histological analyses. Exclusion
criteria were pre-determined before experimentation.
Pattern electroretinogram (PERG)

Mice were anesthetized with ketamine/xylazine (100mg/kg and 20mg/kg) and the pupils
dilated with one drop of 1% tropicamide ophthalmic solution. The mice were kept under dim
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red light throughout the procedure on a built-in warming plate (Celeris, Full-Field and
Pattern Stimulation for the rodent model) to maintain body temperature at 37°C. A black and
white reversing checkerboard pattern with a check size of 1° was displayed on light guide
electrode-stimulators placed directly on the ocular surface of both eyes and centered with the
pupil. The visual stimuli were presented at 98% contrast and constant mean luminance of 50
cd/m2, spatial frequency: 0.05 cyc/deg; temporal frequency: 1Hz. A total of 300 complete
contrast reversals of PERG were repeated twice in each eye and the 600 cycles were
segmented, averaged, recorded. The averaged PERGs were analyzed to evaluate the peak to
trough N1 to P1 (positive wave) amplitude.
Quantification of optic nerve axons in the glaucoma model

For quantification of axons, optic nerves were dissected and fixed overnight in Karnovsky’s
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reagent (50% in phosphate buffer). Semi-thin cross-sections of the nerve were taken at 1.0
mm posterior to the globe and stained with 1% p-phenylenediamine (PPD) for evaluation by
light microscopy. Optic nerve cross sections were imaged at 60x magnification using a
Nikon microscope (Eclipse E800, Nikon, Japan) with the DP Controller v. 1.2.1.108 and the
DP Manager v. 1.2.1.107 software (Olympus). 6–8 non-overlapping photomicrographs were
taken to cover the entire area of each optic nerve cross-section. Using ImageJ (Version 2.0.0-
rc-65/1.51u), a 100 μM × 100 μM square was placed on each 60x image and all axons within
the square (0.01 mm2) were counted using the “threshold” and “analyze particles” functions

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in ImageJ as previously described40,58,59. Damaged axons stain darkly with PPD and were
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not counted. The average axon counts in the 6–8 images were used to calculate the axon
density/mm2 of optic nerve. Individuals performing axon counts were blinded to the
experimental groups.
Quantification of retinal ganglion cells in the glaucoma model

For ganglion cell counting, images of wholemount retinas were acquired using a 63x oil
immersion objective of the Leica TCS SP5 confocal microscope (Leica Microsystems). The
retinal wholemount was divided into four quadrants and two to four images (248.53 μm by
248.53 μm in size) were taken from the midperipheral and peripheral regions of each
quadrant, for a total of twelve to sixteen images per retina. The images were obtained as z-
stacks (0.5 μm), and all Brn3a positive cells in the ganglion cell layer of each image were
counted using an automated counting platform as previously described59,60. Briefly, RGCs
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were counted using the “Cell Counter” plugin (http://fiji.sc/Cell_Counter) in Fiji (ImageJ
Fiji, version 2.0.0-rc-69/1.52n). Each image was loaded into Fiji, and a color counter type
was chosen to mark all Brn3a stained RGCs within each image (0.025 mm2). The average
number of RGCs in the 12–16 images were used to calculate the RGC density per square
millimeter of retina. Two individuals blinded to the experimental groups performed all RGC
counts.
RGC enrichment
Retinas were dissected in AMES solution (oxygenated with 95% O2/5% CO2), digested in
papain, and dissociated to single cell suspensions with manual trituration in ovomucoid
solution. Cells were spun down at 450 × g for eight minutes, resuspended in AMES
+4%BSA to a concentration of 105 cells/ml. Thy1.2-PE-Cy7 antibody (1:2000, Invitrogen
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25–0902-81) was added incubated for 15 min, followed by washing with an excess of media.
Cells were spun down at 450 × g for eight minutes and resuspended again in AMES
+4%BSA at a concentration of ~7 × 106 cells/ml. The live cell marker Calcein Blue (1µl/ml)
was added before filtering cells through 35µm cell strainer into FACS tubes. Over 10,000
high Thy1.2+ and Calcein Blue+ cells were collected using a BD FACS Aria Cell Sorter
with an 130µm nozzle. Frozen cells were sent to GENEWIZ, LLC (South Plainfield, NJ,
USA) for RNA extraction and ultra-low input RNA-seq, or to Zymo Research (Irving, CA)
for DNA extraction and genome-wide reduced representation bisulfite sequencing (RRBS).
RRBS library preparation

RGC DNA from mice at different ages and treatments (Supplementary Table 5) was
extracted using Quick-DNA Plus Kit Microprep Kit. 2–10 ng of starting input genomic DNA
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was digested with 30 units of MspI (NEB). Fragments were ligated to pre-annealed adapters
containing 5’-methyl-cytosine instead of cytosine according to Illumina’s specified
guidelines. Adaptor-ligated fragments ≥ 50 bp in size were recovered using the DNA Clean
& Concentrator™-5 (Cat#: D4003). The fragments were then bisulfite-treated using the EZ
DNA Methylation-Lightning™ Kit (Cat#: D5030). Preparative-scale PCR products were
purified with DNA Clean & Concentrator™-5 (Cat#: D4003) for sequencing on an Illumina
HiSeq using 2×125 bp paired end (PE).

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DNA methylation analysis of mouse RGC

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Reads were filtered using trim galore v0.4.1 and mapped to the mouse genome GRCm38
using Bismark v0.15.0. Methylation counts on both positions of each CpG site were
combined. Only CpG sites covered in all samples were considered for analysis (BioProject
PRJNA655981). Two outliers were excluded by low intercorrelation (< 0.93) within the
batch (Supplementary Table 5). This resulted in a total of 703,583 sites, covered by uniquely
mapped reads. Differential methylation for each CpG site was calculated using two-tailed
Student’s t-tests on every site independently, followed by Benjamini-Hochberg procedure to
adjust for false discovery rate. Delta between groups was calculated as difference between
the means of values in two corresponding groups of samples. Gene ontology analysis of
differentially methylated sites was performed with Cistrome-GO (http://go.cistrome.org).
Due to the repetitive nature of ribosomal DNA sequence, it is not suitable to whole-genome
bisulfite mapping. Thus, to determine the ribosomal DNAm age, reads were mapped to
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ribosomal DNA repeat BK000964 and the coordinates were adjusted accordingly33. 67/72
sites with at least 10 reads per site were covered for the ribosomal DNAm clock, compared
to 102/435 sites of the whole lifespan multi-tissue clock61, or 248/582 and 77,342/ 193,651
sites (ridge) of two entire lifespan multi-tissue clocks62. In agreement with previous
studies57,63,64, DNAm age estimates of neurons tend to be lower than their chronological
age but remain correlated.
DNAm ageing signature

