Fermat's Library | Transient non-integrative expression of nuclear reprogramming factors promotes multifaceted amelioration of aging in human cells annotated/explained version.

### TL;DR ***Slowing or reversing aging is one of the holy grail...

- **Chromatin**: Chromatin is a complex of DNA and protein found in...

> To address all these questions, we devised a platform that could ...

- **Transcriptome**: The transcriptome is the set of all RNA transc...

> Altogether, the analysis of the transcriptomic signatures reveale...

The method employed was able to generate induced pluripotent stem c...

### DNA methylation DNA methylation is a biological process by w...

The results ***demonstrate that transient expression can induce a r...

***The rejuvenation effects were significantly retained after 4 and...

The researches extended their analysis to osteoarthritis - a diseas...

> these results suggest that transient reprogramming partially rest...

ARTICLE

Transient non-integrative expression of nuclear

reprogramming factors promotes multifaceted

amelioration of aging in human cells

Tapash Jay Sarkar

1,2,3

, Marco Quarta

4,5,6,7

✉

, Shravani Mukherjee

, Alex Colville

4,5,6,9

, Patrick Paine

4,5,6,7

Linda Doan

4,5,6,7

, Christopher M. Tran

4,5,6

, Constance R. Chu

8,10

, Steve Horvath

11,12

, Lei S. Qi

Nidhi Bhutani

, Thomas A. Rando

4,5,6

& Vittorio Sebastiano

1,2

✉

Aging is characterized by a gradual loss of function occurring at the molecular, cellular, tissue

and organismal levels. At the chromatin level, aging associates with progressive accumulation

of epigenetic errors that eventually lead to aberrant gene regulation, stem cell exhaustion,

senescence, and deregulated cell/tissue homeostasis. Nuclear reprogramming to plur-

ipotency can revert both the age and the identity of any cell to that of an embryonic cell.

Recent evidence shows that transient reprogramming can ameliorate age-associated hall-

marks and extend lifespan in progeroid mice. However, it is unknown how this form of

rejuvenation would apply to naturally aged human cells. Here we show that transient

expression of nuclear reprogramming factors, mediated by expression of mRNAs, promotes a

rapid and broad amelioration of cellular aging, including resetting of epigenetic clock,

reduction of the inﬂammatory proﬁle in chondrocytes, and restoration of youthful regen-

erative response to aged, human muscle stem cells, in each case without abolishing cellular

identity.

https://doi.org/10.1038/s41467-020-15174-3

OPEN

Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.

Department of Obstetrics

and Gynecology, Stanford University School of Medicine, Stanford, CA 94305, USA.

Department of Applied Physics, Stanford University School of

Humanities and Sciences, Stanford, CA 94305, USA.

Department of Neurology and Neurological Sciences, Stanford University School of Medicine,

Stanford, CA 94305, USA.

Paul F. Glenn Center for the Biology of Aging, Stanford University School of Medicine, Stanford, CA 94305, USA.

Center for

Tissue Regeneration, Repair and Restoration, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304, USA.

Molecular Medicine Research

Institute, Sunnyvale, CA 94085, USA.

Department of Orthopedic Surgery, Stanford University School of Medicine, Sanford, CA 94305, USA.

Department

of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA.

VA Palo Alto Health Care System, Palo Alto, CA 94304, USA.

Department of Human Genetics David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA.

Department of Biostatistics,

Fielding School of Public Health, UCLA, Los Angeles, CA 90095, USA.

Department of Bioengineering, Department of Chemical and Systems Biology,

ChEM-H, Stanford University, Stanford, CA 94305, USA.

✉

email: mquarta@stanford.edu; vsebast@stanford.edu

NATURE COMMUNICATIONS | (2020) 11:1545 | https://doi.org/10.1038/s41467-020-15174-3 | www.nature.com/naturecommunications 1

1234567890():,;

he process of nuclear reprogramming to induced plur-

ipotent stem cells (iPSCs) is characterized, upon comple-

tion, by a profound resetting of the epigenetic landscape of

cells of origin, resulting in reversion of both cellular identity and

age to an embryonic-like state

1–4

Notably, if the expression of the reprogramming factors is only

transiently applied and then stopped (before the so-called Point

of No Return, PNR)

, the cells return to the initiating somatic cell

state. These observations suggest that, if applied for a short

enough time, the expression of reprogramming factors fails to

erase the epigenetic signature deﬁning cell identity; however, it

remains unknown whether any substantial and measurable

reprogramming of cellular age can be achieved before the PNR.

First evidence that transient reprogramming can promote ame-

lioration of aging phenotypes was shown by Ocampo et al., in

progeroid mice carrying a Dox-inducible OSKM cassette

. Yet,

important questions remain open. Murine genetic models of

premature aging only in part recapitulate the complexity of nat-

ural aging, a phenomenon that is characterized by a slow and

progressive accumulation of epigenetic errors. In addition, proof

is lacking that the same rejuvenative effect can be achieved with

naturally aged human cells isolated from elderly individuals,

together with a comprehensive molecular and physiological

analysis of the depth and extension of the rejuvenation in human

cells. To address all these questions, we devised a platform that

could let us test whether transient expression of nuclear repro-

gramming genes has any impact in ameliorating aging pheno-

types in naturally aged human and mouse cells across multiple

cell types and spanning all the hallmarks of aging.

Results

We ﬁrst evaluated the effect of transient expression of repro-

gramming factors on the transcriptome of two distinct cell

types—ﬁbroblasts and endothelial cells—from aged human sub-

jects, and we compared it with the transcriptome of the same cell

types isolated from young donors (Fig. 1a, e). Fibroblasts were

derived from arm and abdomen skin biopsies (25–35 years for the

young control, n = 3, and 60–90 years for the aged group, n = 8),

while endothelial cells were extracted from iliac vein and artery

(15–25 years for the young control, n = 3, and 50–65 years for the

aged group, n = 7). We utilized a non-integrative reprogramming

protocol that we optimized, based on a cocktail of mRNAs

expressing OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG

(OSKMLN)

. Our protocol consistently produces iPSC colonies,

regardless of age of the donors, after 12–15 daily transfections; we

reasoned that the PNR in our platform occurs at about day 5 of

reprogramming, based on the observation that the ﬁrst detectable

expression of endogenous pluripotency-associated lncRNAs

occurs at day 5

. Therefore, we adopted a transient exogenous

expression regimen where OSKMLN was daily transfected for 4

consecutive days, and performed gene expression analysis 2 days

after the interruption (Fig. 1b).

We performed paired-end bulk RNA sequencing on both cell

types for the same three cohorts: young (Y), untreated aged (UA),

and treated aged (TA). First, we compared the quantile normal-

ized transcriptomes of young and untreated aged cells for each

cell type (Y vs. UA) and found that 961 genes (5.85%) in ﬁbro-

blasts (678 upregulated, 289 downregulated, Fig. 1a, c) and 748

genes (4.80%) in endothelial cells (389 upregulated, 377 down-

regulated, Fig. 1e, f) differed between young and aged cells, with

the signiﬁcance criteria of p < 0.05 and a log fold change cutoff

±0.5 (full list of genes in Supplementary Data 1 and 2). We found

these sets of genes were enriched for many of the known aging

pathways, identiﬁed in the hallmark gene set collection in the

Molecular Signatures Database

(Supplementary Data 3 and 4).

When we mapped the directionality of expression above or below

the mean of each gene, we could observe a clear similarity

between treated and young cells as opposed to aged cells for both

ﬁbroblasts and endothelial cells (Fig. 1d, g). We further per-

formed principal component analysis in this gene set space and

determined that the young and aged populations were separable

along the ﬁrst principal component (PC1), which explained

64.8% of variance in ﬁbroblasts and 60.9% of variance in endo-

thelial cells. Intriguingly, the treated cells also clustered closer to

the younger cells than the aged cells, simply along PC1 (Sup-

plementary Fig. 1a, b).

Using the same signiﬁcance criteria deﬁned above, we then

compared the treated and untreated aged populations (TA vs.

UA) (Fig 1a, e, Supplementary Fig. 2 and Supplementary Data 5

and 6) and found that 1042 genes in ﬁbroblasts (734 upregulated

and 308 downregulated) and 992 in endothelial cells (461 upre-

gulated and 531 downregulated) were differentially expressed.

Interestingly, also within these sets of genes, we found enrichment

for aging pathways, within the Molecular Signatures Database

previously described (Supplementary Data 7 and 8). When we

compared the proﬁles young versus untreated aged (Y vs. UA)

and untreated aged versus treated aged (UA vs. TA) in each cell

type, we observed a 24.7% overlap for ﬁbroblasts (odds ratio of

4.53, p < 0.05) and 16.7% overlap for endothelial cells (odds ratio

of 3.84, p < 0.05) with the directionality of change in gene

expression matching that of youth (i.e., if higher in young then

higher in treated aged); less than 0.5% moved oppositely in either

cell types (Supplementary Fig. 1a, b and Supplementary Data 9

and 10).

