Download Immune dysfunctionality of replicative senescent

REGULAR ARTICLE
Immune dysfunctionality of replicative senescent mesenchymal stromal
cells is corrected by IFNg priming
Raghavan Chinnadurai,1 Devi Rajan,2 Spencer Ng,3 Kenneth McCullough,4 Dalia Arafat,5 Edmund K. Waller,3 Larry J. Anderson,2
Greg Gibson,5 and Jacques Galipeau1
1
Department of Medicine, University of Wisconsin Carbone Comprehensive Cancer Center, University of Wisconsin–Madison, Madison, WI; 2Department of Pediatrics,
Division of Bone Marrow Transplantation, Department of Hematology and Oncology, Winship Cancer Institute, and 4Department of Psychiatry and Behavioral Sciences,
Emory University, Atlanta, GA; and 5School of Biology, Georgia Institute of Technology, Atlanta, GA
3
Key Points
• Replication exhausted
human MSCs display
attenuated immunosuppressive properties
partly because of defective kynurenine
production.
• IFNg prelicensing can
rescue replicative
senescence-associated
immune defects of human bone marrow–
derived MSCs.
Industrial-scale expansion of mesenchymal stromal cells (MSCs) is often used in clinical trials,
and the effect of replicative senescence on MSC functionality is of mechanistic interest.
Senescent MSCs exhibit cell-cycle arrest, cellular hypertrophy, and express the senescent
marker b-galactosidase. Although both fit and senescent MSCs display intact lung-homing
properties in vivo, senescent MSCs acquire a significant defect in inhibiting T-cell
proliferation and cytokine secretion in vitro. IFNg does not upregulate HLA-DR on senescent
MSCs, whereas its silencing did not reverse fit MSCs’ immunosuppressive properties.
Secretome analysis of MSC and activated peripheral blood mononuclear cell coculture
demonstrate that senescent MSCs are significantly defective in up (vascular endothelial
growth factor [VEGF], granulocyte colony-stimulating factor [GCSF], CXCL10, CCL2) or down
(IL-1ra, IFNg, IL-2r, CCL4, tumor necrosis factor-a, IL-5) regulating cytokines/chemokines.
Unlike indoleamine 2,3 dioxygenase (IDO), silencing of CXCL9, CXCL10, CXCL11, GCSF, CCL2,
and exogenous addition of VEGF, fibroblast growth factor-basic do not modulate MSCs’
immunosuppressive properties. Kynurenine levels were downregulated in senescent MSC
cocultures compared with fit MSC counterparts, and exogenous addition of kynurenine
inhibits T-cell proliferation in the presence of senescent MSCs. IFNg prelicensing activated
several immunomodulatory genes including IDO in fit and senescent MSCs at comparable
levels and significantly enhanced senescent MSCs’ immunosuppressive effect on T-cell
proliferation. Our results define immune functional defects acquired by senescent MSCs,
which are reversible by IFNg prelicensing.
Introduction
Bone marrow–derived mesenchymal stromal cells (MSCs) are under clinical investigation to test their
use in adoptive immunosuppressive cell therapy for auto- and allo-immune disorders.1 Although earlyphase clinical trials demonstrated that MSC infusion is safe, variations in efficacy is an ongoing issue.2,3
Defining the functionality of in vitro expanded MSCs will inform not only the surrogate measure of in vivo
potency but also the cause and source of clinical inconsistency arising from infused cell preparations. In
bone marrow, native endogenous MSCs are present at a low frequency (0.01%-0.001% of nucleated
cells) and their robust in vitro proliferative potential under standard cell culture conditions make them
as an attractive candidate for cell therapy studies.4 However, the extent of in vitro culture expansion
of MSCs can vary dramatically between low passage manufacture of MSCs, as is typical of many
Submitted 24 February 2017; accepted 15 March 2017. DOI 10.1182/
bloodadvances.2017006205.
© 2017 by The American Society of Hematology
The full-text version of this article contains a data supplement.
628
25 APRIL 2017 x VOLUME 1, NUMBER 11
C
B
0
Apo
G0/G1
S
G2/M
2.5
2.0
1.5
1.0
DA
Y0
Days in culture
Senescent
E
Fit
Propidium iodide
Senescent
SSC
Fit
DA
Y3
0.5
0 7 14 21 28 35 42 49 56 63
D
Senescent
SSC
1
Fit
3.0
DA
Y2
10
OD 570 (fold change)
Doubling time (days)
10
Fit
Senescent
3.5
102
G0/G1
Replicative Senescence
DA
Y1
A
FSC
CD44
CD105
CD73
CD45
G
Fit
Fold change / GAPDH
4
Fit
Sene
3
2
1
Ly96
PERP
RAMP
P16INK4A
Ly96
P16INK4A
Senescent
0
PERP
CD90
RAMP
F
Beta Galactosidase
Figure 1. Phenotypical characteristics of replicative senescent MSCs. (A) MSCs derived from the bone marrow of 2 healthy individuals were culture-passaged at the
indicated time points. Doubling time was calculated based on the cell numbers at seeding and harvesting time points and duration of the culture. (B) MSCs from replicative fit and
senescent phases were seeded at similar density in the 96-well plates. MTT assay was performed at the indicated time points to determine the growths of MSCs. Results are plotted
as mean 6 standard deviation (SD). (C) Cell-cycle analysis was performed on fit and senescent MSCs through propidium iodide staining and subjected to flow cytometry. (D) Size and
granularity of fit and senescent MSCs were determined by forward and side scatter analysis by flow cytometry. (E) Fit and senescent MSCs were subjected to senescence-associated
lysosomal b-galactosidase staining. Dark gray staining represents b-galactosidase staining. Scale bars represent 400 mm. (F) Replicative fit and senescent MSCs were subjected to staining
for MSC markers as defined by the ISCT and acquired through flow cytometry. Open and gray histograms represent marker and isotype controls, respectively. Similar results were obtained in
a repeat experiment with an additional 1 or 2 fit and senescent MSC donor pairs. (G) Fold difference in the P16INK4A, PERP, LAMP, and LY96 mRNA of fit and senescent MSCs, relative to
glyceraldehyde 3-phosphate dehydrogenase (GAPDH), were determined in quantitative sybr green real-time PCR. Cumulative is shown from 2 independent donors.