Sorted RGC samples were split into training and test sets, with n=38 samples used for
candidate CpG selection and biomarker construction and n=23 samples used for validation
(Supplementary Table 6, Supplementary Data and Supplementary Code). The training
samples included those from 1-month-old controls (1 mo; n=6), 6 mo (n=8), 12 mo (n=2),
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18 mo (n=8), 30 mo (n=6), 1 mo GFP-injured (n=4), 12 mo GFP (n=2), and 12 mo OSK

(n=2). To generate a methylation ageing signature, we evaluated DNAm associations with
three traits: age, injury, and OSK treatment. To avoid batch effects, subsamples were
evaluated separately as 6 mo and 18 mo in Batch 1, and 1 mo, 12 mo, and 30 mo in Batch 2
(Supplementary Table 5). Biweight midcorrelation was used to assess age trends for CpGs.
Given that the second batch included samples from developing animals (1-month-old mice)
we applied a transformation to age that has been used in development of human epigenetic
clocks when prenatal and/or developmental samples are included4. This transformation
accounts for non-linear changes during development. Biweight midcorrelation was then
applied to test the associations of CpGs changes after injury and OSK treatment, and results
were compared across the four tests. CpGs were selected if they were consistently in the top
30% of CpGs with the strongest absolute biweight midcorrelations in all four tests and
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showed consistency in the directionality of their associations (e.g. hypomethylated with age
in both batches, hypomethylated with injury, and hypermethylated after OSK). Of the
703,583 CpGs that were considered, 1,226 were selected (723 that trended towards
hypermethylation with age and 503 that trended towards hypomethylation with age, see
Supplementary Table 3). Principal Component Analysis (PCA) was conducted using the
training samples from 1 mo, 12 mo, and 30 mo controls (n = 14). PC1 explained 25% of the
variance in this sample. When applying an elastic net model to train a predictor of age, PCs
beyond PC1 did not increase the robustness of the age prediction65. Thus, PC1 was used to

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represent the ageing signature and was standardized to have a mean = 0 and s.d. = 1.
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Analyses of the ageing signature in independent validation samples were then conducted to
test associations with age, injury, and OSK.
Transcription factor (TF) binding and histone modification enrichment

To identify factors that may mediate epigenetic reprogramming in RGCs, we performed TF
binding enrichment and histone modification enrichment analyses using the genome
coordinates (GRCm38/mm10) of the 1,226 selected CpGs using the CistromeDB Toolkit
(http://dbtoolkit.cistrome.org). A sensitivity analysis was conducted by performing TF and
histone enrichment for five sets of 1,226 random CpGs from the 702,357 unselected sites,
which was then compared against the enrichment for our selected set in in order to identify
TFs and histone marks that were specific to the selected set but not CpGs in general.
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Total RNA extraction and sample quality control

Total RNA was extracted following the Trizol Reagent User Guide (Thermo Fisher
Scientific). 1 µl of 10 mg/ml glycogen was added to the supernatant to increase RNA
recovery. RNA was quantified using a Qubit 2.0 Fluorometer (Life Technologies, Carlsbad,
CA, USA), and RNA integrity was determined using TapeStation (Agilent Technologies,
Palo Alto, CA, USA).
Ultra-low input RNA library preparation and multiplexing

RNA samples were quantified using a Qubit 2.0 Fluorometer (Life Technologies, Carlsbad,
CA, USA), and RNA integrity was ascertained using a 2100 TapeStation (Agilent
Technologies, Palo Alto, CA, USA). RNA library preparations, sequencing reactions, and
initial bioinformatics analysis were conducted at Genewiz (South Plainfield, NJ, USA). A
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SMART-Seq v4 Ultra Low Input Kit for Sequencing was used for full-length cDNA
synthesis and amplification (Clontech, Mountain View, CA), and Illumina Nextera XT
library was used for sequencing library preparation. Briefly, cDNA was fragmented, and
adaptors were added using Transposase, followed by limited-cycle PCR to enrich and add an
index to the cDNA fragments. The final library was assessed by a Qubit 2.0 Fluorometer and
an Agilent TapeStation.
Paired End Sequencing

The sequencing libraries were multiplexed and clustered on two lanes of a flowcell. After
clustering, the flowcell were loaded on the Illumina HiSeq instrument according to
manufacturer’s instructions. Samples were sequenced using a 2×150 Paired End (PE)
configuration. Image analysis and base calling were conducted by the HiSeq Control
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Software (HCS) on the HiSeq instrument. Raw sequence data (.bcl files) generated from an
Illumina HiSeq was converted into fastq files and de-multiplexed using the Illumina
bcl2fastq v2.17 program. One mismatch was allowed for index sequence identification.
RNA-seq analysis
Paired-end reads were aligned with hisat2 v2.1.066 to the Ensembl GRCm38 primary
assembly using splice junctions from the Ensembl release 84 annotation. Paired read counts

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were quantified using featureCounts v1.6.467 using reads with a mapping quality (mapQ)
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value ≥ 20. Differentially-expressed genes for each pairwise comparison were identified
with edgeR v3.2668, testing only genes with at least 0.1 counts-per-million (CPM) in at least
three samples. Gene ontology analysis of differentially expressed genes (BioProject
PRJNA655981) was performed with AmiGO v2.5.1269–71. Gene selection criteria for Fig.
4c, 4d and 9i: genes significantly changed during ageing (q < 0.05, absolute log2 fold-
change > 1), not low expressed (log2(CPM) > - 2) or altered by mock AAV infection (Old vs
Old (−OSK), q > 0.1).
Statistical analysis and figure preparation

Statistical analyses were performed with GraphPad Prism 7/8, using two-tailed Student’s t-
tests, one-way or two-way ANOVA. All of the statistical tests performed are indicated in the
figure legends. The data are presented as mean ± S.E.M., except for the violin plots in
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Extended Data Fig. 8e, f, which show median and quartiles. Statistical analysis of changes to
DNA methylations was performed with SciPy package in python. Figures were prepared
using Keynote and Affinity Designer.
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Extended Data
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Extended Data Fig. 1. Effectiveness and safety of OSK reprogramming.