Next, we used these transcriptomic proﬁles to verify retention

of cell identity after treatment. To this end, using established cell

identity markers, we veriﬁed that none signiﬁcantly changed

upon treatment (Supplementary Data 11). In addition, we could

not detect the expression of any pluripotency-associated markers

(other than the OSKMLN mRNAs transfected in) (Supplemen-

tary Data 11). Altogether, the analysis of the transcriptomic sig-

natures revealed that OSKLMN expression promotes a very rapid

activation of a more youthful gene expression proﬁle, which is

cell-type speciﬁc, without affecting the expression of cell

identity genes.

Epigenetic clocks based on DNA methylation levels are the

most accurate molecular biomarkers of age across tissues and cell

types and are predictive of a host of age-related conditions

including lifespan

3,10–12

. Exogenous expression of canonical

reprogramming factors (OSKM) is known to revert the epigenetic

age of primary cells to a prenatal state

. To test whether transient

expression of OSKMLN could reverse the epigenetic clock of

human somatic cells, we used two epigenetic clocks that apply to

human ﬁbroblasts and endothelial cells: Horvath’s original pan-

tissue epigenetic clock (based on 353 cytosine–phosphate–guanine

pairs), and the more recent skin-and-blood clock (based on 391

CpGs)

3,13

According to the pan-tissue epigenetic clock, transient OSKMLN

signiﬁcantly (two-sided mixed-effect model P value = 0.023)

reverted the DNA methylation age (average age difference = −3.40

years, standard error 1.17). The rejuvenation effect was more

pronounced in endothelial cells (average age difference = −4.94

years, SE = 1.63, Fig. 1i) than in ﬁbroblasts (average age differ-

ence = −1.84, SE = 1.46, Fig. 1h). Qualitatively similar, but less

signiﬁcant results could be obtained with the skin-and-blood

epigenetic clock (overall rejuvenation effect −1.35 years, SE =

0.67, one-sided mixed-effect model P value = 0.042, and average

rejuvenation in endothelial cells and ﬁbroblasts is −1.62 years

and −1.07, respectively).

Prompted by these results, we next analyzed the effect of

transient reprogramming on various hallmarks of cellular

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289 ~16,000 678 734 ~16,000 308

389

~16,000

377

461

~ 16,000

531

Y vs. UA UA vs. TA

Y vs UA UA vs TA

RNA-seq in fibroblasts

RNA-seq in endothelial cells

Y vs UA

fibroblasts

Log 2 (differential)

–log 10(p value)

Y vs UA

endothelail cells

Log 2 (differential)

–log 10(p value)

Young

Above mean

Below mean

Aged Treated

Young Aged Treated

Above mean Below mean

Young, n = 3 Untreated aged, n = 3 Treated aged, n = 3

OSKMNL

(panel b)

OSKMNL

(panel b)

Y vs A signatureY vs A signature

Epigenetic clock

fibroblasts

Epigenetic clock

endothelial cells

Aged Aged

treated

Aged Aged

treated

OSKMNL

RNA seq and

methylome analysis

d0 d1* d2* d3* d4* d5 d6

n = 4 n = 4

–6–4–20246

–6 –4 –2 0 2 4 6

Fig. 1 Transcriptomic and epigenetic clock analysis shows more youthful signature upon transient expression of OSKMNL in human ﬁbroblasts and

endothelial cells. a Venn diagrams show differentially expressed genes in ﬁbroblasts (young, n = 3 individuals; aged and aged treated n = 3 individuals)

deﬁned with at signiﬁcance p value >0.05 and log fold change >0.5. Comparison among the three groups was conducted by ANOVA test. b Schematic of

reprogramming protocol. c Volcano plot showing young versus aged ﬁbroblast differential gene expression. d Heat map of polarity of expression (green =

above, purple = below) the mean for each differential gene. The distribution shows the treated samples transition in expression in this space towards the

direction of the young ﬁbroblasts. Cells in each cohort were subjected to 80 bp paired-end reads of RNA sequencing and quantile normalized. e Venn

diagrams show differentially expressed genes in endothelial cells (young, n = 3 individuals; aged and age-treated n = 3 individuals) deﬁned at signiﬁcance p

value >0.05 and log fold change >0.5. Comparison among the three groups was conducted by ANOVA test. f Volcano plot showing young versus aged

endothelial cells differential gene expression. g Heat map of polarity of expression (green = above, purple = below) the mean for each differential gene.

The distribution shows the treated samples transition in expression in this space towards the direction of the young endothelial cells. h Methylation clock

estimation of patient sample age with and without treatment for ﬁbroblasts; n = 4 individuals. i Methylation clock estimation of patient sample age with

and without treatment for endothelial cells; n = 4 individuals. Statistical analysis of methylation clock was performed by two-sided t-test analysis.

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physiological aging. We employed a panel of 11 established

assays, spanning the hallmarks of aging

(Supplementary

Data 12), and performed most of the analyses using single-cell

high-throughput imaging to capture quantitative changes in

single cells and distribution shifts in the entire population of cells.

All the analyses were performed separately in each individual cell

line (total of 19 ﬁbroblast lines: 3 young, 8 aged, and 8 treated

aged; total 17 endothelial cell lines: 3 young, 7 aged, and 7 treated

aged) (Fig. 2a and Supplementary Figs. 2–5). Statistical analysis

was conducted by randomly sampling 100 cells per sample; the

data was subsequently pooled by group category (see “Materials

and Methods” for a detailed description of the Statistical methods

that were used). Control experiments were performed by adopt-

ing the same transfection scheme using mRNA encoding for

green ﬂuorescent protein (GFP) (Supplementary Figs. 6 and 7).

To extend our previous ﬁndings on epigenetics, we quantita-

tively measured by immunoﬂuorescence (IF) the epigenetic

repressive mark H3K9me3, the heterochromatin-associated

LAP2α

***

H3K9me3 HP1γ

Autophagosome formation

Membrane potential

***

Mitochondrial ROS

***

n.s.

Proteosomal activity

600

400

200

***

Secreted cytokines

Endothelial cells

Young (F)

Aged (F)

Treated (F)

Young (E)

Aged (E)

Treated (E)

Young (F)

Aged (F)

Treated (F)

Young (E)

Aged (E)

Treated (E)

Young (F)

Aged (F)

Treated (F)

Young (E)

Aged (E)

Treated (E)

Young (F)

Aged (F)

Treated (F)

oung (E)

Aged (E)

Treated (E)

Young (F)

Aged (F)

reated (F)

Young (E)

Aged (E)

Treated (E)

Young (F)

Aged (F)

Treated (F)

Young (E)

Aged (E)

Treated (E)

Young (F)

Aged (F)

Treated (F)

Young (E)

Aged (E)

Treated (E)

Young (E)

Aged (E)

Treated (E)

Young (E)

Aged (E)

Treated (E)

oung (E)

Aged (E)

Treated (E)

Young (E)

Aged (E)

Treated (E)

Young (E)

Aged (E)

Treated (E)

Young (E)

Aged (E)

Treated (E)

Cellular physiology of aging

fibroblasts

Cellular physiology of aging

endothelial cells

Young, n = 3 Untreated aged, n = 8 Treated aged, n = 8 Young, n = 3 Untreated aged, n = 8 Treated aged, n = 8

n.s.

IL-18

IL-1A

GROA

IL-22IL-8

IL-9

Fluorescence units

8000 2000

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6000 1500

4000 1000

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protein HP1γ, and the nuclear lamina support protein LAP2 α

(Fig 2b–d). Aged ﬁbroblasts and endothelial cells showed a

decrease in the nuclear signal for all three markers compared with

young cells, as previously reported

; treatment of aged cells

resulted in an increase of these markers in both cell types. Next,

we examined both pathways involved in proteolytic activity of the

cells by measuring formation of autophagosomes, and

chymotrypsin-like proteasomal activity, both reported to decrease

with age

15,16

. Treatment increased both to levels similar to or

even higher than young cells, suggesting that early steps in

reprogramming promote an active clearance of degraded bio-

molecules (Fig. 2e, f).

In terms of energy metabolism, aged cells display decreased

mitochondrial activity, accumulation of reactive oxygen species

(ROS), and deregulated nutrient sensing

2,16,17

. We therefore tested

the effects of treatment on aged cells by measuring mitochondria

membrane potential, mitochondrial ROS, and levels of Sirtuin1

protein (SIRT1) in the cells. Transient reprogramming increased

mitochondria membrane potential in both cell types (Fig. 2g), while

it decreased mitochondrial ROS (Fig. 2h) and increased SIRT1

protein levels in ﬁbroblasts, similar to young cells (Supplementary

Fig. 8). Senescence-associated beta-galactosidase staining showed a

signiﬁcant reduction in the number of senescent cells in aged

endothelial cells but not in ﬁbroblasts (Supplementary Fig. 8). This

decrease was accompanied by a decrease in pro-inﬂammatory

senescence-associated secretory phenotype cytokines again in

endothelial cells and not in ﬁbroblasts (Fig. 2i and Supplementary

Fig. 8)

16,18,19

. Last, in neither cell type did telomere length, mea-

sured by quantitative ﬂuorescence in situ hybridization

2,20

, show

signiﬁcant extension with treatment (Supplementary Fig. 8), sug-

gesting that the cells did not dedifferentiate into a stem-like state in

which telomerase activity would be reactivated, and in agreement

with previous reports where activation of TERT was observed at

later stages of nuclear reprogramming

Next, we assessed the perdurance of these effects and found

that most were signiﬁcantly retained after 4 and 6 days from the

interruption of reprogramming (Supplementary Figs. 9 and 10).