academic-sponsored trials relative to large-scale expansion of
MSC-like cells typical of industry-sponsored studies. Considering
that an average cell dose for most clinical studies is ;1 to 2 million
MSCs per kilogram of body weight, most academic cell processing
centers will typically manufacture 5 to 10 doses from an allogeneic
donor, whereas industrial-scale expansion of MSC-like cells can
be as high as 10 000 to 1 000 000 doses per donor. Considering
that advanced-phase clinical trials using industrial-scale expanded
MSCs have not met primary efficacy end points, it is worthwhile,
considering the possibility that prolonged culture expansion and
companion replicative senescence may affect the potency of
25 APRIL 2017 x VOLUME 1, NUMBER 11
MSC-like cells when compared with low-passage products. In
support of this hypothesis, it was observed retrospectively that
late passage MSCs were clinically shown to be less effective in
ameliorating graft-versus-host disease than early passage cells.3,5
Prolonged culture expansion leads to the replicative exhaustion/
senescence of MSCs like any other primary somatic cells in culture
as defined by the “Hayflick limit.”6 Cellular senescence is an antitumorigenesis process that replicating cells use in conjunction with
cell death programs to prevent malignant transformation. In vitro expanded human MSCs neither exhibit chromosomal aberrations nor
undergo tumorigenic transformation while they acquire replicative
SENESCENT MSCS DISPLAY IMPAIRED IMMUNE SUPPRESSION
629
B
A
No MSC
77.2
29.8
71.2
P<0.05
100
Senescent
Ki67
10.1
FIT
80
% CD3+ Ki67+
NS
60
40
20
0
No MSC
CD3
Fit
Senescent
+ MSC
C
D
No MSC
0.0287
4.73
Senescent
32.7
IFNγ
22.9
FIT
Senescent
Fit
40
%CD3+CFSEdim IFNγ+
NS
30
20
10
0
0
CFSE
1:40 1:20 1:10
1:5
MSC:PBMC ratio
FIT
No MSC
32.8
82.8
Ki67
94.3
Senescent
Fit
+Senescent
21
F
100
80
% CD3+Ki67+
E
60
40
20
Sene
Fit+Sene
P<0.05
G
Fit
No MSCs
0
CD3
+ MSCs
P<0.05
NS
Human gDNA CT-1 values
0.040
0.038
0.036
0.034
0.032
0.030
None
Fit
Senescent
Figure 2. Replicative senescent MSCs display attenuated immunosuppressive and intact lung-homing properties. Replicative fit and senescent MSCs were cultured
with aCD3aCD28 Dynabeads-stimulated PBMCs. Four days post culture, T-cell proliferation was measured by Ki67 intracellular staining. (A) Representative fluorescence-activated
cell sorting (FACS) plot and (B) cumulative effect of fit and senescent MSCs’ (n 5 5 donor pairs) effect on T-cell proliferation (% CD31Ki671) is shown. CFSE-labeled PBMCs were
cocultured with replicative fit and senescent MSCs and were stimulated with aCD3aCD28 Dynabeads. On the fourth day, intracellular cytokine staining was performed to determine
the percentage of cytokine-secreting proliferated T cells CD31CFSEdim IFNg T cells. (C) Representative FACS plot and (D) dose-dependent effect are shown. Individual or mixed fit
and senescent MSCs at 1:1 ratio were cultured with aCD3aCD28 Dynabeads-stimulated PBMCs and 4 days post culture, T-cell proliferation was measured as indicated above. (E)
Representative FACS plot and (F) cumulative effect are shown. Similar results were obtained in a repeat experiment with an additional 1 or 2 fit and senescent MSC donor pairs. (G)
630
CHINNADURAI et al
25 APRIL 2017 x VOLUME 1, NUMBER 11
senescence as part of extensive culture expansion procedures.7-11
Replicative senescence on MSCs has been shown to be associated
with reduced telomere length, increased P16INK4A expression, alterations in differentiation potential, global gene expression profile,
microRNA expression, DNA methylation pattern, and reactive oxygen
species accumulation.12-18 Thus, replicative senescence induces
alterations in MSC functionality, but the effect of these alterations on
MSCs’ interaction with immune responders and responsiveness to
inflammatory cues is largely unknown. An important unanswered
question is: Are MSCs’ interactions with immune responders different based on their replicative history and relative immunologic
fitness? In the present study, we have characterized early- and latepassage MSCs for their comparative and distinct immunomodulatory properties and suppressive mechanisms through phenotype,
transcriptome, and immune functional analysis. In addition, to improve MSCs’ function, we investigated whether cytokine prelicensing
rescues senescence-induced defects in MSC immunosuppressive
functions.
appropriate isotype controls (BD Biosciences, San Jose, CA). Mean
fluorescent intensity and histogram analysis for the marker expression
was performed with Flow Jo software.
MSC and T-cell coculture
Coculture of MSCs and T cells has been described previously.22
Briefly, 6 IFNg-activated, fit (low passage), or senescent (high
passage) MSCs were seeded onto 96-well plates and cocultured with
carboxyfluorescein succinimidyl ester (CFSE)labeled or –nonlabeled
random donor human PBMCs with indicated ratio. Dynabeads Human
T-Activator CD3/CD28 (Life Technologies AS, Norway) was used to
stimulate the T cells. For intracellular cytokine staining, Brefeldin A was
added at the concentration of 10 mg/mL (Sigma-Aldrich, St. Louis,
MO) for 12 to 14 hours, and intracellular flow cytometry staining was
performed with BD Cytofix and Cytoperm procedure with the antibodies CD3APCCy7 and IFNgAPC. Ki67 proliferation assay was
performed after 4 days according to manufacturer instructions (BD
Biosciences).
Tracking of human MSCs in mice
Materials and methods
Bone marrow MSC isolation and passage
MSCs were obtained from the bone marrow of consenting healthy
individuals compliant with Emory University Institutional Review
Board guidelines. Harvested bone marrow was separated by Ficoll
density gradient and plated on Minimum Essential Medium Eagle a
modification (a-MEM) containing 15% to 20% fetal calf serum and
100 U/mL penicillin/streptomycin (200 000 cell/cm2). Three days
post–flask seeding, nonadherent cells were removed and adhered
MSCs were allowed to expand for an additional 7 days (passage 0).
Subsequently, MSCs were passaged weekly and replated at a
seeding density of 1000 cells/cm2. During early passage, cells were
split in 80% confluence or at every seventh day. Doubling time was
calculated using a formula as described previously.19 Fit MSCs are
derived from passage 2 to 4 (doubling time 1.6 6 0.2 days) and
senescent MSCs are derived from passages 10 to 15 (doubling
time 10.9 6 3.1 days).
Tracking of human MSCs in C57BL/B6 mice was performed as
described previously.23 Fit or senescent MSCs (0.5 3 106 cells/
mouse) were infused in to C57BL/B6 mice (8-10 weeks old; The
Jackson Laboratories) through tail vein. Twenty-four hours post infusion,
the mice were killed and the lungs were collected and total genomic
DNA was extracted using a QIAamp DNA Mini Kit (QIAGEN). 100 ng
of DNA were used for real-time polymerase chain reaction (PCR) in
Applied Biosystems 7500 fast real-time PCR using RT2 SYBR Green
ROX qPCR master mix and mouse RT2 qPCR (QIAGEN) and human
gDNA primers (SA Biosciences).
siRNA knockdown on human MSCs
Fit or senescent MSCs were seeded on to 96-well plates at a
density of 5000 cells/cm2. Cells were cultured with either medium
containing human platelet lysate or fetal calf serum. MTT assays
were performed as described previously.20 Senescence-associated
b-galactosidase activity was determined using X-gal staining as
described previously.21 Cell-cycle analysis was performed in flow
cytometer using standard propidium iodide staining in ice-cold
methanol-fixed fit and senescent MSCs.