a, Experimental outline for testing the effects of OSKM and OSK on gene expression in
fibroblasts from young and old transgenic (TG) mice. b and c, Expression of OSKM (b,
R26rtTA; Col1a1OSKM, n = 3 biological replicates each condition) and OSK (c, R26rtTA;
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Col1a1OKS-mCherry, n = 3 and 8 biological replicates) rescue age-associated transcriptional

changes without inducing Nanog mRNA. mo, month(s). qPCR primers are in Supplementary
Table 7. d, AAV-ubiquitinC (UbC)-rtTA and AAV-TRE-Luc vectors for measuring tissue
distribution. e, Luciferase imaging of WT mice 2 mo after intravenous injection (retro-
orbital) of AAV9-UbC-rtTA;TRE-Luc (1.0 × 1012 gene copies total). Doxycycline (Dox)
was delivered in drinking water (1 mg/mL) for 7 days to +Dox mice. f, Luciferase imaging
of the eye (Ey), brain (Br), pituitary gland (Pi), heart (He), thymus (Th), lung (Lu), liver
(Li), kidney (Ki), spleen (Sp), pancreas (Pa), testis (Te), adipose (Ad), muscle (Mu), spinal

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cord (SC), stomach (St), small intestine (In), and cecum (Ce) 2 mo after retro-orbital
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injection of AAV9-UbC-rtTA;TRE-Luc followed by treatment with Dox for 7 days. The

luciferase signal was primarily in the liver. Imaging the same tissues with a longer exposure
time (right panel) with the liver removed, revealing a strong signal in the pancreas. g,
Toxicity and safety studies in young and old mice after in vivo delivery of OSK-expressing
AAVs. For h and i, at age 5 mo, mice were intravenously injected with AAV9-rtTA;TRE-
OSK (3 and 7 × 1011 gene copies each AAV/mouse). After 1 mo, mice remained untreated
(−Dox) or treated with Dox (+Dox) for 18 mo. WT mice were not injected with AAV. For j–
n, at age 21 mo, mice were injected intravenously with 5 × 1011 gene copies of AAV9-rtTA
and 7 × 1011 of either AAV9-TRE-GFP (GFP) or TRE-OSK (OSK) per mouse. After 1 mo,
GFP, OSK, and non-injected WT mice were treated with Dox for 10 mo. h, Sox2 expression
in the liver of WT mice 2 mo post-intravenous delivery of OSK-expressing AAV9s with or
without a mo of Dox induction, and in the liver of OSK transgenic (TG) mice, 129S1/
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C57BL/6J mixed background. Uncropped scans are shown in Supplementary Figure 1. i,

Body weight of WT mice, OSK transgenic mice, and AAV-mediated OSK-expressing mice
with or without doxycycline in the first 4 weeks (left panel, n = 5, 3, 6, 4, 4, and 3 mice) and
following 17 mo (right panel, n = 5, 3, 6, and 4 mice). j, Examples of liver sections from WT
or GFP mice showing the infection of AAV9. Scale bar, 100 µm. k, Klf4 and GFP protein
levels in livers of WT, GFP, and OSK mice at 32 mo of age. * indicates high OSK
expression, + indicates induced protein expression levels in livers of OSK transgenic mice.
Uncropped scans in Supplementary Figure 1. l, Tumor incidence in WT, GFP, and OSK
mice at age 32 mo after 10 mo of Dox induction. m and n, Liver tumor scores (m) and white
blood cell counts (n) for WT, GFP, and OSK groups at age 32 mo after 10 mo of Dox
induction. OSK mice were defined as either high expression (indicated by * in panel k) or
low expression (WT, n = 11 mice; GFP, n = 10 mice; OSK high, n = 7 mice; OSK low, n = 8
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mice). For m and n, there was no difference between the groups using one-way ANOVA. All
data are presented as mean ± s.e.m.
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Extended Data Fig. 2. Normal architecture and absence of tumors in the retina after long-term
OSK expression mediated by AAV2 delivery.
a and b, Representative wholemount retina display of RBPMS (a RGC marker) and Klf4
immunofluorescence showing (a) expression from the AAV2 Tet-Off system can be turned
off by doxycycline in drinking water (2 mg/mL, 3 d), and (b) expression from the AAV2
Tet-On system can be turned on by doxycycline (2 mg/mL, 2 d). Scale bars in a and b, 1
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mm. n = 4 retinas each condition. c, Corresponding retinal wholemount images stained for
RBPMS and Klf4 are shown for each group tested (i) no injection, n = 6; (ii) −OSK
(intravitreal injection of AAV2-rtTA;TRE-OSK without Dox induction), 10 mo post-
injection, n = 3; (iii) +OSK (intravitreal injection of AAV2-tTA;TRE-OSK with Dox
induction), 15 mo post-injection, n = 6. All retinas are from 16-month-old animals, showing
similar expression within the group. Scale bar, 100 µm. d, Volume intensity projection of en-
face OCT (optical coherence tomography) retinal image with a white line indicating the
location of e. e, Representative retinal cross section B-scan images. White box indicates the

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location of the high magnification scans in f (retinal layers: GCL = ganglion cell layer, INL
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= inner nuclear layer, ONL = outer nuclear layer, and choroid). Videos of complete retinal
cross section B-scan images of the entire globe are in Supplementary Videos. g, Low- and
high-power representative images of H&E-stained cross-sections of corresponding eyes,
verifying retinal layers. h, Quantitative measurements of retinal thickness, there was no
difference between the groups at any location using two-way ANOVA with Bonferroni
correction (n = 6, 3, and 6, respectively). i, Immunosuppressed NOD scid gamma mice
received a subretinal injection of ~10,000 human retinoblastoma tumor cells. The OCT
image shows a small retinal tumor and increased retinal thickness 14 days post-injection,
demonstrating the ability of the OCT scan to detect tumors, * Rb (retinoblastoma).
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Extended Data Fig. 3. Polycistronic OSK induces long-distance axon regeneration post-injury
without RGC proliferation.

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a, Proliferating cells in the optic nerve (e.g. glial cells) in BrdU-injected mice as a positive
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control (n = 2 nerves). BrdU staining co-localized with Ki67, a proliferation marker. b,