We then examined how rapidly these physiological rejuvenative

changes manifest by repeating the same sets of experiments in

ﬁbroblasts and endothelial cells that were transfected for just 2

consecutive days. Remarkably, we observed that most of the

rejuvenative effects could already be seen after 2 days of treat-

ment, although most were more moderate (Supplementary

Figs. 11 and 12).

Collectively, this data demonstrates that transient expression of

OSKMLN can induce a rapid, persistent amelioration, and

reversal of cellular age in human somatic cells at the tran-

scriptomic, epigenetic, and cellular levels. Importantly, these data

demonstrate that the process of cellular rejuvenation is engaged

very early, rapidly, and broadly in the reprogramming process.

These epigenetic and transcriptional changes occur before any

epigenetic reprogramming of cellular identity takes place, a novel

ﬁnding in the ﬁeld.

With these indications of a beneﬁcial effect on cellular aging,

we next investigated whether transient expression of OSKMNL

could also reverse the inﬂammatory phenotypes associated with

aging. After obtaining preliminary evidence of this reversal in

endothelial cells (Fig. 2j), we extended our analysis to osteoar-

thritis, a disease strongly associated with aging and characterized

by a pronounced inﬂammatory spectrum affecting the chon-

drocytes within the joint

. We thus isolated chondrocytes from

cartilage of six 60–70-year-old patients undergoing total joint

replacement surgery owing to their advanced-stage OA, and

compared the results of treatment with chondrocytes isolated

from three young individuals (Fig. 3a). Transient OSKMLN

expression was performed for 2 or 3 days, and the analysis per-

formed after 2 days from interruption of reprogramming, though

the more consistent effect across patients was with longer treat-

ment. Treatment showed a signiﬁcant reduction in intracellular

mRNA levels of RANKL and iNOS2, as well as in levels of

inﬂammatory factors secreted by the cells (Fig. 3b–d). In addition,

we observed increased cell proliferation (Fig. 3e), increased ATP

production (Fig. 3f), and decreased oxidative stress as revealed by

reduced mitochondrial ROS and elevated RNA levels of anti-

oxidant SOD2 (Fig. 3g, h), a gene that has been shown to be

downregulated in OA

. Finally, when we checked for retention

of cellular identity, we observed that the treatment did not affect

the expression level of SOX9 (a transcription factor core to

chondrocyte identity and function) and signiﬁcantly increased the

level of expression of COL2A1 (the primary collagen in articular

cartilage) (qRT-PCR in Fig 3i, j), suggesting retention of chron-

drogenic cell identity. Together, these results show that transient

expression of OSKMLN can promote a partial reversal of gene

expression and cellular physiology in aged OA chondrocytes

toward a healthier, more youthful state, suggesting a potential

new therapeutic strategy to ameliorate the OA disease process.

Stem cell loss of function and regenerative capacity represents

another important hallmark of aging

. We thus wanted to assess

Fig. 2 Transient OSKMNL expression reverts aged physiology toward a more youthful state in human ﬁbroblasts and endothelial cells. a Fibroblasts

(F) and endothelial cells were obtained from otherwise healthy young and aged individuals. Young untreated cells (n = 3 distinct individuals for both

ﬁbroblasts and endothelial cells, dark blue), aged untreated cells ( n = 8 individuals for ﬁbroblast, n = 7 individuals for endothelial cells, red), and aged

treated cells (n = 8 for ﬁbroblast, n = 7 for endothelial cells, light blue) were analyzed for a panel of 11 different hallmarks of aging. Most of the assays were

performed by high-throughput imaging on 500–1000 cells per sample to allow population-wide studies with single-cell resolution (Supplementary Figs. 2–

5). 100 cells per sample (i.e., individuals) were randomly selected and pooled per treatment group to do a statistical comparison across the three groups

(young ﬁbroblasts n = 300; aged ﬁbroblasts n = 800; aged treated ﬁbroblasts n = 800; young endothelial cells n = 300; aged endothelial cells n = 700;

aged treated endothelial cells n = 700). Pairwise statistical analysis was done by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. b Quantiﬁcation of

single-nucleus levels of trimethylated H3K9, a repressive mark of gene expression. Both cell types show signiﬁcant elevation of the mark towards the

youthful distribution. c Quantiﬁcation of single-nucleus levels of heterochromatin marker HP1γ by immunocytochemistry showing a trend toward youth

upon treatment. d Quantiﬁcation of the inner nuclear membrane polypeptide LAP2α, a regulator of nuclear lamina by regulating the binding of lamin B1 and

chromatin. This again shows a trend toward youth after cells are treated. e Results of live cells imaging with ﬂorescent marker of autophagosome formation

in single cells. f Cleavage of ﬂuorescent-tagged chymotrypsin-like substrate elevated in treated and young ﬁbroblasts and endothelial cells corresponding

to increased proteasome 20S core particle activity. g Individual cell mitochondria membrane potential measurements also showing more active

mitochondria as a result of transient reprogramming. Quantiﬁcation of pro-inﬂammatory factors secreted by the cells in each cohort. h Individual cell

mitochondria ROS measurements also showing less accumulated ROS as a result of transient reprogramming. i Inﬂammatory cytokine proﬁling in

endothelial cells, with a signiﬁcant elevation and depression speciﬁcally in aged and treated endothelial cells, respectively. In b–h data are represented as

box–whisker plots with median, and bars represent whiskers with distribution variability 10th–90th percentile. In f–j data are represented as mean values

and bars represent SD.

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COL2A1

Aged OA

chondrocytes

Untreated

Young

chondrocytes

RANKL INOS2 Secreted cytokines

Cell proliferation

1.3

240

–2

–4

220

200

180

160

140

1.2

1.1

1.0

AT P

ROS SOD2

SOX9

515

–5

–10

–5

–10

oung healthy

Treated

Young healthy

reated

Young healthy

Treated

Young healthy

Treated

oung healthy

Treated

Young healthy

Treated

oung healthy

Treated

Young healthy

Treated

Log fold change over OA mean

Fluorescence units

Treated

Untreated

2000

1500

1000

500

–5

–10

–5

–10

bc d

efg h

MIP1A IL-6 IFNA MCP3

***

n.s.

***

n.s.

Fig. 3 Transient OSKMNL expression mitigates inﬂammatory phenotypes in diseased chondrocytes. a Workﬂow summarizing the strategy adopted to

mitigation of age-related disease. Chondrocytes were obtained from six distinct aged patients diagnosed late stage Osteoarthritis (OA) patients from

cartilage biopsies. Healthy cells (blue), aged OA cells (red) and transiently reprogrammed OA cells (light blue) were evaluated for OA speciﬁc phenotypes.

b qRT-PCR evaluation shows treatment diminishes of intracellular RNA levels of NF-κB ligand RANKL. c qRT-PCR evaluation shows treatment drops levels

of iNOS for producing nitric oxide as a response and to propagate inﬂammatory stimulus. d Cytokine proﬁling of chondrocyte secretions shows an increase

pro-inﬂammatory cytokines in OA chondrocytes that diminishes with treatment. e Cell proliferation rate as measured by cell-tracking dye. f Measurement

of ATP concentration using glycerol based ﬂuorophore shows elevation of ATP levels with treatment. g Live single-cell image of cells up taking superoxide

triggered ﬂuorescent dyes shows diminished signal after treatment. h qRT-PCR evaluation of RNA levels of antioxidant SOD2, elevated with treatment.

i qRT-PCR levels of chondrogenic identity and function transcription factor SOX9 is retained after treatment. j qRT-PCR shows elevation RNA levels for

extracellular matrix protein component. Young samples n = 3 individuals; aged OA samples treated and untreated n = 6 individuals. Pairwise statistical

analysis was done by one-way ANOVA. For ROS (g) analysis was conducted by high-throughput imaging on 500–1000 cells per sample to allow

population-wide studies with single-cell resolution. One-hundred cells per sample were randomly selected to do a statistical comparison across the three

groups. Statistical analysis was then done by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. Statistical analysis by one-way ANOVA was conducted

for all the other assays.

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the effect of transient reprogramming on the age-related changes

in somatic stem cells that impair regeneration. First, we tested the

effect of transient reprogramming on mouse-derived skeletal

muscle stem cells (MuSCs). We treated MuSCs for 2 days while

they were kept in a quiescent state using an artiﬁcial niche

.We

conducted initial experiments with young (3 month) and aged

(20–24 months) murine MuSCs isolated by FACS (Fig. 4a).