MSCs were seeded in 96-well plates at a concentration of 5000
cells per well one day before transfection with nontargeting
control siRNA or HLA-DR, CXCL9, CXCL10, CXCL11, granulocyte colony-stimulating factor (GCSF), CCL2, kynureninase
(KYN), kynurenine 3-monooxygenase (KMO), IDO SMART Pool
siRNA (Dharmacon, Lafayette, CO). During transfection, the cells
were conditioned with serum-free 10 mM (4-(2-hydroxyethyl)-1piperazineethanesulfonic acid) (HEPES) containing a-MEM for
30 minutes. 2 mL of 100 uM–specific/control siRNA solution (A) or
3 mL Dharmafect 1 reagent (B) was added to 250 mL a-MEM–
containing 10 mM HEPES. A and B were mixed and incubated at
room temperature for 30 minutes. 50 ml of the siRNA transfection
cocktail was added to each well. The cells were then incubated
for 5 hours and the transfection medium was replaced with
MSC culture medium. After 12 hours, MSCs were cultured with
Dynabead-activated PBMCs on the next day of transfection.
Phenotyping of MSCs by flow cytometry
Secretome analysis
Fit or Senescent MSCs (6 IFN-g activated) were subjected to flow
cytometry analysis for the expression of HLA-ABC APC, HLADR
PerCP, CD80 PE, CD86 PE, B7H1 PE, B7DC PE, CD105 PE,
CD44 PE, CD73 PE, CD90 APC CD45 PE, CD119 PE, and
Supernatants were collected from fit and senescent MSCs cocultured with activated PBMCs for 4 days and stored at 280 °C. Thawed
supernatants were centrifuged at 1500 rpm for 3 minutes to eliminate
cell debris and analyzed by magnetic bead–based multiplex luminex
Assays to determine replicative senescence
Figure 2. (continued) Fit or senescent MSCs (0.5 3 106/animal) were injected IV into C57BL/B6 mice via the tail vein. At 24 hours post-infusion, the animals were killed and the lungs
were excised to extract total gDNA for real-time PCR amplification of human gDNA and mouse gDNA. Human gDNA threshold cycle (CT) values were normalized with mouse gDNA
values. Cumulative inverse CT values with mean 6 SD were shown from 2 independent experiments (n 5 6 animals per group), performed with 2 unique fit and senescent MSC
donors pairs. P , .05 was considered statistically significant based on 2-tailed Student t tests.
25 APRIL 2017 x VOLUME 1, NUMBER 11
SENESCENT MSCS DISPLAY IMPAIRED IMMUNE SUPPRESSION
631
A
B
Fit
Fit
Senescent
Senescent
IFNγ (ng/ml)
5
0.5
0.05
0
CD119
pSTAT1
D
Fit
100
E
Senescent
104
HLA-DR mRNA
fold change / GAPDH
10
1
Fit
Senescent
103
102
101
100
10-1
+IFNγ
0
0.5
G
B7-1
NS
Isotype
Cont siRNA
5000
4000
3000
2000
1000
0
No Stim
HLADR siRNA
HLADR
Isotype
H
Control
MSC
PDL1
No MSC
HLADR KO
MSC
46.1
57.3
PDL2
Ki67
91.4
CD3
I
J
60
40
20
0
60
40
20
N
A
R
D
LA
H
C
siR
N
SC
iR
M
ts
on
o
A
0
C
N
80
N
o
M
on S C
ts
H
iR
LA
N
D
A
R
siR
N
A
+ IFNγ
100
%CD3+CD8+ Ki67+
Isotype Control
No IFNγ
%CD3+CD4+ Ki67+
100
80
Figure 3.
632
CHINNADURAI et al
50
+IFNγ
Mean fluorescence intensity
F
B7-2
5
IFNγ (ng/ml)
25 APRIL 2017 x VOLUME 1, NUMBER 11
HLADR siRNA
-IFNγ
+IFNγ
HLA DR
-IFNγ
0.1
Cont siRNA
HLADR MFI (fold change)
Senescent
HLA ABC
Fit
HLADR siRNA
C
Cont siRNA
Isotype
HLADR
assays for cytokines, chemokines, and certain growth factors (supplemental Figures 1 and 3) (human cytokine 30-plex panel, Life Technologies) according to the manufacturer’s instructions using Luminex
xMAP (multi-analyte profiling) technology. Kynurenine enzyme-linked
immunosorbent assay was performed as described in the manufacturer’s instructions (US Biological Sciences).
Fluidigm nanoscale 48*48 PCR array
Quantitative reverse transcription (RT)-PCR was performed using
Fluidigm 48 3 48 nanofluidic arrays.20 Briefly, 6 IFNg (20 ng/mL)stimulated cDNA samples from fit and senescent MSCs were preamplified with 14-cycle PCR reaction for each sample with the
combination of 100 ng cDNA with pooled primers as described
by TaqMan Pre-Amp Mastermix (Fluidigm BioMark) manufacturer
protocols. Two-thousand three-hundred-four parallel quantitative RTPCR reactions were performed for each primer pair on each sample
on a 48 3 48 array. Amplification was detected in Eva Green
detection assay on a Biomark I machine based on standard Fluidigm
protocols. PCR data were normalized and analyzed with SAS/JMP
Genomics software.
IDO assays
Fit or senescent MSCs (6 IFNg-activated) were lysed and total
RNA was extracted using an RNeasy plus mini kit (QIAGEN).
Normalized RNA was used to convert cDNA using Quantitect
reverse transcription kit (QIAGEN). Sybr green (Perfecta Sybr
green fast mix, Quanta Biosciences) real-time PCR was performed
with IDO primer pairs as described previously.22 IDO protein were
detected using primary rabbit anti-human IDO1 (1:1,000; EMD
Millipore Corporation, Billerica, MA) or rabbit anti-human b-actin
(1:1000; Cell Signaling Technology, Inc, Danvers, MA), and secondary horseradish peroxide–coupled goat anti-rabbit IgG h1l
(1:10 000; Bethyl Laboratories, Inc, Montgomery, TX). ECL detection system (Amersham Pharmacia Biotech, Piscataway, NJ)
was used to detect immunoreactive blots. IDO activity was blocked
using 1-methyl-DL-tryptophan (1 mM concentration) (Sigma-Aldrich)
in MSC and T-cell coculture.
Statistical analysis
Data were analyzed with the GraphPad Prism 5.0 software. For
the comparison of 2 groups, paired Student t test was applied. A
2-sided P value , .05 was considered statistically significant.