Representative Retina wholemount staining shows OSK-expressing RGCs do not stain for
BrdU the first or second week after crush injury, n = 4 retinas. Scale bars, 100 µm. c,
Imaging of optic nerves showing regenerating and sprouting axons with or without OSK
AAV treatment, 12 weeks post-crush (wpc), n = 2 nerves. Scale bars, 200 µm. d, Whole
nerve imaging showing CTB- labeled regenerative axons at 16 wpc in wild-type mice with
intravitreal injection of AAV2-tTA;TRE-OSK (n = 2 nerves). Scale bars, 200 µm. e, Survival
of RBPMS-positive cells in the RGC layer transduced with different AAV2s, 16 dpc (n = 6,
4, 4, 4, 4, 4, 8, and 4 eyes). All data are presented as mean ± s.e.m. f and g, Representative
immunofluorescence (f) and sub-population proportion (g) of wholemount retinas
transduced with a polycistronic AAV vector expressing Oct4, Sox2, and Klf4 in the same
cell. White arrows designate triple-positive cells. n = 3 retinas. Scale bars, 100 µm. h and i,
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Immunofluorescence (h) and sub-population proportion (i) of wholemount retinas

transduced with AAVs separately encoding Oct4, Sox2, and Klf4. Red, blue, and green
arrows designate single-positive cells, with a white arrow marking a triple-positive cell, and
other arrows marking double-positive cells. n = 3 retinas. Scale bar, 100 µm. One-way
ANOVA with Bonferroni correction in e, with comparisons to d2EGFP shown.
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Extended Data Fig. 4. Regenerative and pro-survival effects of OSK are RGC-specific and cell-
autonomous.
a, Effect of OSK expression on RGC survival in young (1 mo, n = 8), adult (3 mo, n = 5),
and old mice (12 mo, n = 8) after optic nerve crush injury compared to expression of
d2EGFP as a negative control (n = 6, 5, and 6, respectively). b, Axon regeneration after OSK
expression compared to d2EGFP controls in young (1 mo, n = 5 and 6), adult (3 mo, n = 6),
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and old (12 mo, n = 4 and 5) mice, 2 wpc. c, RGC number of 12-month-old mice in intact, 2
or 5 wpc retinas with GFP (AAV2-tTA;TRE-d2EGFP, n = 7, 6, and 6, respectively) or OSK
(AAV2-tTA;TRE-OSK, n = 5, 8, and 6, respectively) expression. d, Axon regeneration in
12-month-old mice with OSK AAV or control AAV (d2EGFP) treatment, 5 wpc (n = 5
nerves). e, Schematic of retinal structure showing Vglut2-Cre mice selectively expressing
Cre in excitatory neurons such as RGCs, while Vgat-Cre mice selectively express Cre in
inhibitory amacrine and horizontal cells. f, Schematic of the FLEx (flip-excision) CRE-
switch system. AAV2-FLEx-tTA is inverted by Cre to express tTA and therefore induces

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OSK only in Cre-positive cells. g, Confocal image stack demonstrating delivery of AAV2-
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FLEx-tTA;TRE-OSK to intact Vglut2-Cre transgenic retinas, resulting in RGC-specific

OSK expression (upper) and robust axon regeneration in the optic nerve (lower). White
arrows indicate RBPMS+ (AP2-)-labeled RGCs that express Klf4 (green). n = 4 independent
replicates. h, Confocal image stack demonstrating delivery of AAV2-FLEx-tTA;TRE-OSK
to intact Vgat-Cre transgenic retinas, resulting in amacrine-specific OSK expression (upper)
and poor axon regeneration in the optic nerve (lower). White arrows indicate AP2+
(RBPMS-)-labeled amacrine cells that express Klf4 (green). n = 4 independent replicates. i,
Representative image of AAV-expressing or non-expressing RGCs in intact and crushed
retinas 2 wpc with AAVs expressing d2EGFP or OSK. d2EGFP: AAV2-tTA;TRE-d2EGFP,
n = 6 retinas; OSK: AAV2-tTA;TRE-OSK, n = 8 retinas. j, RGC survival rate (crushed /
intact) of d2EGFP- (n = 6 eyes) or Klf4-expressing cells (n = 8 eyes) and their surrounding
non-expressing cells indicating a cell-autonomous pro-survival effect of OSK-expressing
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RGCs after crush, 2 wpc. k, Frequency of d2EGFP- or Klf4-positive RGCs pre- or 2 weeks
post-injury (n = 4, 6, 6, and 8 eyes). Two-way ANOVA with Bonferroni correction in a–d, j;
one-way ANOVA with Bonferroni correction in k. Scale bars, 100 µm in g, h, and i. All data
are presented as mean ± s.e.m.
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Extended Data Fig. 5. OSK activates Stat3 in the absence of mTOR activation or global
demethylation.
a, Representative images of retinal wholemounts transduced with AAV2-tTA (−OSK) or
AAV2-tTA;TRE-OSK (+OSK) in the presence or absence of crush injury after 3 days.
Retinal wholemounts immunostained for pStat3, Klf4, and RBPMS. n = 2 retinas each
condition. b, Representative images of retinal wholemounts transduced with d2EGFP- or
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OSK-encoding AAV2 in the presence or absence of a crush injury. Retinal wholemounts

immunostained for RBPMS and mTOR activation marker phosphorylated S6 (pS6). n = 4
retinas each condition. c, Percent of pS6-positive RGCs in intact and crushed samples (n = 4
retinas each condition). d, Representative images of d2EGFP in retina expressed from the
Tet-On AAV system. No GFP expression was observed in the absence of Dox. GFP
expression reached peak levels 2 days after Dox induction and remained at a similar level at
day 5 after induction. n = 2 retinas each condition. e, Representative images of retinal
d2EGFP expression using the Tet-Off AAV system with various durations of Dox treatments

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(2 mg/mL). Once pre-treated with Dox to suppress expression (on Dox), GFP was sparse
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even on day 8 after Dox withdrawal, lower than peak expression (Never Dox). n = 2 retinas
each condition. f, Axon regeneration at 2 or 4 wpc in response to OSK induction either pre-
or post-injury (n = 4, 5, 5, 4, and 4 eyes, respectively). g, Correlation between ribosomal
DNAm age (mo) and chronological age (mo) of sorted mouse RGCs (1 mo, n = 6; 12 mo, n
= 2; 30 mo, n = 5), with the light blue region representing the confidence interval. P value of
the linear regression is calculated by two-sided F-test of overall significance. In agreement
with previous studies, DNAm age estimates of neurons tend to be lower than their
chronological age but remain correlated (see Methods). h, Average DNA methylation levels
across the mouse genome in RGCs from different ages and treatments, based on 703,583
shared CpG sites from RRBS of all samples (combined strands), n = 6, 8, 2, 8, 6, 4, 8, 8, 6,
5, 6, 4, and 5, respectively. i, Correlation of DNAm at each CpG site versus age (x-axis; 1
mo, 12 mo, 30 mo) and versus injury (y-axis; intact, injured GFP). Heatmap represents the
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number of sites located in each block of value coordinates. Pearson’s correlation coefficient,
r = 0.34, P < 1e−200. j, Hierarchical clustered heatmap of methylation levels of 4,106 CpGs
that significantly changed in RGCs after crush injury (Intact vs Injured GFP, q < 0.05) and
the effect of OSK. k, Top biological processes associated with the 698 CpGs that were
significantly altered by both injury and OSK. Two-way ANOVA with Bonferroni correction
in c and f. Scale bars, 100 µm in a, b, d, e. All data are presented as mean ± s.e.m.
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Extended Data Fig. 6. Protective and regenerative effect of OSK is dependent on Tet1/2.
a, Mouse Tet mRNA levels with or without OSK expression in RGCs (n = 6 biological
replicates each condition). * Unpaired one-tailed t-test. b, Representative images of retinal
wholemounts transduced with AAV2-tTA;TRE-OSK in combination with a AAV2-shRNA-
YFP (yellow fluorescent protein) having either a scrambled sequence (sh-Scr) or a hairpin
sequence to knockdown Tet1 (sh-Tet1) or Tet2 (sh-Tet2) expression, at titer ratio 5:5:1.
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Retinal wholemounts immunostained for Klf4. n = 3 retinas each condition. c,