Treatment of aged MuSCs reduced both time of ﬁrst division,

approaching the faster activation kinetics of quiescent young

MuSCs

25,26

, and mitochondrial mass

(Supplementary Fig. 13a,

b). Moreover, treatment partially rescued the reduced ability of

single MuSCs to form colonies

25,28

(Supplementary Fig. 13c). We

further cultured these cells and observed that treatment did not

change expression of the myogenic marker MyoD but instead

improved their capacity to differentiate into myotubes (Supple-

mentary Fig. 14a–d), suggesting that transient reprogramming

does not disrupt the myogenic fate but can enhance the myogenic

potential.

Next, we wanted to test MuSC function and potency to

regenerate new tissue in vivo. To do this, we transduced young,

aged, or transiently reprogrammed aged MuSCs with a lentivirus-

expressing luciferase and GFP, and then transplanted the cells

into injured tibialis anterior (TiA) muscles of immunocompro-

mised mice. Longitudinal bioluminescence imaging (BLI) initially

showed that muscles transplanted with treated aged MuSCs

showed the highest signal (day 4, Fig 4b, c), but became com-

parable with muscles with young MuSCs by day 11 post trans-

plantation; conversely muscle with untreated aged MuSCs

showed lower signals at all time points post transplantation

(Fig. 4b, c). IF analysis further revealed higher numbers of donor-

derived (GFP

) myoﬁbers in TiAs transplanted with treated

compared with untreated aged MuSCs (Fig. 4d, e). Moreover, the

GFP

myoﬁbers from treated aged cells exhibited increased

cross-sectional areas when compared with their untreated coun-

terparts, and in fact even larger than the young controls (Fig. 4f).

Together, these results suggest improved tissue regenerative

potential of transiently reprogrammed aged MuSCs. After

3 months, all mice were subjected to autopsy, and no neoplastic

lesions or teratomas were discovered (Supplementary Table 1).

To test potential long-term beneﬁts of the treatment, we induced

a second injury 60 days after cell transplantation, and again

observed that TiA muscles transplanted with transiently repro-

grammed aged MuSCs yielded higher BLI signals (Fig. 4g).

Sarcopenia is an age-related condition that is characterized by

loss of muscle mass and force production

29,30

. Similarly, in mice

muscle functions show progressive degeneration with age

31,32

We wanted to test whether transient reprogramming of aged

MuSCs would improve a cell-based treatment in restoring phy-

siological functions of muscle of older mice. To test this, we ﬁrst

performed electrophysiology to measure tetanic force production

in TiA muscles isolated from young (4 months) or aged

(27 months) immunocompromised mice. We found that TiA

muscles from aged mice have lower tetanic forces compared with

young mice, suggesting an age-related loss of force production

(Fig. 4h). Next, we isolated MuSCs from aged mice

(20–24 months). After treating aged MuSCs, we transplanted

them into cardiotoxin-injured TiA muscles of aged (20 months)

immunocompromised mice. We waited 30 days to give enough

time to the transplanted muscles to fully regenerate. We then

performed electrophysiology to measure tetanic force production.

Muscles transplanted with untreated aged MuSCs showed forces

comparable with untransplanted muscles from aged control mice

(Fig. 4h). Conversely, muscles that received treated aged MuSCs

showed tetanic forces comparable with untransplanted muscles

from young control mice (Fig. 4h and Supplementary Fig. 15a).

These results suggest that transient reprogramming in

combination with MuSC-based therapy can restore physiological

function of aged muscles to that of youthful muscles.

Last, we wanted to translate these results to human MuSCs. We

repeated the study, employing operative samples obtained from

patients in different age ranges (10–80 years old), and transdu-

cing them with GFP- and luciferase-expressing lentiviral vectors

(Fig. 4a). As in mice, transplanted, transiently reprogrammed,

aged human MuSCs resulted in increased BLI signals compared

with untreated MuSCs from the same individual, and comparable

with those observed with young MuSCs (Fig. 4i and Supple-

mentary Fig. 16a, b). Interestingly, the BLI signal ratio between

contralateral muscles with treated and untreated MuSCs was

higher in the older age group (60–80 years old) than in the

younger age groups (10–30 or 30–55 years old), suggesting that

ERA restores lost functions to younger levels in aged cells

(Fig. 4j). Taken together, these results suggest that transient

reprogramming partially restores the potency of aged MuSCs to a

degree similar to that of young MuSCs, without compromising

their fate, and thus has potential as a cell therapy in regenerative

medicine.

Nuclear reprogramming to iPSCs is a multi-phased process

comprising initiation, maturation, and stabilization

. Upon

completion of such a dynamic and complex epigenetic repro-

gramming, iPSCs are not only pluripotent but also youthful.

While proof of principle that transient reprogramming can exert

a systemic rejuvenation in a genetic model of aging (progeroid

mice), the proof that a multispectral cellular rejuvenation could

be achieved in a cell-autonomous fashion in human cells isolated

from naturally aged individuals was missing. Here we demon-

strate that a non-integrative, mRNAs-based platform of transient

cellular reprogramming can very rapidly reverse a broad spec-

trum of aging hallmarks in the initiation phase, when epigenetic

erasure of cell identity has not yet occurred. We show that the

process of rejuvenation occurs in naturally aged human and

mouse cells, with restoration of lost functionality in diseased cells

and aged stem cells while preserving cellular identity. Future

studies are required to elucidate the mechanism that drives the

reversal of the aged phenotype during cellular reprogramming,

uncoupling it from dedifferentiation process

34,35

. Our results are

novel and represent a signiﬁcant step toward the goal of reversing

cellular aging, and have potential therapeutic implications for

aging and aging-related diseases.

Methods

Human ﬁbroblast isolation and culture. Isolation was performed at Coriell

Institute on healthy patients and from Alzheimer patient samples at Stanford

Hospital, in accordance to the methods and protocols approved by the Institutional

Review Board of Stanford University, biopsied for skin mesial aspect of mid-upper

arm or abdomen using 2-mm punch biopsies from both male and female patients

60–70 years old (n = 8) and 25–35 years (n = 3). Cells were cultured out from these

explants and maintained in Eagle’s Minimum Essential Medium with Earl’s salts

supplemented with nonessential amino acids, 10% fetal bovine serum, and 1%

Penicillin/Streptomycin. Cells were cultured at 37 °C with 5% CO

Human endothelial cell isolation and culture. Isolation was performed at Coriell

Institute from iliac arteries and veins, and muscle biopsies from Stanford Hospital,

in accordance to the methods and protocols approved by the Institutional Review

Board of Stanford University, from otherwise healthy 45–60 years old (n = 7).

Tissue was digested with collagenase and cells released from the lumen were used

to initiate cultures. Plates for seeding were coated with 2% gelatin, then washed

with PBS before use. Cells were maintained in Medium 199 supplemented with 2

L-glutamine, 15% fetal bovine serum, 0.02 mg/ml Endothelial Growth Sup-

plement, 0.05 mg/ml Heparin, and 1% Penicillin/Streptomycin. Cells were cultured

at 37 °C with 5% CO

Human articular chondrocyte isolation and culture. In accordance to the

methods and protocols approved by Institutional Review Board of Stanford Uni-

versity, the human OA chondrocytes were derived from discarded tissues of OA

patients (50–72 years of age, n = 6) undergoing total knee arthroplasty. The

samples were surgical waste and were fully deidentiﬁed prior to procurement,

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NATURE COMMUNICATIONS | (2020) 11:1545 | https://doi.org/10.1038/s41467-020-15174-3 | www.nature.com/naturecommunications 7

Aged

Young

Aged

Young

Untreated

Treated

d0 d11 d60 d71

1st muscle

injury

2nd muscle

injury

Luciferase and GFP

lentiviral infection

Host

immunocompromised

Aged untreated

Aged treated

14711

10e3

Days

Treated

Untreated

20 Mo mouse

Bioluminescence

(photons cm

–2

–1

)

Bioluminescence

(photons cm

–2

–1

)

Bioluminescence

(photons cm

–2

–1

)

Bioluminescence

(ratio treted/untreated)

Tetanic force (mN mm

)

GFP regenerated fibers (#)

Fibers (%)

Aged

untreated

Aged

treated

Aged

untreated

Aged

treated

Fiber area (μn

)

Aged treated

61 65 67 71

Aged untreated

Young untreated

Aged treated

Aged untreated

Young untreated

Aged treated

Aged untreated

Young untreated

Age 60–80

Age 30–55

Age 10–30

Days

Young

untretaed

Young

non-

transplanted

14711

47911

Aged

non-

transplanted

bc d

1500

0.8

0.6

0.4

0.2

100

400

700

1000

1300

1600

1900

2200

2500

2800

3100

3400

3700

4000

1000

500

1200

800

400

hi j

Muscle

Satellite cells

Fig. 4 Transient OSKMNL expression restores aged muscle stem cell potency. a Schematic showing the experimental design of partially reprogrammed

aged mouse and human MuSCs. b Representative images of bioluminescence measured from mice 11 days after transplantation and injury in TiA muscles

of treated/untreated Luciferase

mouse MuSCs. c Quantiﬁed results of bioluminescence in b at different time points following transplantation and injury