Results
Characteristics of senescent human MSCs
We passaged MSCs (1000 cells/cm2) derived from the bone marrow
of healthy donors under standard cell culture conditions using fetal
calf serum as a growth supplement. We observed that prolonged
culture expansion increases the doubling time and induces replicative
senescence of human MSCs (Figure 1A). Short-term growth kinetics
using MTT assay demonstrated that senescent MSCs do not replicate
efficiently compared with Fit MSCs (Figure 1B). Cell cycle analysis
using propidium iodide staining demonstrated that senescent MSCs
are in G0/G1 cycle (Figure 1C). Forward and side scatter analysis
by flow cytometry demonstrated that senescent MSCs acquire a
hypertrophic phenotype as evidenced by increase in cell size and
granularity (Figure 1D). Detection of X-gal staining at pH 6.0 is specific
for endogenous b-galactosidase activity within acidic lysosomal compartment and is considered as senescence-associated b-galactosidase
activity.21,24 Our results show that senescent-associated b-galactosidase
activity in replication exhausted MSCs (Figure 1E). The International
Society for Cell Therapy (ISCT) has defined consensus minimal
criteria for MSCs25 and found that there is no distinction in the
phenotypical markers (CD901CD441CD1051CD731CD45–) expressed by fit and senescent MSCs (Figure 1F). Senescent MSCs
have been shown to express higher level of mRNA for genes such
as P16INK4A, PERP, RAMP, and LY96,13,17 and we found that
P16INK4A and PERP mRNA were upregulated (;twofold) by senescent MSCs (Figure 1G).
Replicative senescent MSCs exhibit attenuated T-cell
immunosuppressive properties in vitro and maintain
intact lung-homing properties in vivo
We analyzed the veto effect of fit and senescent MSCs on activated
T-cell proliferation in vitro. Our results demonstrate that fit MSC
populations inhibit CD31Ki671 T-cell proliferation more efficiently
than senescent counterparts (%CD31Ki671:No MSC [70 6 11],
fit MSC [29 6 11], senescent MSC [60 6 17]) (Figure 2A-B). Next
we investigated the effect of fit and senescent MSCs on cytokine
production by proliferating (CFSE-diluted) T cells. We found that
senescent MSCs fail to inhibit IFNg production by CFSE-diluted
T-cell populations, whereas fit MSCs exhibit inhibition (Figure 2C-D).
To determine whether senescent MSCs added to fit MSC in a
heterogeneous cell culture system lead to a dominant-negative
effect on T-cell suppression, we mixed fit and senescent MSCs in a
1:1 ratio and investigated the immunosuppressive effect of this
Figure 3. Senescence-associated defective HLA-DR upregulation does not modulate immunosuppressive properties of MSCs. (A) Fit and senescent MSCs were
subjected to staining of the receptor for IFNg (CD119) and acquired through flow cytometry. (B) Fit and senescent MSCs stimulated with indicated concentrations of IFNg for
15 minutes. P-STAT1 (Y701) Phosflow was performed subsequently and acquired through flow cytometry. Similar results were obtained in a repeat experiment with an additional
1 or 2 fit and senescent MSC donor pairs. (C) Fit and senescent MSCs were stimulated with IFNg for 48 hours and subsequently stained with the antibodies to the surface markers
for HLA-ABC, HLA-DR, B7-1, B7-2, B7H1, and B7DC for flow cytometry. (D) Cumulative HLA-DR MFI fold change derived from 2 independent fit and senescent MSC donor
pairs were shown. (E) Fit and senescent MSCs were stimulated with IFNg for 48 hours. Expression level of HLA-DR mRNA relative to GAPDH was evaluated by the quantitative
SYBR green real-time PCR. d-d CT method was applied to calculate the fold induction of HLA-DR over the unstimulated control. Control or HLA-DR siRNA-transfected MSCs
were stimulated with IFNg for 48 hours. Cells were trypsinized, stained for HLA-DR or appropriate isotype control antibodies, and analyzed by flow cytometry. (F) Representative
histogram. (G) Cumulative mean fluorescent intensity of HLA-DR and isotype control antibody stains were shown on 2 independent fit and senescent MSC donor pairs. HLA-DR or
control siRNA-transfected MSCs were cultured with aCD3aCD28 Dynabeads-stimulated PBMCs. Four days post culture, T-cell proliferation was measured by Ki67 intracellular
staining. (H) Representative FACS plot and effect of MSCs on (I) CD31CD41 and (J) CD31CD81 T-cell proliferation are shown. Similar results were obtained in a repeat
experiment with another MSC donor.
25 APRIL 2017 x VOLUME 1, NUMBER 11
SENESCENT MSCS DISPLAY IMPAIRED IMMUNE SUPPRESSION
633
Coculture
C
B
Transwell
Activated PBMCs
40
20
10
5
0
20000
10000
15000
10000
5000
0
N
F
S
N
F
o
1500
10000
N
150000
20000
10000
50
1.0
0.5
N
IDO
1.0
0.5
1.0
0.5
0.0
on
t
0.0
G
C
C
FGF-Basic
100
80
60
40
20
0
1000
200
40
ng/ml
VEGF
100
80
60
40
20
0
% CD3+ Ki67+
% CD3+ Ki67+
H
0
0 1:8 1:4 1:2
0 1:8 1:4 1:2
MSC:PBMC ratio
MSC:PBMC ratio
Figure 4.
CHINNADURAI et al
F
1.5
ID
L1
XC
S
CCL2
CCL2 mRNA
GCSF mRNA
on
t
C
KO
L1
0
C
F
1.5
0.0
XC
KO
0.5
C
C
XC
L9
C
C oM
C ont SC
X
s
C CL iRN
XC 9
s A
C L1 iRN
XC 0
A
L1 siR
G 1 s NA
C iR
S
C F s NA
C iR
L2 N
A
ID SiR
O NA
siR
N
A
70
60
50
40
30
20
10
0
N
GCSF
0.0
on
t
0.0
on
t
0.0
0.5
1.0
100
0
N
1.5
KO
0.5
1.0
S
CXCL11
1.5
CXCL11 mRNA
CXCL10 mRNA
1.0
F
IDO mRNA
CXCL10
1.5
150
0
N
t
S
S
P<0.05
C
F
KO
CXCL9
1.5
50000
SF
N
1
F
100000
0
S
F
E
FGF basic (pg/ml)
30000
N
L2
5000
S
200
on
10000
200000
0
F
F
P<0.05
40000
CCL2 (pg/ml)
15000
0
N
C
200
0
S
t
400
CXCL10 (pg/ml)
GCSF (pg/ml)
600
N
F
P<0.05
20000
200
0
S
P<0.05
800
400
C
P<0.05
F
C
N
on
D
S
C
F
500
0
0
N
1000
O
20000
KO
30000
600
IL10 (pg/ml)
2000
KO
1000
P<0.05
P<0.05
40000
IL-5 (pg/ml)
TNF (pg/ml)
1500
0
634
S
N
IL-1ra(pg/ml)
2000
500
VEGF (pg/ml)
20000
M
Se
ne
Fi
t
ne
Fi
t
Se
SC
M
o
N
1000
CXCL9 mRNA
30000
P<0.05
2500
2000
% CD3+ Ki67+
25000
0
P<0.05
P<0.05
G
40000
0
3000
IL2R (pg/ml)
IFN (pg/ml)
IL-10 mRNA
fold induction in M
% CD3+ Ki67+
60
P<0.05
P<0.05
15
80
CCL4 (pg/ml)
100
SC
Fi
t
F 1
Se it 2
n
Se e 1
ne
2
A
25 APRIL 2017 x VOLUME 1, NUMBER 11
S
mixed population. We observed that mixture of fit and senescent
MSCs (akin to what could be found as part of clinical cell
preparations) inhibits T cells as well as fit MSCs alone, suggesting
that senescent MSCs do not lead to a dominant-negative effect on
fit cells (Figure 2E-F) in inhibiting T-cell proliferation, suggesting a
loss-of-function defect in senescent MSCs. Our published results
demonstrate that cell preparation and handling methods such
as freeze-thawing compromise human MSCs’ lung-binding properties in vivo.23,26 Here we have investigated the effect of replicative senescence on human MSCs’ in vivo lung tropic properties
following tail vein injection in C57BL/B6 mice. Our cumulative
results of 2 independent experiments using quantitative human
genomic DNA PCR demonstrated that both fit and senescent
MSCs exhibit comparable short-term lung-homing properties
(Figure 2G).