Quantification of shRNA-YFP AAV transduction in OSK-expressing RGCs (n = 3 retinas
each condition). d, Mouse Tet mRNA levels with sh-Scr (n = 5), sh-Tet1 (n = 4), or sh-Tet2
(n = 5) YFP AAV2 in RGCs in the presence of OSK expression. e and f, Quantification of
axon regeneration (e, n = 4 eyes each condition) and RGC survival (f, n = 10, 7, and 9 eyes)
at 2 wpc in retinas co-transduced with AAV2- tTA;TRE-OSK;shRNA. g, Mouse Stat3
mRNA levels after sh-Scr (n = 5), sh-Tet1 (n = 4), or sh-Tet2 (n = 5) knockdown in RGCs in

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the presence of OSK expression. h, Cre-dependent Tomato expression in RGCs after

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intravitreal AAV2-Cre injection of Tomato reporter mice (Rosa-CAG-lox-STOP-lox-

Tomato), and the co-expressed frequency of Cre and Klf4 (n = 3 eyes). i and j, RGC survival
(i) and representative longitudinal sections of regenerating axons in longitudinal sections (j)
in response to OSK expression (AAV2-tTA;TRE-OSK, n = 5 each condition) compared to
no expression (Saline, n = 3 and 4) , 16 days after crush injury in Tet2flox/flox mice injected
with saline (Tet2 WT) or AAV2-Cre (Tet2 cKO). Scale bars, 100 µm in b, h, and j. Two-way
ANOVA in a, d, and i; unpaired two-tailed Student’s t-test in g; one-way ANOVA in e and f.
All data are presented as mean ± s.e.m.
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Extended Data Fig. 7. OSK-induced axon regeneration and survival require non-global active
DNA demethylation through TDG.
a, Representative images of retinal wholemounts transduced with sh-Scr-H2B-GFP or sh-
TDG-H2B-GFP AAV2s for 4 weeks, demonstrating TDG knockdown increased levels of 5-

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hydroxymethylcytosine (5-hmC). n = 4 retinas each condition. b, Representative retinal

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wholemount images and images of longitudinal sections through the optic nerve showing
CTB-labeled regenerative axons in wild-type mice, 16 dpc after an intravitreal injection of
AAV2-tTA ;TRE-OSK in combination with AAV2-sh-Scr (sh-Scr) or AAV2-sh-TDG (sh-
TDG) at titer ratio 5:5:1. n = 4 retinas each condition. c and d, Quantification of
regenerating axons (c) and RGC survival (d) in OSK-treated mice 16 dpc with AAVs
carrying sh-Scr or sh-TDG (n = 4 nerves each condition). e, Representative image of retinal
wholemounts transduced with AAV2-tTA;TRE-OSK. Retinal wholemounts were
immunostained for 5-methylcytosine (5-mC) and Klf4, showing a lack of global
demethylation in OSK expressing RGCs. n = 3 retinas. f, Representative images of retinal
wholemounts transduced with AAV2 vectors encoding the HA-Tet1 catalytic domain (Tet1-
CD) or its catalytic mutant (Tet1-mCD) for 4 weeks, demonstrating overexpression of Tet1-
CD decreases global 5-mC levels. n = 3 retinas each condition. g and h, Quantification of
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axon regeneration (g) and RGC survival (h) at 2 wpc in retinas transduced without or with
AAV2 vectors encoding HA-Tet1 CD mutant or HA-Tet1 CD (n = 3, 4, and 3 eyes). i, A
schematic diagram illustrating passive demethylation and TDG-dependent active DNA
demethylation. Scale bars, 100 µm in a b, e, and f. One-way ANOVA with Bonferroni’s
multiple comparison test in c, g, and h; unpaired two-tailed Student’s t-test in d. There was
no difference between the groups in g and h using two-way ANOVA and one-way ANOVA,
respectively. All data are presented as mean ± s.e.m.
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Extended Data Fig. 8. OSK induces axon regeneration and reversal of DNAm age in human
neurons.
a, mRNA levels of murine Oct4, Sox2, and Klf4 in human neurons transduced with vectors
packaged by AAV-DJ, a recombinogenic hybrid capsid that is efficient for in vitro
transduction. −OSK: AAV-DJ-tTA (n = 3); +OSK: AAV-DJ-tTA;TRE-OSK (n = 3). b,
Percentage of cells in S phase by PI-staining (n = 4). c, FACS profiles of G1, S, and G2
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phases in undifferentiated SH-SY5Y cells and differentiated cells transduced with −OSK
and +OSK vectors. d, Experimental outline for testing axon regeneration in human neurons
post-vincristine (VCS) damage. e and f, DNA methylation (DNAm) age of human neurons
without damage (intact), and 1 or 9 days post-VCS damage in the absence (e) or presence (f)
of OSK expression, measured using the skin and blood clock suited to in vitro studies (see
Methods). The linear regression P value in e (P = 0.55) indicates non-linear DNAm age
changes, and in f (P = 0.008) indicates a continuous decrease in DNAm age (n = 3, 3, and 6).
g, Average DNAm levels among 850,000 probes from the EPIC array in human neurons