(n = 10). d Representative immunoﬂuorescence of GFP expression in TiA muscle cross-sections of mice imaged and quantiﬁed in c and d, isolated 11 days

after transplantation (Scale bar = 500 μm). e Quantiﬁcation of immunoﬂuorescence staining in d (n = 5). f Quantiﬁcation of the cross-sectional area of

donor-derived GFP + ﬁbers in TiA muscles that were recipients of transplanted MuSCs (n = 5). g Results of bioluminescence imaging of TiA muscles

reinjured after 60 days (second injury) after MuSC transplantations (n = 6). The second injury was performed to test whether the bioluminescence signal

increased as a consequence of activating and expanding luciferase

/GFP

MuSCs that were initially transplanted and that had engrafted under the basal

lamina. h Tetanic force measurements of aged muscles injured and transplanted with aged MuSCs. TiA muscles were dissected and electrophysiology

ex vivo for tetanic measurement performed. Baseline of force production of untransplanted muscles was measured in young (4 months, blue broken line)

and aged (27 months, red broken line) mice. Treated aged MuSCs were transplanted into TiA muscles of aged mice and force production measured

30 days later (n = 5). i Quantiﬁed results of bioluminescence measured from mice 11 days after transplantation in TiA muscles of treated Luciferase

human MuSCs. j Variation in ratio of bioluminescence between treated and untreated MuSCs obtained from healthy donors of different age groups.

Signiﬁcance is calculated with one-sided student’s t test, pairwise between treated and aged, and group wise when comparing with young patients (age

groups. 10–30: n = 5 individuals; 30–55: n = 7 individuals; 60–80: n = 5 individuals). *P < 0.05, **P < 0.01, ***P < 0.001, color of the asterisks matches the

population being compared with.

ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15174-3

8 NATURE COMMUNICATIONS | (2020) 11:1545 | https://doi.org/10.1038/s41467-020-15174-3 | www.nature.com/naturecommunications

hence no prior patient consent was required. Cartilage pieces were shaved off bone

by scalpel, taking care to avoid any fat, then digested with collagenase in DMEM/

F12 media (supplemented with 25 mg/ml ascorbate, 2 mM

L-glutamine, 1% peni-

cillin/streptomycin antibiotics, and 10% fetal bovine serum) for 1–2 days until

shavings were substantially dissolved. Supernatant from cultures was strained,

ﬁltered, and centrifuged, and the cells were then resuspended in fresh media. The

chondrocytes were cultured in high-density monolayer at 37 °C with 5% CO

Mice. C57BL/6 male and NSG mice were obtained from Jackson Laboratory.

NOD/MrkBomTac-Prkdcscid mice were obtained from Taconic Biosciences. Mice

were housed and maintained in the Veterinary Medical Unit at the Veterans Affairs

Palo Alto Health Care Systems. The Administrative Panel on Laboratory Animal

Care of Stanford University approved animal protocols.

Human skeletal muscle specimens. The human muscle biopsy specimens were

taken after patients (10–30 years, n = 2; 30–55 years, n = 2; 60–80 years, n = 3)

gave informed consent as part of a human studies research protocol that was

approved by the Stanford University Institutional R eview Board. Sample processing

for cell analysis began within 1–12 h of specimen isolation. In all studies, standard

deviation reﬂects variability in data derived from studies using true biological

replicates (i.e., unique donors). Data were not correlated with donor identity.

MuSC isolation and puriﬁcation. Muscles were harvested from mouse hind limbs

(n = 4) and mechanically dissociated to yield a fragmented muscle suspension. This

was followed by a 45–50-min digestion in a Collagenase II-Ham’s F10 solution

(500 U ml

−1

, Invitrogen). After washing, a second digestion was performed for

30 min with Collagenase II (100 U ml

−1

) and Dispase (2 U ml

−1

, ThermoFisher).

The resulting cell suspension was washed, ﬁltered, and stained with VCAM-biotin,

CD31-FITC, CD45-APC, and Sca-1-Paciﬁc-Blue antibodies, all at dilutions of

1:100. Human MuSCs were puriﬁed from fresh operative samples. Operative

samples were carefully dissected from adipose and ﬁbrotic tissue and a dissociated

muscle suspension prepared as described for mouse tissue. The resulting cell

suspension was then washed, ﬁltered, and stained with anti-CD31-Alexa Fluor 488,

anti-CD45-Alexa Fluor 488, anti-CD34-FITC, anti-CD29-APC, and anti-NCAM-

Biotin antibodies. Unconjugated primary antibodies were then washed and the cells

were incubated for 15 min at 4 °C in streptavidin-PE/Cy7 to detect NCAM-biotin.

Cell sorting was performed on calibrated BD-FACSAria II or BD FACSAria III

ﬂow cytometers equipped with 488-, 633-, and 405-nm lasers to obtain the MuSC

population. A small fraction of sorted cells was plated and stained for Pax7 and

MyoD to assess the purity of the sorted population.

mRNA transfection. Cells were transfected using either mRNA-In (mTI Global

Stem) for ﬁbroblasts and chondrocytes, to reduce cell toxicity, or Lipofectamine

MessengerMax (ThermoFisher) for endothelial cells and MuSCs, which were more

difﬁcult to transfect, using the manufacturer’s protocol. For ﬁbroblast and endo-

thelial cells, serum free Pluriton medium with bFGF was used for transfection,

while muscle stem cells and chondrocytes were kept in their original media—the

former lacking serum and the later requiring serum to prevent the natural ded-

ifferentiation of chondrocytes in culture. Culture medium was changed for ﬁbro-

blasts and endothelial cells 4 h after transfection, but not for chondrocytes or

MuSCs as overnight incubation was needed to produce a signiﬁcant uptake of

mRNA. Efﬁciency of delivery was conﬁrmed by both GFP mRNA and immu-

nostaining for individual factors in OSKMLN cocktail, the former also being used

as a transfection control with the same protocol.

Immunocytochemistry. Cells were washed with HBSS/CA/MG and then ﬁxed

with 15% paraformaldehyde in PBS for 15 min. Cells were then blocked for 30 min

to 1 h with a blocking solution of 1% BSA and 0.3% Triton X-100 in PBS for

ﬁbroblasts, endothelial cells, and 20% donkey serum/0.3% Triton in PBS for

MuSCs. Primary antibodies were then applied in blocking solution and allowed to

incubate overnight at 4 °C. The following day, the cells were washed with HBSS/

CA/MG or PBST for MuSCs before switching to the corresponding Alexa Fluor-

labeled secondary antibodies and incubated for 2 h. The cells were then washed

again and stained with DAPI for 30 min and switched to HBSS/CA/MG for ima-

ging or Fluoview for MuSCs.

Autophagosome formation staining. Cells were washed with HBSS/Ca/Mg and

switched to a staining solution containing a proprietary ﬂuorescent autophagosome

marker (Sigma). The cells were then incubated at 37 °C in 5% CO

for 20 min,

washed two times using HBSS/Ca/ Mg, and stained for 15 min using CellTracker

Deep Red cell labeling dye. Cells were then switched to HBSS/Ca/Mg for single-cell

imaging using the Operetta High -Content Imaging System (Perkin Elmer).

Proteasome activity measurement. Wells were ﬁ rst stained with PrestoBlue Cell

Viability dye (Life Technologies) for 10 min. Well signals were read using a

TECAN ﬂuorescent plate reader as a measure of cell count. Then cells were washed

with HBSS/Ca/Mg before switching to original media containing the

chymotrypsin-like ﬂuorogenic substrate LLVY-R110 (Sigma), which is cleaved by

proteasome 20S core particle. Cells were then incubated at 37 °C in 5% CO

for 2 h

before signals were again read on the TECAN ﬂuorescent plate reader. Readings

were then normalized by PrestoBlue cell count.

Mitochondrial membrane potential staining. Tetramethylrhodamine Methyl

Ester Perchlorate (Thermo) was added to cell culture media. This dye is seques-

tered by active mitochondria based on their membrane potential. Cells were

incubated for 30 min at 37 °C in 5% CO

and washed two times with HBSS/Ca/Mg

before staining for 15 min using CellTracker Deep Red. Finally, cells were imaged

in fresh HBSS/Ca/Mg using the Operetta High-Content Imaging System (Perkin

Elmer).

Mitochondrial ROS measurement. Cells were washed with HBSS/Ca/Mg and

then switched to HBSS/Ca/Mg containing MitoSOX (Thermo), a live-cell-

permeant ﬂuorogenic dye that selectively targeted to mitochondria and ﬂuoresces

when oxidized by superoxide. Cells were incubated for 10 min at 37 °C in 5% CO

Cells were then washed twice with HBSS/Ca/Mg, and stained for 15 min using

CellTracker Deep Red. Finally, cells were imaged in fresh HBSS/Ca/Mg using the

Operetta High-Content Imaging System (Perkin Elmer).