Senescence-associated defect in HLA-DR
upregulation is dispensable for MSCs’
immunosuppressive properties
The immunosuppressive potential of MSCs upon transfusion in vivo
is dependent on its response to IFNg.27 We have compared fit
and senescent MSCs for IFNg-mediated activation of upstream
signaling cascade and downstream key effector molecule expression. Both fit and senescent MSCs express the IFNg receptor 1
(CD119) on their surface (Figure 3A). IFNg activates the JAK-STAT
signaling pathway in MSCs,28 and Phosflow analysis demonstrated
that short-term IFNg activation phosphorylates STAT1 in both fit
and senescent MSCs at similar levels (Figure 3B). IFNg upregulates
HLA-ABC, B7H1 (PDL1), and B7DC (PDL2) molecules on the
surface of both fit and senescent MSCs (Figure 3C). Neither
costimulatory molecules B7-1 (CD80) nor B7-2 (CD86) were
upregulated by IFNg on fit and senescent MSCs (Figure 3C).
However, senescent MSCs do not upregulate HLA-DR upon
stimulation with IFNg in contrast to what occurs in fit MSCs
(Figure 3C-D). To determine whether the blunted upregulation of
HLA-DR on the surface of senescent MSCs is caused by cytosolic
retention or at the transcriptional level, we have performed real-time
PCR analysis for the transcripts of HLA-DR. Our results demonstrate that HLA-DR mRNA was not efficiently unregulated by IFNg
(Figure 3E). To further investigate the functional role of HLA-DR
in maintaining the veto properties of MSCs, we used a siRNA
knockdown approach (Figure 3F-G). Our results demonstrate that,
compared with control siRNA, HLA-DR siRNA–transfected MSCs
are unaffected in regard to their inhibitory effects on CD31CD41
and CD31CD81 T-cell proliferation (Figure 3H-J).
The secretome arising from interaction between
senescent MSCs and immune responders is distinct
from that of fit MSCs
To compare contact- and noncontact-dependent senescent MSCs’
interaction with immune responders, we have cocultured fit or
senescent MSCs with activated PBMCs in a 2-chamber transwell
assay system. Our results demonstrate that senescent MSCs fail to
inhibit T-cell proliferation in the transwell system, suggesting that
senescent MSCs are defective in secreting immunosuppressive
factors (Figure 4A). To define the role of senescent MSCs in
deriving suppressive macrophages, we have investigated IL-10
mRNA expression in macrophages cocultured with fit or senescent
MSC cultures with PBMCs. Our results demonstrate that both fit
and senescent MSCs upregulate IL-10 mRNA in macrophages
(Figure 4B). Although prior studies have investigated senescent
MSCs’ secretome in resting status,29 their interaction with PBMCs
is largely unknown, and we have further analyzed the supernatant of
fit and senescent MSCs cocultured with activated PBMCs using
multiplex Luminex assay with a 30-plex-cytokine profile. Our results
identified 3 different patterns of cytokine profile following fit or
senescent MSC cocultured with PBMCs. First, fit MSCs are more
potent than senescent MSCs at quenching cytokine production by
PBMCs. IL-1ra, IFNg, IL-2R, CCL4, tumor necrosis factor-a (TNFa), IL-5, and IL-10 were all substantially downregulated in activated
PBMC cocultures with fit MSCs, whereas senescent counter led to
a statistically significant attenuated effect (Figure 4C). Second,
MSC-sourced vascular endothelial growth factor (VEGF), GCSF,
CXCL10, and CCL2 were significantly upregulated in activated
PBMC cultures with fit MSCs, whereas senescent MSCs displayed
a significant defect in upregulation of these factors (Figure 4D)
Finally, MSC-sourced fibroblast growth factor basic secretion
(FGF-basic) is modestly increased in activated PBMC cultures
Figure 4. Differential secretome of activated PBMCs cocultured with fit and senescent MSCs. Replicative fit and senescent MSCs were cultured in the presence and
absence of aCD3aCD28 Dynabeads-stimulated PBMCs in 2-chamber transwell plates. Four days post culture, T-cell proliferation was measured by Ki67 intracellular staining. (A)
Relative effect of fit and senescent MSCs’ effect on T-cell proliferation (% CD31Ki671) in coculture and transwell culture are shown. Similar results were obtained in a repeat
experiment with an additional 2 fit and senescent MSC donor pairs. (B) CD141 purified macrophages were cultured on transwell and in the bottom PBMCs were activated with
2 independent fit or senescent MSC pairs. 48 hours later, macrophages in the transwell were harvested and IL-10 mRNA was measured with GAPDH mRNA as endogenous
housekeeping control. Fold induction of IL-10 mRNA was derived through d-d CT method. Replicative fit and senescent MSCs were cultured in the presence and absence of
aCD3aCD28 Dynabeads-stimulated PBMCs. Four days post culture, supernatant was collected and human cytokine 30-plex panel Luminex assays were performed according to
the manufacturer’s instructions. Fit and senescent MSC pairs from 4 independent donors were tested against different PBMC donors. Cytokine levels were shown in pg/mL. F, fit
MSCs and activated PBMCs; N, no MSCs and activated PBMCs; S, senescent MSCs and activated PBMCs. (C) IL-1ra, IFNg, IL-2r, CCL4, TNFa, IL-5, and IL-10 were decreased
efficiently by fit but not senescent MSCs. (D) VEGF, GCSF, CXCL10, and CCL2 were upregulated by fit MSCs cocultured with activated PBMCs and the senescent MSCs display
attenuated upregulation. (E) FGF-basic was substantially increased by senescent MSCs compared with fit MSCs. (F) Control, CXCL9, CXCL10, CXCL11, GCSF, CCL2, and
IDO siRNA-transfected MSCs were cultured with aCD3aCD28 Dynabeads-stimulated PBMCs in 2-chamber transwell plates for 3 to 4 days. Expression level of appropriate
silenced mRNA relative to GAPDH was evaluated by quantitative SYBR green real-time PCR. d-d CT method was applied to calculate the fold induction of silenced gene over the
control siRNA-transfected MSCs. (G) CXCL9, CXCL10, CXCL11, GCSF, CCL2, IDO, and control siRNA-transfected MSCs were cultured with aCD3aCD28 Dynabeadsstimulated PBMCs. Four days post culture, T-cell proliferation was measured by Ki67 intracellular staining. Proliferation of T cells (CD31Ki671) in the presence and absence of
siRNA transfected MSCs is shown. Similar results were obtained in a repeat experiment with an additional independent MSC donor. (H) VEGF and FGF were added exogenously to
fit MSCs cocultured with activated PBMCs at indicated ratios. Four days later, CD31 T-cell proliferation was measured by Ki67 intracellular staining. Similar results were obtained in
a repeat experiment with an additional independent MSC donor.