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without damage (intact), and 1 or 9 days post-VCS damage in the absence or presence of
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OSK expression (n = 3, 3, and 6). h and i, Representative images (h, similar results were
confirmed in two series of experiments) and quantification (i) of neurite area at different
time points after VCS damage (n = 6, 7, 5, 5, 7, and 5; 2 independent experiments). Cells
were not passaged after damage to avoid cell-cell contact for quantifying maximum axon
regeneration. j-l, Human Tet1–3 mRNA level with scrambled shRNA (sh-Scr) or sh-Tet2
AAV in human neurons in the presence or absence of OSK expression (n = 4; 2 independent
experiments). m, Representative images of human neurons in each AAV treated group, 9
days post-VCS damage. Similar results were confirmed in three series of experiments. n-p,
Neurite area (n), axon number (o), and axon length (p) in each AAV-treated group 9 days
post-VCS damage (n = 20, 21, 24, and 23; 3 independent experiments). q, Mouse Oct4
mRNA levels (from OSK AAV) in human neurons with sh-Scr or sh-Tet2 AAV and in
presence or absence of OSK AAV (n = 4). r, The effect of mTOR inhibition by rapamycin
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(Rap, 10 nM) on axon regeneration of differentiated neurons with or without OSK (n = 18,
19, 13, and 11; 2 independent experiments). s, S6 phosphorylation levels in human neurons
5 days after treatment with rapamycin (Rap, 10 nM). Similar results were seen in two
independent experiments. Uncropped scans in Supplementary Figure 1. t, Neurite area of
neurons expressing Tet1 catalytic domain (Tet1-CD) or its catalytic mutant (Tet1-mCD) 9
days post-VCS damage (n = 24, 28, and 21; 2 independent experiments). One-way ANOVA
with Bonferroni’s multiple comparison test in b, e, f, g, and t; two-way ANOVA with
Bonferroni’s multiple comparison test in a, i, j–l, and n–r. All bar graphs are presented as
mean ± s.e.m.
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Extended Data Fig. 9. Vision restoration and regenerative effect of OSK rely on functional
improvement of existing RGCs.
a, Axon density and representative photomicrographs of PPD-stained optic nerve cross-
sections, 4 weeks after microbead or saline injection (baseline, n = 5 eyes each condition).
Scale bars, 25 µm. b, Quantification of RGCs and representative confocal microscopic
images from retinal flat-mounts stained with anti-Brn3a (red), an RGC-specific marker, and
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DAPI (4′,6-diamidino-2-phenylindole, blue), a nuclear stain, 4 weeks after microbead or

saline injection (baseline, n = 5 eyes each condition). Scale bar, 100 µm. c, Axon density and
representative micrographs from PPD-stained optic nerve cross-sections, 4 weeks after
AAV2 or PBS injection (treated, n = 9, 7, 6, and 8 eyes). Scale, 50 µm. d, Quantification of
RGCs and representative confocal microscopic images 4 weeks post-PBS or AAV injection
(treated, n = 7, 5, 6, and 5 eyes). Scale bar, 100 µm. e, PERG measurement at different ages
4 weeks after −OSK (n = 16, 14, and 11 eyes) or +OSK treatment (n = 20, 12, and 14 eyes).
Similar results from 2 independent experiments are combined. f, Visual acuity in 18-month-

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old mice treated with −OSK (n = 11 eyes) or +OSK AAV (n = 14 eyes) for 4 weeks. g and h,
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Axon (g, n = 4, 6, 10, and 9 nerves) and RGC (h, n = 5, 4, 10, and 8 retinas) density in 4-
and 12-month-old-mice, 4 weeks after −OSK or +OSK AAV injection. i, Scatter plot of
OSK-induced changes and age-associated changes in mRNA levels in RGCs, with
differentially expressed genes labelled. Gene selection criteria are in Methods. j,
Hierarchical clustered heatmap showing relative mRNA levels of age-associated sensory
perception genes in FACS-sorted RGCs from untreated young (5 mo) or old mice (12 mo) or
old mice treated with either −OSK or +OSK AAV. Sensory genes were extracted from the
mouse Sensory Perception (GO:0007600) category the Gene Ontology database, including
genes that were differentially expressed (q < 0.05) in 12- versus 5-month-old mice,
excluding genes that were induced by the empty virus infection (q < 0.1). −OSK: AAV2-
rtTA;TRE-OSK for c–h, AAV2-TRE-OSK for i and j; +OSK: AAV2-tTA;TRE-OSK for c-j.
Unpaired two-tailed Student’s t-test in a, b, and f; one-way ANOVA with Bonferroni’s
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multiple comparison test in c and d; two-way ANOVA with Bonferroni correction in e, g,

and h. All data are presented as mean ± s.e.m.
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Extended Data Fig. 10. OSK expression in old RGCs restores youthful epigenetic signatures.
a and b, Top biological processes based on transcriptome data that were either lower
expressed in old compared to young RGCs and reversed by OSK (a), or higher expressed in
old RGCs compared to young and reversed by OSK (b). c, Heatmap showing relative mRNA
levels of genes involved in the negative regulation of neural projection development, among
the 464 differentially expressed genes during ageing. Efemp1 is a gene whose accumulation
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during ageing is suspected to play a role in diseases of the retina. d, RGC Efemp1 mRNA
levels measured by qPCR (relative to GAPDH) compared between young, old, and old
treated with −OSK or + OSK AAV. Old RGCs with sh-Scr, sh-Tet1, or sh-Tet2 knockdown
combined with +OSK AAV are included for comparison (n = 7, 6 ,5, 4, 5, 5, and 6 eyes). e,
Principal Component 1 value of 1 mo, 12 mo, and 30 mo RGC training samples in the PCA
analysis. Values are standardized to have a mean=0 and s.d.=1 (n = 6, 2, and 6). f, DNAm
ageing signatures of 6-week-old RGCs isolated from axon-intact retinas infected with GFP-

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expressing AAV, or from axon-injured retinas infected with GFP- or OSK- expressing AAV
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at 4 dpc (n = 4, 4, and 8 eyes). g, Top biological processes associated with the 1,226
signature CpG sites. h and i, Transcription factor (TF) binding (h) and histone modifications
(i) specifically enriched at the 1,226 signature CpG sites, compared to five sets of randomly
selected CpGs. j, Correlation of Tet1 and Tet2 knockdown-induced changes in methylation
(5-mC and 5-hmC together) at the selected CpGs. r = 0.4, P = 2.53e-45. k, Delta value of
ribosomal DNAm age (mo) of 12-month-old RGCs infected for 4 weeks with +OSK (n = 5
retinas). Values are relative to the average of RGCs infected with −OSK AAV. l, Ribosomal
DNAm age (mo) of 12-month-old OSK-treated RGCs infected for 4 weeks with sh-Tet1 or
sh-Tet2 (n = 4, 5 retinas). Values are relative to the average of RGCs infected with sh-Scr. m,
PERG amplitudes in old mice (12 mo) treated with −OSK, +OSK, or +OSK together with
either sh-Scr or sh-Tet1/sh-Tet2-mediated knockdown for 4 weeks (n = 8, 7, 5, 6, and 6
eyes). n, Working model. The loss of youthful epigenetic information during ageing and
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injury (including genome-wide changes to DNA methylation, acceleration of the DNAm

