### TL;DR
***Slowing or reversing aging is one of the holy grail...
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- **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
8
, 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
13
,
Nidhi Bhutani
8
, 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
1
Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
2
Department of Obstetrics
and Gynecology, Stanford University School of Medicine, Stanford, CA 94305, USA.
3
Department of Applied Physics, Stanford University School of
Humanities and Sciences, Stanford, CA 94305, USA.
4
Department of Neurology and Neurological Sciences, Stanford University School of Medicine,
Stanford, CA 94305, USA.
5
Paul F. Glenn Center for the Biology of Aging, Stanford University School of Medicine, Stanford, CA 94305, USA.
6
Center for
Tissue Regeneration, Repair and Restoration, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304, USA.
7
Molecular Medicine Research
Institute, Sunnyvale, CA 94085, USA.
8
Department of Orthopedic Surgery, Stanford University School of Medicine, Sanford, CA 94305, USA.
9
Department
of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA.
10
VA Palo Alto Health Care System, Palo Alto, CA 94304, USA.
11
Department of Human Genetics David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA.
12
Department of Biostatistics,
Fielding School of Public Health, UCLA, Los Angeles, CA 90095, USA.
13
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():,;
T
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)
5
, 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
6
. 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)
7
. 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
8
. 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
9
(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
9
as
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
3
. 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
ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15174-3
2 NATURE COMMUNICATIONS | (2020) 11:1545 | https://doi.org/10.1038/s41467-020-15174-3 | www.nature.com/naturecommunications
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
Young, n = 3 Untreated aged, n = 3 Treated aged, n = 3
OSKMNL
(panel b)
OSKMNL
(panel b)
Y vs A signatureY vs A signature
ab
cd
e
fg
h
80
80
60
40
20
70
60
50
40
i
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
**
4
3
2
1
0
–6–4–20246
4
3
2
1
0
–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.
NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15174-3 ARTICLE
NATURE COMMUNICATIONS | (2020) 11:1545 | https://doi.org/10.1038/s41467-020-15174-3 | www.nature.com/naturecommunications 3
physiological aging. We employed a panel of 11 established
assays, spanning the hallmarks of aging
14
(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
40
600
400
200
50
40
30
20
10
0
30
20
10
0
*
*
*
***
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)
Y
oung (E)
Aged (E)
Treated (E)
Young (F)
Aged (F)
T
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)
Y
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.
*
**
n.s.
IL-18
IL-1A
GROA
IL-22IL-8
IL-9
Fluorescence units
Fluorescence units
Fluorescence units
Fluorescence units
Fluorescence units
Fluorescence units
Fluorescence units
Fluorescence units
*
**
*
*
**
*
**
a
b
8000 2000
2000
1000
800
600
400
200
0
1500
1000
500
0
4000
4000
3000
2000
1000
0
3000
2000
1000
0
6000 1500
4000 1000
2000 500
0
100
1000
800
600
400
200
0
1000
65 80
60
40
20
0
60
55
50
45
40
35
800
600
400
200
0
150
100
50
0
90
80
70
60
0
cd
e
f
gh
i
ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15174-3
4 NATURE COMMUNICATIONS | (2020) 11:1545 | https://doi.org/10.1038/s41467-020-15174-3 | www.nature.com/naturecommunications
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
2
; 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
21
.
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
22
. 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
23
. 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
14
. 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.
NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15174-3 ARTICLE
NATURE COMMUNICATIONS | (2020) 11:1545 | https://doi.org/10.1038/s41467-020-15174-3 | www.nature.com/naturecommunications 5
COL2A1
Aged OA
chondrocytes
Untreated
Young
chondrocytes
RANKL INOS2 Secreted cytokines
Cell proliferation
1.3
4
240
6
4
2
0
–2
–4
220
200
180
160
140
3
2
1
0
1.2
1.1
1.0
AT P
ROS SOD2
SOX9
515
10
5
0
–5
–10
0
–5
–10
Y
oung healthy
OA
Treated
Young healthy
OA
T
reated
Young healthy
OA
Treated
Young healthy
OA
Treated
Y
oung healthy
O
A
Treated
Young healthy
OA
Treated
Y
oung healthy
OA
Treated
Young healthy
OA
Treated
Log fold change over OA mean
Log fold change over OA mean
Log fold change over OA mean
Log fold change over OA mean
Log fold change over OA mean
Fluorescence units
Fluorescence units
Fluorescence units
Treated
Untreated
a
10
5
2000
1500
1000
500
60
40
20
0
0
–5
–10
5
0
–5
–10
bc d
efg h
ij
MIP1A IL-6 IFNA MCP3
*
*
*
*
**
*
**
*
***
***
n.s.
***
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.
ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15174-3
6 NATURE COMMUNICATIONS | (2020) 11:1545 | https://doi.org/10.1038/s41467-020-15174-3 | www.nature.com/naturecommunications
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
24
.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
27
(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
33
. 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
2
.
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
mM
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
2
.
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,
NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15174-3 ARTICLE
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
3
10
9
10
8
10
7
14711
2
10e3
9
8
7
6
5
4
Days
Treated
Untreated
20 Mo mouse
Bioluminescence
(photons cm
–2
s
–1
)
Bioluminescence
(photons cm
–2
s
–1
)
Bioluminescence
(photons cm
–2
s
–1
)
Bioluminescence
(ratio treted/untreated)
Tetanic force (mN mm
2
)
GFP regenerated fibers (#)
Fibers (%)
Aged
untreated
Aged
treated
Aged
untreated
Aged
treated
Fiber area (μn
2
)
Aged treated
60
10
8
10
7
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
Days
Days
Young
untretaed
Young
non-
transplanted
10
7
7
6
5
4
3
2
1
0
10
6
1
14711
47911
Aged
non-
transplanted
a
bc d
e
1500
1
0.8
0.6
0.4
0.2
0
100
400
700
1000
1300
1600
1900
2200
2500
2800
3100
3400
3700
4000
1000
500
0
1200
800
400
0
fg
hi j
**
**
*
*
**
**
Muscle
Satellite cells
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
2
.
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
2
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
2
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
2
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
2
.
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
2
. 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.
2
. 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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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
36
. 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
37
. For human MuSC sorting scheme, we followed the
same strategy previously published
38
.
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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.
11
). We
focused on two epigenetic clocks that apply to fibroblasts and endothelial cells. In
our primarily analysis, we used the pan-tissue epigenetic clock
3
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
3
. 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
13
.
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
3
.
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
41
.
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
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> 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