25 APRIL 2017 x VOLUME 1, NUMBER 11
SENESCENT MSCS DISPLAY IMPAIRED IMMUNE SUPPRESSION
635
A
B
C
P<0.05
Tryptophan
Kynurenine
HAAO
QPRT
KMO
Sene
3’-Hydroxy
Anthranilic acid
KAT3
KAT4
GAPDH
HAAO
+PBMCS
KYN
Quinolinic acid
IDO
F it
QPRT
Se
ne+
F it+
PB
NAD+
MC
S
Fit
KYN
PB
0
KMO
Kynurenic acid
Anthranilic acid
2
KAT2
3’-Hydroxy
Kynurenine
ne
4
Se
KYN
KAT1, KAT2
KAT3, KAT4
MC
S
6
19.338
21.422
23.505
25.589
27.673
29.757
31.806
33.854
35.903
37.951
40
KAT1
IDO
CT Values
Kynurenine (ng/ml)
8
D
F+P= Fit+PBMCs
S+P= Sene+PBMCs
KAT1
KAT2
KAT3
KAT4
KYN
QPRT
IDO
KMO
HAAO
F+P S+P
F+P S+P
F+P S+P
F+P S+P
F+P S+P
F+P S+P
F+P S+P
F+P S+P
F+P S+P
CT values
40
30
20
10
E
F
KMO
KYN
% CD3+ Ki67+
0.5
40
20
SC
o
M
SC
N
KO
C
C M
on S
ts C
KY iRN
N
A
KM siR
N
O
A
s
ID iRN
O
A
siR
N
A
0
on
tM
M
SC
SC
KO
KY
N
on
t
C
1.0
0.0
M
M
SC
M
KO
on
t
KM
O
C
0.5
0.0
SC
0.0
1.0
60
ID
O
0.5
IDO mRNA
KYN mRNA
1.0
80
1.5
1.5
1.5
Fold reduction
over control
KMO mRNA
IDO
G
Donor 2
80
100
60
40
20
N
F
Donor 3
S
80
60
40
20
- + - + - +
N
F
Donor 4
S
80
60
40
F
S
80
N= No MSC
60
F= Fit MSC
40
S=Senescent
MSC
0
0
- + - + - +
100
N
20
20
0
0
100
% CD3+ Ki67+
S
% CD3+ Ki67+
% CD3+ Ki67+
100
F
% CD3+ Ki67+
Donor 1
N
- + - + - +
- + - + - +
Kynurenine
Figure 5.
636
CHINNADURAI et al
25 APRIL 2017 x VOLUME 1, NUMBER 11
with fit MSCs, whereas this effect is substantially and significantly
upregulated by senescent MSCs (Figure 4E). We did not see a
significant difference in other cytokines (IL-7, IL-13, IL-17, CXCL9,
CCL3, IL-15, IL-6, IL-1b, RANTES, eotaxin, IL-12, IL-8, IL-4,
granulocyte macrophage-CSF, IFNa, human growth factor) between cultures containing fit vs senescent MSCs (supplemental
Figure 1). To examine the role of CXCL9, CXCL10, CXCL11,
GCSF, CCL2, and IDO in MSCs’ immunosuppressive properties,
we used an siRNA knockdown approach (Figure 4F). Our results
demonstrate that, unlike IDO, knocking down of other cytokines
individually does not reverse fit MSCs’ inhibitory effect on T cells
(Figure 4G). In addition, the exogenous addition of VEGF and
FGF-basic do not inhibit fit MSCs’ inhibitory potential on T cells
(Figure 4H), suggesting that upregulation of these factors was a
bystander effect of MSC/PBMC interaction, which does not directly
affect T-cell proliferation.
Senescent MSCs display a defect in
kynurenine production
To test the cell biochemical activity of IDO in MSCs, we measured
the levels of kynurenine accumulation in the culture supernatant of
fit and senescent MSCs cocultured with activated PBMCs. Our
results demonstrated that kynurenine levels were downregulated
in senescent MSC cultures with PBMCs compared with fit MSC
counterparts (Figure 5A). To further decipher the role of catabolic
pathway of tryptophan in senescent MSCs, we investigated the
levels of RNA encoding for enzymes downstream of tryptophan
catabolism in fit and senescent MSCs cocultured with activated
PBMCs30 (Figure 5B). Our results demonstrated that KYN, a
major enzyme in kynurenine degradation pathway, is substantially
upregulated on MSCs upon culture with PBMCs and clustered
with IDO in grouping analysis (Figure 5C; supplemental Figure 2).
However, KYN and the RNA of other enzymes involved in kynurenine
degradation pathway are not differentially expressed in senescent MSCs compared with fit MSCs (Figure 5D). To define the
role of KYN in MSCs’ immunobiology, we performed an siRNA
knockdown approach (Figure 5E). We also silenced kynurenine
3-monooxygenase (KMO), which is an additional primary kynureninemetabolizing enzyme, along with IDO as controls (Figure 5E). Our
results demonstrated that, in contrast to IDO, both KYN and
KMO–silenced MSCs inhibit T-cell proliferation as efficiently
as control siRNA–transfected MSCs (Figure 5F). To test whether
add-back of kynurenine to senescent MSC restores inhibitory
properties, we added kynurenine exogenously in senescent MSC
coculture with PBMCs. Our results demonstrated that PBMCs
in the coculture of senescent MSCs were efficiently inhibited by
exogenous kynurenine (Figure 5G).
IFNg stimulation restores senescent MSCs’
immunosuppressive properties
We have published a quantitative PCR array platform of .40 genes
informed by published reports of MSCs’ functional responsiveness
to IFNg.20 Our results are consistent with our previous observation20 and demonstrate that IFNg upregulates IDO, CXCL9,
CXCL10, CXCL11, PD-L1, and ICAM-1 on MSCs. In addition,
we observed that there is no significant difference in the global
pattern of RNA transcriptional response to IFNg observed between
fit and senescent MSCs other than HLA-DR (Figure 6). These
results suggested that IFNg stimulation would correct the defective
immunosuppressive properties of replicative senescent MSCs,
consistent with published data showing that IFNg stimulation
enhances MSCs’ immunosuppressive properties.20,22,31,32 We
tested whether IFNg activation rescues senescent MSCs’ immunosuppressive properties and demonstrated that IFNg-stimulated
senescent MSCs inhibit T-cell proliferation as efficiently as actively
growing fit MSCs (Figure 7A-B). However, IFNg prelicensed fit
MSCs exhibit significantly superior T-cell suppressor activity than
all the MSC populations tested. Next we investigated the IDO
response of fit and senescent MSCs. Our results demonstrated that
IFNg stimulation upregulates IDO RNA and protein on fit and
senescent MSCs at comparable levels (Figure 7C-D). Blocking of
IDO catalytic activity with 1-methyl tryptophan (1MT) negates the
suppressive effect of IFNg prelicensed senescent MSCs on T-cell
proliferation (Figure 7E), suggesting that IDO plays a central role in
conferring IFNg rescue effects on the immunosuppressive properties of senescent MSCs.