clock, and disruption of youthful gene expression patterns) causes a decline in tissue
function and regenerative capacity. OSK-mediated reprogramming recovers youthful
epigenetic information, reverses the DNAm clock, restores youthful gene expression
patterns, and improves tissue function and regenerative capacity, a process that requires
active DNA demethylation by TET1/TET2 and TDG. The PRC2 complex may serve to
recruit TET1 and TET2 to specific sites in the genome, and DNA methylation by DNA
methyltransferases (DNMTs) may be important as well. One-way ANOVA with
Bonferroni’s multiple comparison test in d, e, f and m. All data are presented as mean ±
s.e.m.
Supplementary Material
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Refer to Web version on PubMed Central for supplementary material.
Acknowledgements
We greatly thank A.Wagers, R. Mostoslavsky, Y. Shi, A. Das, A. Pogoutse, C. Petty, A. Coffey, B. Zhang, P.
Dmitriev, K. Booher, E. Chen, J. Wang, D. Vogel, M. Thompson, A. Jacobi and S. Hou for advice and assistance.
We thank Y. Weng, H. Song and F. Wang for reagents and animals. The work was supported by the Harvard
Medical School Epigenetics Seed Grant and Development Grant, The Paul F. Glenn Foundation for Medical
Research, a kind gift from Edward Schulak, NIH awards R01AG019719 and R37AG028730 (to D.A.S),
R01EY026939 and R01EY021526 (to Z.H.), R01AG067782 and R01GM065204 (to V.N.G.), and R01AG065403
(to M.E.L and V.N.G.). We thank Boston Children’s Hospital Viral Core, which is supported by
NIH5P30EY012196, and Schepens Eye Institute Core facilities, supported by NEI-P30EY003790. X.T. was
supported by NIH award K99AG068303 and by NASA Postdoctoral Fellowship 80NSSC19K0439; D.L.V. was
supported by NIH training grant T32AG023480; J.-H.Y. was partially supported by National Research Foundation
of Korea (2012R1A6A3A03040476); B.R.K. was partially supported by the St Vincent de Paul Foundation and by
NEI awards R24EY028767 and R01EY025794; and M.S.G.-K. by NEI award R21EY030276. We thank Paul F.
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Glenn for his mentorship and support of ageing research. This paper is dedicated to Honghua Lu, grandfather to
Y.L., who passed away bravely fighting ageing during preparation of this manuscript, and to Dr. Michael
Bonkowski, our beloved friend and co-author of this paper, who passed away only days before it was published.
Mike we are going to miss you terribly.
Data and code availability

RRBS data for DNA methylation analysis and RNA-seq data are available in the Biosample
database (NCBI) and under BioProject PRJNA655981. Illumina Human Methylation EPIC

Lu et al. Page 36
array data is available in the GEO database (NCBI) and under GSE147436. The code for
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determining methylation ageing signatures is provided as Supplementary Code. All other

relevant data that support the findings of this study are available from the corresponding
author upon reasonable request.
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Figure 1. AAV2-delivered polycistronic OSK promotes axon regeneration and RGC survival
following optic nerve injury.
a, Schematic of the Tet-On and Tet-Off dual AAV vectors. b, Schematic and representative
retinal wholemount/cross sections (n = 10), two weeks after intravitreal injection of AAV2-
tTA;TRE-OSK showing Klf4 (green) expression in the RGCs (RBPMS+, blue). Scale bars, 1
mm and 100 µm, respectively. c, Experimental outline of the optic nerve crush study using
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the Tet-Off system. 555-CTB was used for anterograde axonal tracing. wks, weeks. d,
Number of axons 16 days after crush injury at distances distal to the lesion in mice treated
with either AAV2 encoding destabilized enhanced green fluorescent protein (d2EGFP),
Oct4, Sox2, Klf4, Oct4-Sox2, or OSK on three monocistronic AAV2s or a single
polycistronic AAV2 (n = 5, 4, 4, 4, 4, 4, and 7 eyes). Suppression of polycistronic OSK
expression by Dox (n = 5 eyes) is shown as OSK Off in d and e. e, Representative images of
longitudinal sections through optic nerves after receiving intravitreal injections of AAV2-
tTA;TRE-OSK in the presence (n = 5) or absence of doxycycline (n = 7). CTB-labeled axons

Lu et al. Page 41
are shown 16 days post crush (dpc). Asterisks indicate optic nerve crush site. f,
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Representative confocal images of longitudinal sections through the optic nerve showing
CTB-labeled axons in 12-month-old mice, 5 weeks post-crush (wpc), with AAV2-mediated
expression of either GFP or OSK. Representative results from n = 5 eyes. g, At 16 dpc, the
number of axons at multiple distances distal to the lesion site in transgenic Cre lines that
selectively express OSK either in RGCs (Vglut2-CRE) or amacrine cells (Vgat-CRE) with
intravitreal injection of AAV2-FLEx-tTA;TRE-OSK (n = 4 eyes each condition). Details in
Extended Data Fig. 4e–h. Scale bars, 200 µm. One-way ANOVA with Bonferroni correction
in d and g, with comparisons to d2EGFP shown in f. All data are presented as mean ± s.e.m.
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Lu et al. Page 42
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Figure 2. DNA demethylation is required for OSK-induced axon regeneration post injury.
a, Strategies for induction of OSK pre- and post-injury. b, RGC survival per mm2 at 2 and 4
wpc in response to OSK induction either pre- or post-injury (n = 4 eyes except for Post-inj
On group, 2 wpc, n = 7 eyes). c, Representative longitudinal sections through the optic nerve
showing CTB-labeled axons at 4 wpc, with or without post-injury induction of OSK.
Asterisks indicate optic nerve crush site. Representative results from n = 4 eyes. Scale bars,
200 µm. d, ribosomal DNAm age (mo, month) of 6-week-old RGCs isolated from axon-
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intact retinas infected with or without GFP-expressing AAV, or from axon-injured retinas
infected with GFP- or OSK-expressing AAV at 4 dpc (n = 6, 4, 8, and 8 eyes). DNA
methylation age estimates of neurons tend to be lower than their chronological ages but
remain correlated (see Extended Data Fig. 5g and Methods). e, Hierarchical clustered
heatmap of DNA methylation levels of CpGs that significantly changed in RGCs after crush
injury (GFP vs Injured GFP, q < 0.05) and the effect of OSK at 4 dpc. f, Axon regeneration
in response to OSK expression (AAV2-tTA; TRE-OSK), 16 dpc in Tet2flox/flox mice infected
with saline (Tet2 WT, n = 3 each condition) or AAV2-Cre (Tet2 cKO, n = 4 each condition).
One-way ANOVA with Bonferroni’s multiple comparison test in b, d, and f. All bar graphs
are presented as mean ± s.e.m.
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Lu et al. Page 43
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Figure 3. Four weeks of OSK expression reverses vision loss after glaucomatous damage has
already occurred.
a, Experimental outline of the induced glaucoma studies using 8-wk-old mice. wks, weeks.
b, Intraocular pressure (IOP) measured weekly by rebound tonometry for the first 4 weeks
post-microbead/saline injection (saline, n = 10 mice; microbeads, n = 29 mice). c, High-
contrast visual stimulation to measure optomotor response. Reflexive head movements were
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tracked in response to the rotation of a moving stripe pattern that increased in spatial
frequency (cyc/deg, cycle per degree). d, Optomotor response of each mouse before
treatment and 4 weeks after intravitreal injection of AAVs (n = 10, 7, 12, and 12 mice;
similar results from 3 independent experiments combined). e, Representative PERG
waveforms in response to a reversing contrast checkerboard pattern, recorded from the same
eye both at pre-injection baseline and at 4 weeks after −OSK (upper graph) or +OSK (lower
graph) AAV injection. f, Mean PERG amplitudes of recordings from each mouse in d at
baseline before treatment and 4 weeks after intravitreal AAV injection (n = 10, 7, 12, and 12