SAβGal histochemistry. Cells were washed twice with PBS then ﬁxed with 15%

Paraformaldehyde in PBS for 6 min. Cells were rinsed three times with PBS before

staining with X-gal chromogenic substrate, which is cleaved by endogenous Beta

galactosidase. Plates were kept in the staining solution, Paraﬁlmed, to prevent from

drying out, and incubated overnight at 37 °C with ambient CO

. The next day, cells

were washed again with PBS before switching to a 70% glycerol solution for

imaging under a Leica bright-ﬁeld microscope.

Fixed and live-cell imaging. Samples were imaged using ﬂuorescent microscopes

—the Operetta High-Content Imaging System (Perkin Elmer) or the BZ-X700

(Keyence)—and either a 10× or 20× air objective. Harmony (Operetta) or Volocity

(BZ-X700) imaging software was used to adjust excitation and emission ﬁlters and

came with preprogrammed Alexa Fluor ﬁlter settings which were used whenever

possible. All exposure times were optimized during the ﬁrst round of imaging, and

then kept constant through all subsequent imaging.

Image analysis . Columbus (Operetta) or Image J (BZ-X700) was used for image

analysis. Columbus software was to identify single cells utilizing DAPI of Cell-

Tracker Re d to delineate nuclear and cell boundaries and calculate the signal

statistics for each cell. Image J was used for muscle ﬁbers to calculate the percentage

of area composed of collagen by using the color threshold plug-in to create a mask

of only the area positive for collagen. That area was then divided over the total area

of the sample, which was found using the free draw tool. All other ﬁber analyses

were performed using Volocity software and manually counting ﬁbers using the

free draw tool.

Statistics. Statistical analysis for physiological hallmarks of aging was done as

described previously in Miller et al.

. Brieﬂy, 100 cells were randomly selected from

each experimental group (data depicted in Supplementary Figs. 2–5), and they were

then pooled in a unique population of 800 cells for aged ﬁbroblasts (100 cells × 8

individuals for both aged and aged treated); 300 cells for young ﬁbroblasts (100

cells × 3 individuals); 700 cells for aged endothelial cells (100 cells × 7 individuals

for both aged and aged treated); 300 cells for young endothelial cells (100 cells × 3

individuals). Box distribution plots display the ﬂuorescence intensity quantiﬁcation

of 100 cells from each patient. Distributions were compared by statistical analysis

by using multiple-comparison ANOVA. Arbitrary units for frequency distributions

of different cell types should not be compared because staining was performed at

different times. Matlab 2017 (MathWorks) was used for data presentation and

analysis.

Cytokine proﬁling. This work was performed together with the Human Immune

Monitoring Center at Stanford University. Cell media was harvested and spun at

400 rcf for 10 min at room temperature. The supernatant was then snap frozen

with liquid nitrogen until analysis. Analysis was done using the human 63-plex kit

(eBiosciences/Affymetrix). Beads were added to a 96-well plate and washed in a

Biotek ELx405 washer. Samples were added to the plate containing the mixed

antibody-linked beads and incubated at room temperature for 1 h followed by

overnight incubati on at 4 °C with shaking. Cold and room temperature incubation

steps were performed on an orbital shaker at 500–600 rpm. Following the overnight

incubation, plates were washed in a Biotek ELx405 washer and then biotinylated

detection antibody added for 75 min at room temperature with shaking. Plates

were washed as above and streptavidin-PE was added. After incubation for 30 min

at room temperature, wash was performed as above and reading buffer was added

to the wells. Each sample was measured in duplicate. Plates were read using a

Luminex 200 instrument with a lower bound of 50 beads per sample per cytokine.

Custom assay Control beads by Radix Biosolutions were added to all wells.

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NATURE COMMUNICATIONS | (2020) 11:1545 | https://doi.org/10.1038/s41467-020-15174-3 | www.nature.com/naturecommunications 9

Antibodies. The following antibodies were used in this study. The source of each

antibody is indicated. Rabbit::H3K9me3 (Abcam #ab8898 1:4000), LAP2α (Abcam

#ab5162 1:500), SIRT1 (Abcam #ab7343 1:200); Rabbit: Mouse: HP1γ (Millipore

Sigma #05-690 1:200), Lamin A/C (Abcam #ab40567 1:200), GFP (Invitrogen,

#A11122, 1:250); Luciferase (Sigma-Aldrich, #L0159, 1:200); Collagen I (Cedarlane

Labs, #CL50151AP, 1:200); HSP47 (Abcam, #ab77609, 1:200), Laminin (Abcam,

#AB11576, 1:1000), anti-CD31-Alexa Fluor 488 (clone WM59; BioLegend;

#303110, 1:75), anti-CD45-Alexa Fluor 488 (clone HI30; Invitrogen; #MHCD4520,

1:75), anti-CD34-FITC (clone 581; BioLegend; #343503, 1:75), anti-CD29-APC

(clone TS2/16; BioLegend; #303008, 1:75) and anti-NCAM-Biotin (clone HCD56;

BioLegend; #318319, 1:75), anti-CD31-Alexa Fluor 488 (clone WM59; BioLegend;

#303110, 1:75), anti-CD45-Alexa Fluor 488 (clone HI30; Invitrogen; #MHCD4520,

1:75), anti-CD34-FITC (clone 581; BioLegend; #343503, 1:75), anti-CD29-APC

(clone TS2/16; BioLegend; #303008, 1:75), and anti-NCAM-biotin (clone HCD56;

BioLegend; #318319, 1:75).

RNA sequencing and data analysis. Cells were washed and digested by TRIzol

(Thermo). Total RNA was isolated using the Total RNA Puriﬁcation Kit (Norgen

Biotek Corp) and RNA quality was assessed by the RNA analysis screentape (R6K

screentape, Agilent); RNA with RIN > 9 was reverse transcribed to cDNA. cDNA

libraries were prepared using 1 μg of total RNA using the TruSeq RNA Sample

Preparation Kit v2 (Illumina). RNA quality was assessed by an Agilent Bioanalyzer

2100; RNA with RIN > 9 was reverse transcribed to cDNA. cDNA libraries were

prepared using 500 ng of total RNA using the TruSeq RNA Sample Preparation Kit

v2 (Illumina) with the added beneﬁt of molecular indexing. Prior to any PCR

ampliﬁcation steps, all cDNA fragment ends were ligated at random to a pair of

adapters containing a 8-bp unique molecular index. The molecular indexed cDNA

libraries were than PCR ampliﬁed (15 cycles) and then QC’ed using a Bioanalzyer

and Qubit. Upon successful QC, they were sequenced on an Illumina Nextseq

platform to obtain 80-bp single-end reads. The reads were trimmed by 2 nt on each

end to remove low-quality parts and improve mapping to the genome. The 78-nt

reads that resulted were compressed by removing duplicates, while keeping track of

how many times each sequence occurred in each sample in a database. The unique

reads were then mapped to the human genome using exact matches. This misses

reads that cross exon–exon boundaries, as well as reads with errors and SNPs/

mutations, but it does not have substantial impact on estimating the levels of

expression of each gene. Each mapped read was then assigned annotations from the

underlying genome. In case of multiple annotations (e.g., a miRNA occurring in

the intron of a gene), a hierarchy based on heuristics was used to give a unique

identity to each read. This was then used to identify the reads belonging to each

transcript and coverage over each position on the transcript was established. This

coverage is nonuniform and spiky. Therefore, we used the median of this coverage

as an estimate of the expression value of each gene. In order to compare the

expression levels in different samples, quantile normalization was used. Further

data analysis was done in Matlab. Ratios of expression levels were then calculated

to estimate the log (base 2) of the fold change. Student’s t test was used to

determine signiﬁcance with a p < 0.05 cutoff. Molecular Signatures Database

categorization was done using Broad Institute online tools https://software.

broadinstitute.org/gsea/msigdb/.

Gene expression analysis. Total RNA was puriﬁed using the RNeasy Plus Mini

kit (Qiagen), and cDNA was prepared with the First-strand cDNA synthesis kit

(Applied Biosystems). The quantitative polymerase chain reaction was performed

using VeriQuest Mastermix (ThermoFisher Scientiﬁc) for SYBR Green and Taq-

man primer sets, respectively. The relative gene expression was analyzed by the

ΔΔCt method and normalized to glyceraldehyde-3-phosphate dehydrogenase

(GAPDH). The Taqman probes for human GAPDH (Hs02758991); COL2A1

(Hs00264051); SOX9 (Hs00165814); MMP3 (Hs00233962) and MMP13

(Hs00233992) were purchased from Applied Biosystems. The SYBR green primer

sequences used are: Human SOD2 (F)-5′GGC CTA CGT GAA CAA CCT GA3′;

Human SOD2 (R)-5′TGG GCT GTA ACA TCT CCC TTG3′; Human iNOS (F)-5′

GTC CCG AAG TTC TCA AGG CA3′; Human iNOS (R)-5′

GTT CTT CAC TGT

GGG GCT TG3′; Human RANKL (F)-5′CAG GTT GTC TGC AGC GT3′ and

Human RANKL (R)-5′GAT CCA TCT GCG CTC TGA AAT A3′; Human

GAPDH (F)- 5′TGT CCC CAC TGC CAA CGT GTC3′; Human GAPDH (R)-5′

AGC GTC AAA GGT GGA GGA GTG GGT3′.