Discussion
Several industry-sponsored advanced clinical trials have investigated the clinical utility of bulk-produced culture-expanded random
donor allogeneic MSCs for the treatment of various immune and
tissue injury disorders. Considering that large-scale culture expansion
invariably leads to some measure of replicative senescence in a final
Figure 5. Exogenous addition of kynurenine corrects senescence-associated impaired immunosuppressive properties of MSCs. (A) Replicative fit and senescent
MSCs were cultured in the presence and absence of aCD3aCD28 Dynabeads-stimulated PBMCs. Four days post culture, supernatant was collected and kynurenine levels were
performed according to the manufacturer’s instructions. Cumulative of 6 donor pairs were shown. (B) Catabolism of kynurenine pathway and the enzymes that facilitate this cascade
is shown with a cartoon adapted from reference 30. IDO, kynurenine aminotransferases (KAT), kynurenine 3-monooxygenase (KMO), KYN, 3-hydroxyanthranilate 3,4-dioxygenase
(HAAO), and quinolinate phosphoribosyl transferase (QPRT). Fit and senescent MSCs were cultured in the presence and absence of aCD3aCD28 Dynabeads-stimulated PBMCs
in 2-chamber transwell plates. Four days later, MSCs were harvested and the expression level of mRNA of kynurenine catabolic enzymes relative to GAPDH was evaluated by
quantitative SYBR green real-time PCR. (C) Representative heat map with CT values (red 5 high expression, blue 5 low expression) and (D) cumulative comparison between fit and
senescent MSC pairs from 3 unique donors is shown. F1P, fit MSCs 1 activated PBMCs; S1P, senescent MSCs 1 activated PBMCs. (E) Control, KYN, KMO, and IDO siRNAtransfected MSCs were cultured with aCD3aCD28 Dynabeads-stimulated PBMCs in 2-chamber transwell plates for 3 to 4 days. Expression level of appropriate silenced mRNA
relative to GAPDH was evaluated by quantitative SYBR green real-time PCR. d-d CT method was applied to calculate the fold induction of silenced gene over the control siRNAtransfected MSCs. KYN, KMO, and IDO, and control siRNA-transfected MSCs were cultured with aCD3aCD28 Dynabeads-stimulated PBMCs. Four days post culture, T-cell
proliferation was measured by Ki67 intracellular staining. (F) Proliferation of T cells (CD31Ki671) in the presence and absence of siRNA transfected MSCs is shown. Similar results
were obtained in a repeat experiment with an additional independent MSC donor. (G) Kynurenine (500 uM) was added exogenously in the coculture of aCD3aCD28 Dynabeadsstimulated PBMCs with and without fit and senescent MSCs. Four days post culture, T-cell proliferation was measured by Ki67 intracellular staining. F, fit MSCs 1 activated PBMCs; N,
no MSCs 1 activated PBMCs; S, senescent MSCs 1 activated PBMCs. Kynurenine’s effect on T-cell proliferation with 4 independent fit and senescent MSC donor pairs is shown.
25 APRIL 2017 x VOLUME 1, NUMBER 11
SENESCENT MSCS DISPLAY IMPAIRED IMMUNE SUPPRESSION
637
CT Values
TG FB
CC R10
G AL-1
CXCR1
AN G IO PO IETIN 2
CXCR4
HLA-G 5
CC L5
CX3CR 1
CXCL10
CXCL11
CXCL9
IDO
IDO
HLADR
CIITA
TR AIL
TIM P-1
VEG F
CD 46
IL6
TLR 4
PD L2
CO X-2
VC AM -1
CD 55
CXCL12
KG F
TIM P-2
G APDH
CC L2
TLR 3
CC L7
ICAM -1
PD L1
PD L1
ULBP-3
TSG -6
CC R7
A20
HG F
PI9
CXCR6
BC I2
IL8
3.8513
5.9223
7.9934
10.064
12.135
14.207
17.472
20.737
24.003
F
F
S
S
0ng/ml
F
F
S
S
20ng/ml
Figure 6. Molecular genetic responses of fit and senescent MSCs to IFNg. Fit and senescent MSC pairs derived from 2 independent donors were stimulated with
20 ng/mL IFNg for 48 hours, and total cDNA were generated from RNA. Transcriptional profiles of .40 genes were investigated in Fluidigm nanoscale qPCR 48*48 array
plates (n 5 3/sample). Heat map of fit (F) and senescent (S) MSCs stimulated with 620 ng/mL IFNg showing the expression genes (red 5 high expression, blue 5 low
expression). Heat map was generated using JMP software.
cell product, we investigated whether senescent MSCs deploy
comparable potency and functionalities germane to immune suppression to their early passage, “fit” counterpart. Using the ISCT
guidelines,33 we have shown that senescent MSCs are plastic638
CHINNADURAI et al
adherent, despite displaying cell-cycle arrest in standard culture
conditions, and express surface molecules such as CD105, CD90,
CD73, and CD44, and lack expression of hematopoietic markers akin
to and indistinguishable from fit MSCs. However, replication-exhausted
25 APRIL 2017 x VOLUME 1, NUMBER 11
A
Fit
No MSC
Senescent
-IFN
-IFN
+IFN
17.8
1.37
+IFN
45.7
2.63
Ki67
91.1
CD3
B
C
IDO fold change / GAPDH
IFN Fit
IFN Senescent
100
Donor #1
% CD3+ Ki67+
Fit
Senescent
80
60
40
20
0
1:16
1:8
Fit
Senescent
0
1:4
0.05
100
80
60
40
0.5
5
50
ng/ml IFN
Donor #2
% CD3+ Ki67+
0
107
106
105
104
103
102
101
100
10-1
D
Fit
Senescent
IFN
20
(ng/ml)
0
1:16
1:8
1:4
0
1:16
1:8
1:4
0" 0.5" 5" 50"
0" 0.5" 5" 50"
100
Donor #3
% CD3+ Ki67+
0
80
60
40
20
E
- 1MT
100
0
1:4
20
IFN Fit
40
IFN Sene
60
0
No MSC
Fit
Sene
Donor #5
100
80
40
IFN Fit
1:8
IFN Sene
1:16
Fit
0
60
Sene
40
80
No MSC
60
% CD3+Ki67+
80
20
% CD3+ Ki67+
+ 1MT
100
Donor #4
% CD3+ Ki67+
0
20
0
0
1:16
1:8
1:4
MSC:PBMC ratio
Figure 7. IFNg prelicensed senescent MSCs display enhanced immunosuppressive activity through IDO. aCD3aCD28 Dynabeads-stimulated PBMCs were cocultured
in the presence and absence 6 IFNg prelicensed (20 ng/mL for 48 hours) fit and senescent MSCs. Four days post culture, T-cell proliferation was measured by Ki67 intracellular
staining. (A) Representative FACS plot and (B) dose-dependent T-cell inhibitory effect of 6 IFNg prelicensed fit and senescent MSC pairs from 5 independent donors is
shown. (C) Fit and senescent MSCs were stimulated with IFNg for 48 hours. Expression level of IDO mRNA relative to GAPDH was evaluated by quantitative SYBR green real-time
PCR. d-d CT method was applied to calculate the fold induction of IDO over the unstimulated control. (D) IDO expression at protein level is shown by western blot analysis,
and actin was used as an internal control. Similar results were obtained in a repeat experiment with an additional independent MSC donor. (E) 6 IFNg prelicensed fit and senescent
MSCs were cocultured with activated PBMCs in the presence and absence of IDO blocker, 1MT. Four days after, T-cell proliferation was measured by flow cytometry. Similar results
were obtained in a repeat experiment with another MSC donor pair.