Lu et al. Page 44
mice; similar results from 3 independent experiments combined). −OSK (not induced):
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AAV2-rtTA;TRE-OSK; +OSK (induced): AAV2-tTA;TRE-OSK. Two-way ANOVA with

Bonferroni correction between groups was used for b; Two-way mixed ANOVA with
Bonferroni correction between groups was used for the overall effect of time and treatment
for d, and f; before and after treatments were analyzed using a paired two-tailed Student’s t-
test in d and f. Data in b, d, and f are presented as mean ± s.e.m.
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Lu et al. Page 45
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Figure 4. Restoration of youthful vision, transcriptome and DNAm ageing signature in old mice.
a, Experimental outline for testing the effect of reprogramming in old mice. b, Visual acuity
in young (4 mo) and old mice (12 mo) after 4 weeks of −OSK or +OSK treatment (n = 16,
20, 14, and 12 eyes; similar results from 2 independent experiments combined). mo,
month(s). c and d, Hierarchical clustered heatmap (c) and scatter plot (d) showing mRNA
levels of 464 genes that were differentially expressed between young and old RGCs and the
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effect of OSK. RGCs were sorted from untreated young (5 mo, n = 5), old (12 mo, n = 6), or
treated old retinas (−OSK, n = 5; +OSK, n = 4). Gene selection criteria for c and d are
described in the methods. e, DNAm ageing signatures of 12-mo-old RGCs infected for 4
weeks with: −OSK, or +OSK (n = 4 and 3 retinas). f, Visual acuity in old mice (12 mo)
treated for 4 weeks with: −OSK, +OSK, or +OSK together with sh-Scr, sh-Tet1, or sh-Tet2
(n = 8, 7, 5, 6, and 6 eyes). −OSK: AAV2-rtTA;TRE-OSK for b, AAV2-TRE-OSK for c and
d, AAV2-tTA;TRE-d2EGFP for e; +OSK: AAV2-tTA;TRE-OSK for b–f. Two-way ANOVA

Lu et al. Page 46
with Bonferroni correction in b; Kruskal-Wallis test for e; one-way ANOVA with Bonferroni
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correction in f, with comparisons to the −OSK group. All bar graphs are presented as mean
± s.e.m.
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Glaucoma Reversed by OSK Study

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Reprogramming to recover youthful epigenetic information and

Karolina Chwalek1, Luis A. Rajman1, George M. Church7, Konrad Hochedlinger8, Vadim N.

6.Department of Pathology, Yale School of Medicine, New Haven, CT, USA;

Massachusetts General Hospital, MA, USA;

The metaphor of the epigenetic landscape, first invoked by Waddington to explain

Nature. Author manuscript; available in PMC 2021 June 02.

Ageing is generally considered a unidirectional process akin to an increase in entropy.

Reset of ageing signatures, not identity

OSK stimulates axon regeneration

Nature. Author manuscript; available in PMC 2021 June 02.

body, resulting in Dox-responsive gene expression in around 40% of RGCs, as estimated by

Nature. Author manuscript; available in PMC 2021 June 02.

OSK counters injury-induced DNAm ageing

Regeneration requires DNA demethylation

Nature. Author manuscript; available in PMC 2021 June 02.

Axon regeneration in human neurons

Recovery of vision in mice with glaucoma

Restoration of vision in old mice

Nature. Author manuscript; available in PMC 2021 June 02.

Nature. Author manuscript; available in PMC 2021 June 02.

Production of adeno associated viruses (AAVs)

Nature. Author manuscript; available in PMC 2021 June 02.

Systemic delivery of AAV9

Cell culture of ear fibroblasts

AAV2 intravitreal injection

Optical Coherence Tomography (OCT)

Creation of retinoblastoma tumors

making a corneal incision, followed by a subretinal injection of 10,000 retinoblastoma tumor

Optic nerve crush

Nature. Author manuscript; available in PMC 2021 June 02.

In vivo doxycycline induction or suppression

Quantification of axon regeneration for the optic nerve crush model

Wholemount optic nerve preparation

Nature. Author manuscript; available in PMC 2021 June 02.

5-Bromo-2′-deoxyuridine (BrdU) labelling

(1:20,000, Sigma-Aldrich, A3854).

RGC survival and phospho-S6 signal

Human neuron differentiation and regeneration assay

Nature. Author manuscript; available in PMC 2021 June 02.

Cell cycle analysis

Human neuron DNA methylation analyses

(based on 391 CpGs) was previously published57.

Microbead-induced elevated intraocular pressure (IOP)

Nature. Author manuscript; available in PMC 2021 June 02.

Pattern electroretinogram (PERG)

Quantification of optic nerve axons in the glaucoma model

Nature. Author manuscript; available in PMC 2021 June 02.

Quantification of retinal ganglion cells in the glaucoma model

RRBS library preparation

Nature. Author manuscript; available in PMC 2021 June 02.

DNA methylation analysis of mouse RGC

DNAm ageing signature

18 mo (n=8), 30 mo (n=6), 1 mo GFP-injured (n=4), 12 mo GFP (n=2), and 12 mo OSK

Nature. Author manuscript; available in PMC 2021 June 02.

Transcription factor (TF) binding and histone modification enrichment

Total RNA extraction and sample quality control

Ultra-low input RNA library preparation and multiplexing