ATP assay. ATP in the chondrocytes was measured using colorimetric assay and

the ATP assay kit (ab83355; Abcam, Cambridge, MA) following the manufacturer’s

instructions. Cells were washed in cold phosphate buffered saline and homogenized

and centrifuged to collect the supernatant. The samples were loaded with assay

buffer in triplicate. ATP reaction mix and background control (50 µL) was added to

the wells and incubated for 30 min in dark. The plate was read at OD 570 nm using

SpectraMax M2e (Molecular Devices, Sunnyvale, CA). The mean optical density

was used to estimate of the intracellular ATP concentration relative to the

standard curve.

Cell proliferation assay. Cell viability was assayed using the PrestoBlue Cell

Viability (Life Technologies) reagent consecutively for 3 days post transfection in

accordance with the manufacturer’s instructions. PrestoBlue reagent was added to

the cell culture medium, and the cells were incubated at 30 °C for 30 min.

Absorbance of the PrestoBlue was measured daily using SpectraMax M2e (Mole-

cular Devices, Sunnyvale, CA).

EDU staining. Staining was done according to the manufacturer’s protocol using

the Click-iT EdU kit. Cells were labeled with Edu after switching to growth media.

Cells were allowed to grow 1 or 2 days before ﬁxation with 4% paraformaldehyde

and permeabilization with 0.5% Triton X-100 in PBST. Cells were the incubated in

Click-It reaction cocktail for 30 min before washing in PBS and imaging.

MitoTracker staining and ﬂow cytometry analysis. Cells were washed twice with

Ham’s F10 (no serum or pen/strep). Subsequently, MuSCs were stained with

MitoTracker Green FM (ThermoFisher, M7514) and DAPI for 30 min at 37 °C,

washed three times with Ham’s F10, and analyzed using a BD FACSAria III ﬂow

cytometer.

Myogenic colony-forming cell assay for MuSCs. Single treated and control

MuSCs were deposited into wells of collagen- and laminin-coated plates at one cell

per well by BD FACSAria III ﬂow cytometer. Collagen/laminin coating was

accomplished by overnight incubation of the plates rocking at 4 °C with a 1:1

mixture of laminin (10 μg/ml ThermoFisher 23017-015) and collagen (10 μg/ml

Sigma C8919) in PBS. Coated wells were washed three times with PBS before use.

The cells were cultured in grow media, F10 medium supplemented with 20% horse

serum, and 5 ng/ml basic ﬁbroblast growth factor (bFGF; PeproTech 100-18B).

After 6 days of culture, plates were ﬁxed with 4% paraformaldehyde (Electron

Microscopy Services 15710), stained with DAPI (Invitrogen D1306), and scored by

microscopy to determine the number of myogenic colony-forming cells, deﬁned by

wells that contained at least eight cells.

Myogenic/fusion index. Myogenic analysis was completed as previously descri-

bed

. After MuSCs underwent reprogramming or control treatment, cells were

cultured in grow media. To induce differentiation, myoblast cultures were main-

tained in DMEM supplemented with 2% horse serum. The myogenic/fusion index

was determined as the percentage of myonuclei in myotubes (deﬁned as cells with

three or more nuclei) compared with the total number of nuclei in the ﬁeld.

Lentiviral transduction. Luciferase and GFP protein reporters were subcloned into

a third-generation HIV-1 lentiviral vector (CD51X DPS, SystemBio). To transduce

freshly isolated MuSCs, cells were plated on a Poly-D-Lysine (Millipore Sigma, A-

003-E) and ECM coated eight-well chamber slide (Millipore Sigma, PEZGS0896)

and were incubated with 5 μl of concentrated virus per well and 8 μg/mL polybrene

(Santa Cruz Biotechnology, sc-134220). Plates were spun for 5 min at 3200 g, and

for 1 h at 2500 g at 25 °C. Cells were then washed with fresh media two times,

scraped from plates, and resuspended in the ﬁnal volume according to the

experimental conditions.

Bioluminescence imaging. Bioluminescent imaging was performed using the

Xenogen IVIS-Spectrum System (Caliper Life Sciences). Mice were anesthetized

using 2% isoﬂurane at a ﬂow rate of 2.5 l/min Intraperitoneal injection of

D-

Luciferin (50 mg/ml, Biosynth International Inc.) dissolved in sterile PBS was

administered. Immediately following the injection, mice were imaged for 30 s at

maximum sensitivity (f-stop 1) at the highest resolution (small binning). Every

minute, a 30-s exposure was taken until the peak intensity of the bioluminescent

signal began to diminish. Each image was saved for subsequent analysis.

Bioluminescence image analysis. Analysis of each image was performed using

Living Image Software, version 4.0 (Caliper Life Sciences). A manually generated

circle was placed on top of the region of interest and resized to completely sur-

round the limb or the speciﬁed region on the recipient mouse. Similarly, a back-

ground region of interest was placed on a region of a mouse outside the

transplanted leg.

Tissue harvesting. TiA muscles were carefully dissected away from the bone,

weighed, and placed into a 0.5% PFA solution for ﬁxation overnight. The muscles

were then moved to a 20% sucrose solution for 3 h or until muscles reached their

saturation point and began to sink. The tissues were then embedded and frozen in

Optimal Cutting Temperature (OCT) medium and stored at −80 °C until sec-

tioning. Sectioning was performed on a Leica CM3050S cryostat that was set to

generate 10-μm sections. Sections were mounted on Fisherbrand Colorfrost slides.

These slides were stored at −20 °C until immunohistochemistry could be

performed.

Flow cytometry. For mouse MuSC sorting scheme, we followed the same gating

strategy previously published

. For human MuSC sorting scheme, we followed the

same strategy previously published

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Histology. TiA muscles were ﬁxed for 5 h using 0.5% electron-microscopy-grade

paraformaldehyde and subsequently transferred to 20% sucrose overnight. Muscles

were then frozen in OCT, cryosectioned at a thickness of 10 μm, and stained. For

colorimetric staining with Hematoxylin and Eosin (Sigma) or Gomori Trichrome

(Richard-Allan Scientiﬁc), samples were processed according to the manufacturer’s

recommended protocols.

Ex vivo for ce measurement. To measure the force, we isolated the TiA in a bath

of oxygenated Ringer’s solution and stimulated it with plate electrodes. Immedi-

ately after euthanasia, the distal tendon of the TiA, the TiA, and the knee (proximal

tibia, distal femur, patella, and associated soft tissues) were dissected out and placed

in Ringer’s solution (Sigma) maintained at 25 °C with bubbling oxygen with 5%

carbon dioxide. The proximal tibia was sutured to a rigid wire attached to the force

transducer, and the distal tendon was sutured to a rigid ﬁxture. No suture loops or

slack was present in the system. The contralateral limb was immediately dissected

and kept under low passive tension in oxygenated Ringer’s solution bath until

measurement. Supramaximal stimulation voltage was found, and the active force-

length curve was measured in a manner similar to the in vivo condition. After

measurement, the muscle was dissected free and the mass measured. An Aurora

Scientiﬁc 1300-A Whole Mouse Test System was used to gather force

production data.

DNA methylation data. The human Illumina Inﬁnium EPIC 850K chip was

applied to n = 16 DNA samples (corresponding to two treatment levels (before/

after treatment) of four ﬁbroblasts and four endothelial cells). The raw image data

were normalized using the “preprocessQuantile” normalization method imple-

mented in the “minﬁ” R package

39,40

Epigenetic clock analysis. Several DNAm-based biomarkers have been proposed

in the literature, which differ in terms of their applicability (most were developed

from blood), and in terms of their biological interpretation (reviewed in ref.

). We

focused on two epigenetic clocks that apply to ﬁbroblasts and endothelial cells. In

our primarily analysis, we used the pan-tissue epigenetic clock

because it applies

to all sources of DNA (with the exception of sperm). A previously deﬁned

mathematical algorithm is used to combine the methylation levels of 353 CpG into

an age estimate (in units of years), which is referred to as epigenetic age or DNAm

age

. In our secondary analysis, we used the skin-and-blood epigenetic clock (based

on 391 CpGs) because it is known to lead to more accurate DNAm age estimates in

ﬁbroblasts, keratinocytes, buccal cells, blood cells, saliva, and endothelial cells

We used the online version of the epigenetic clock software to arrive at DNA

methylation age estimates from n = 16 samples collected from n = 8 individuals

Although the chronological age range was relatively narrow (ranging from 47 to 69

years, median age = 55), the two DNAm age estimates exhibited moderately high

correlations with chronological age (r = 0.42 and r = 0.63, P = 0.0089 for the pan-

tissue- and the skin-and-blood clock, respectively).

Two samples (before and after rejuvenation treatment) were generated from

each of n = 8 individuals. To properly account for the dependence structure in the

data, we used linear mixed effects models to regress DNAm age (dependent

variable) on treatment status, chronological age, and individual identiﬁer (coded as

random effect). Toward this end, we used the “lmer ” function in the “lmerTest” R

package

Reporting summary. Further information on research design is available in

the Nature Research Reporting Summary linked to this article.

Data availability

The data that support the ﬁndings of this study are available from the corresponding

author upon request. The data used for the methylation clock analysis will be available

through the following GSE number starting April 08 2020: GSE142439. RNASeq data

have been deposited to the Sequence read Archive (SRA), and will be available upon

publication through the following SRA number: PRJNA598923.

Received: 7 August 2019; Accepted: 20 February 2020;

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Acknowledgements

We thank the members of the Sebastiano, Bhutani, and Rando laboratories for comments

and discussions; we thank Jens Durruthy for valuable technical information on mRNA-

based transient reprogramming. We thank Tony Wyss-Coray and his lab for providing

primary ﬁbroblast lines from patients diagnosed with AD, and Helen Blau and her lab for

technical consulting on telomere-length evaluation. This work was supported by the

AFAR Junior Investigator Award to V.S., by grants from the National Institutes of Health

(NIH/NIAMS) (R01 AR070865 and R01 AR070864) to N.B., the Glenn Foundation for

Medical Research and by grants from the National Institutes of Health (NIH) (P01

AG036695, R01 AG23806 (R37 MERIT Award), R01 AG057433, and R01 AG047820),

and the Department of Veterans Affairs (BLR&D and RR&D Merit Reviews) to T.A.R.;

and by the CalPoly funding Award # TB1-01175.

Author contributions

V.S., T.A.R., T.J.S., N.B., and M.Q. conceived, designed, and supervised the experiments

reported. T.J.S. performed the Fibroblast and Endothelial in vitro experiments. T.J.S.

performed the processing for the Fibroblast and Endothelial RNA sequencing experi-

ments. N.B. and C.C. obtained the cartilage surgical specimens and S.M and T.J.S per-

formed the chondrocyte isolation and in vitro experiments. P.P., C.M.T., and A.C.

performed the MuSC isolation, lentivirus infection design, and experiments. T.J.S., P.P.,

C.M.T., and A.C. performed the MuSCs in vitro experiments. M.Q., P.P., A.C., and L.D.

performed the MuSCs in vivo experiments. T.J.S., S.M., N.B., M.Q., C.C., S.H., L.S.Q.,

T.A.R., and V.S. analyzed data and wrote the paper.

Competing interests

The authors declare no competing interests.

Additional information

Supplementary information is available for this paper at https://doi.org/10.1038/s41467-

020-15174-3.

Correspondence and requests for materials should be addressed to M.Q. or V.S.

Peer review information Nature Communications thanks the anonymous reviewer(s) for

their contribution to the peer review of this work. Peer reviewer reports are available.

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Discussion

> To address all these questions, we devised a platform that could let us test whether transient expression of nuclear reprogramming genes has any impact in ameliorating aging phenotypes in naturally aged human and mouse cells across multiple cell types and spanning all the hallmarks of aging. The researches extended their analysis to osteoarthritis - a disease strongly associated with aging and characterized by a pronounced inflammatory spectrum affecting the chondrocytes. Their ***data shows that transient expression of can promote a partial reversal of gene expression and cellular physiology in aged chondrocytes toward a healthier, more youthful state, suggesting a potential new therapeutic strategy to ameliorate the OA disease process.*** > these results suggest that transient reprogramming partially restores the potency of aged MuSCs to a degree similar to that of young MuSCs, without compromising their fate, and thus has potential as a cell therapy in regenerative medicine. ### DNA methylation DNA methylation is a biological process by which methyl groups are added to the DNA molecule. Methylation can change the activity of a DNA segment without changing the sequence. DNA methylation is essential for normal development and is a common epigenetic signaling tool that cells use to lock genes in the "off" position. It is associated with a number of key cellular processes, including embryonic development, genomic imprinting, X-chromosome inactivation, and preservation of chromosome stability. Learn more here: ["The Role of Methylation in Gene Expression"](https://www.nature.com/scitable/topicpage/the-role-of-methylation-in-gene-expression-1070/) > Altogether, the analysis of the transcriptomic signatures revealed that OSKLMN expression promotes a very rapid activation of a more youthful gene expression profile, which is cell-type specific, without affecting the expression of cell identity genes. The method employed was able to generate induced pluripotent stem cells for all donors after 12-15 days of transfections. Researchers observed that the point of no return occurred at day 5 of transfections. The regimen adopted was then: - 4 consecutive days of transfections. - the gene expression was measured 2 days after the interruption of the intervention. ***The rejuvenation effects were significantly retained after 4 and 6 days from the interruption of reprogramming.*** The results ***demonstrate that transient expression can induce a rapid, persistent amelioration, and reversal of cellular age in human somatic cells at the transcriptomic, epigenetic, and cellular levels.*** The data also shows that the process of ***cellular rejuvenation is engaged very early, rapidly, and broadly in the reprogramming process.*** These epigenetic and transcriptional changes occur before any epigenetic reprogramming of cellular identity takes place. - **Chromatin**: Chromatin is a complex of DNA and protein found in eukaryotic cells. Its function is to package long molecules into more compact and denser structures. This structure plays an important role in reinforcing the DNA during cell division, preventing DNA damage, and regulating gene expression and DNA replication. - **Epigenetics**: Epigenetics refers to non-genetic influences on gene expression. It is the study of how your behaviors and environment can cause changes that affect the way your genes work. Unlike genetic changes, epigenetic changes are reversible and do not change your DNA sequence, but they can change how your body reads a DNA sequence. - **Pluripotency**: Pluripotent cells can give rise to all of the cell types that make up the body, ***embryonic stem cells are pluripotent.*** - **Transient Expression**: Transient expression is the temporary expression of genes which happens after a nucleic acid - DNA encoding an expression cassette - has been introduced into eukaryotic cells. - **Chondrocytes**: are the cells responsible for cartilage formation, and they are crucial for the process of endochondral ossification, which is useful for bone development. - **Messenger RNA (mRNA)**: mRNA is a single-stranded RNA molecule that is complementary to one of the DNA strands of a gene. The mRNA is an RNA version of the gene that leaves the cell nucleus and moves to the cytoplasm where proteins are made. - **Transcriptome**: The transcriptome is the set of all RNA transcripts in an individual or a population of cells. - **Fibroblasts**: Fibroblasts are the most common cells of connective tissue in animals. They synthesize the extracellular matrix and collagen and produce the structural framework for animal tissues. - **Endothelial cells**: Endothelial cells form the barrier between vessels and tissues. They control the flow of substances and fluid into and out of a tissue. Endothelial cells line blood vessels and lymphatic vessels, they are found exclusively in vascularized tissue. ### TL;DR ***Slowing or reversing aging is one of the holy grails of science.*** Recent studies have made great strides and presented novel results towards the goal of reversing human cellular aging. This paper was published in 2020 and demonstrates that transient expression can **induce a rapid, persistent amelioration, and reversal of cellular age in human somatic cells.** When applied for a short enough time, the expression of reprogramming factors fails to erase the epigenetic signature defining cell identity which constitutes novel finding in the field. A few bullet points worth reading: - At the chromatin level, aging associates with progressive accumulation of epigenetic errors that eventually lead to aberrant gene regulation, stem cell exhaustion, senescence, and deregulated cell/tissue homeostasis. - Nuclear reprogramming to pluripotency can revert both the age and the identity of any cell to that of an embryonic cell. - Recent evidence shows that transient reprogramming can ameliorate age-associated hallmarks and extend lifespan in progeroid mice. - Epigenetic clocks based on DNA methylation levels are the most accurate molecular biomarkers of age across tissues and cell types and are predictive of a host of age-related conditions including lifespan - According to the pan-tissue epigenetic clock, transient OSKMLN significantly reverted the DNA methylation age. The rejuvenation effect was more pronounced in endothelial cells than in fibroblasts. - Collectively, this data demonstrates that transient expression of OSKMLN can induce a rapid, persistent amelioration, and reversal of cellular age in human somatic cells at the tran- scriptomic, epigenetic, and cellular levels. Importantly, these data demonstrate that the process of cellular rejuvenation is engaged very early, rapidly, and broadly in the reprogramming process. These epigenetic and transcriptional changes occur before any epigenetic reprogramming of cellular identity takes place, a novel finding in the field. - Transient cellular reprogramming can very rapidly reverse a broad spectrum of aging hallmarks in the initiation phase, when epigenetic erasure of cell identity has not yet occurred. We show that the process of rejuvenation occurs in naturally aged human and mouse cells, with restoration of lost functionality in diseased cells and aged stem cells while preserving cellular identity

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