25 APRIL 2017 x VOLUME 1, NUMBER 11
SENESCENT MSCS DISPLAY IMPAIRED IMMUNE SUPPRESSION
639
MSCs display classical senescence-associated protein and genetic
markers such as b-galactosidase and P16INK4A, respectively.13,17
Consistent with previous reports,13 we have observed hypertrophic
phenotype changes in senescent MSCs, which have been shown to
be a major limitation for the use of MSC transplantation for cartilage
repair.34-37
Considering that IFNg plays a dominant role in activation of
MSCs,38,39 analysis of MSCs’ responsiveness to IFNg is evolving
commonly used method to define their immune plasticity.40,41
Here, we have shown that senescent MSCs are responsive to
IFNg as defined by STAT1 phosphorylation. However, downstream effector pathways display significant variations as IFNg
upregulates MHC class I, PDL-1, and PDL-2, but not HLA-DR in
senescent MSCs. Indeed, a senescence-associated defect in
MHC class II upregulation by IFNg and impaired GM-CSF–
dependent proliferation was demonstrated in macrophages
derived from aged mice.42-44 In addition, upregulation of MHC
class II is also affected in cell-cycle–arrested macrophages, which
corroborates with our findings with growth-arrested senescent
MSCs.45 These results suggest that MHC class II induction defect
is a common feature of telomere-shortened senescent cells of
both hematopoietic and nonhematopoietic origins. Although we
demonstrate that the role of HLA-DR in MSCs in inhibiting T-cell
proliferation is dispensable, IFNg-induced defect in HLA-DR
expression could serve as a surrogate marker for loss of MSCs’
inhibitory potency and replication exhaustion.
Replicative senescent human fibroblasts and epithelial cells secrete
proinflammatory cytokines such as IL-6, IL-8, and GM-CSF as part
of the senescence-associated secretory phenotype (SASP).46
Using a 30-plex cytokine analysis, we did not observe an altered
secretome by resting senescent MSCs (supplemental Figure 3).
However, we have observed that human MSCs are exquisitely
responsive to proximate activated PBMCs. We have shown here
that relative to fit MSCs, their senescent counterpart cocultured
with allogeneic immune responders PBMCs deployed a blunted
upregulation MSC-sourced cytokines such as VEGF, GCSF,
CXCL10, and CCL2 and were less effective at downregulation
of PBMC-sourced cytokines such as IL-1ra, IFNg, IL-2r, CCL4,
TNFa, IL-5, and IL-10. Importantly, we have found that senescent
MSCs deploy a blunted secretome response upon interaction with
immune responders, with substantially reduced expression of
VEGF, CCL2, GCSF, and CXCL10. To better understand the
contribution of these and other effector molecules by fit MSCs, we
knocked down CXCL9, CXCL10, CXCL11, GCSF, and CCL2
individually without any meaningful effect on veto properties of
MSCs. In contrast, knockdown of IDO abolished MSC suppressor
function. These data suggest that IDO remains the key effector
pathway for blocking PBMC proliferation in vitro. However, we have
found that senescent MSCs are defective in kynurenine production
upon interaction with PBMCs. Considering that senescenceassociated aging affects cellular amino acid metabolism, this may
provide an explanation for defective tryptophan catabolism by
senescent MSCs.47-52 It has also been demonstrated that IDO in
senescent MSCs undergo proteasomal degradation, which could
additionally account for defective kynurenine production and
associated impaired immune suppression.53 Consistent with our
previous study, we also show here that IL-10 expression is increased in macrophages cocultured with MSCs.54 We also show
that senescent MSCs upregulate IL-10 production by macrophages
640
CHINNADURAI et al
like fit MSCs despite exhibiting poor IDO activity. Published data
suggest that MSC and macrophage interactions are complex and
involve additional effector pathways other than IDO.55
IFNg induces cellular senescence in human endothelial cells
through p53-dependent DNA damage.56 Although it is unknown
whether such a mechanism occurs in MSCs, IFNg leads to a
cytostatic nonproliferative response and affects the differentiation
potential of MSCs.20,57 Nevertheless, IFNg rescue of senescentassociated defective immunosuppressive properties suggests that
IFNg prelicensing may serve as a remedy for MSCs’ utilization as
cellular pharmaceutical for immune disorders. We also compared
.40 transcripts before and after treatment with IFNg that are
important for MSCs’ immune function, homing, and regenerative
properties in fit and senescent counterparts. The majority of the
genes are highly upregulated both on fit and senescent MSCs,
suggesting that IFNg prelicensing can partially correct the defective
immunosuppressive properties of senescent MSCs.
In most industrial clinical trials, cryobanked MSCs are used. High
prefreeze senescence cell content has been shown to correlate
with poor post-thaw function of MSCs,58 and thus both replicative
senescence and thawing from cryopreservation may be additive
detrimental determinants of MSC function and potency. In the
present study, we have used MSCs that are cultured in fetal calf
serum growth–supplemented media akin to the most prevalent
culture methods in support of clinical trials. We have found that
addition of human platelet lysate does not rescue proliferation of
MSCs with pre-established senescence, suggesting that although
replicative senescence can be postponed or prevented at some
extent with the appropriate culture expansion medium, it cannot be
reversed (supplemental Figure 4).19
Acquired senescence in culture-adapted MSCs may provide
insight on why industry-sponsored clinical trials using heavily
expanded MSCs do not show efficacy, yet meet standards of
safety.59 Our data support the notion that replication fit MSCs
are distinct and superior to senescent MSCs in their ability to
suppress T-cell proliferation, and that culture conditions, including
use of licensing cytokines, may provide a remedy to mitigate this
defect.
Acknowledgments
The authors thank Shala Yuan and Marco Garcia for technical
assistance.
The study was supported by a grant from ACTSI/
ImmunoEngineering Pilot Award. Research reported in this publication
was supported in part by developmental funds from the Winship
Cancer Institute of Emory University (R.C.). This work was directly
supported by National Institutes of Health, National Institute of
Diabetes and Digestive and Kidney Diseases award R01DK109508.
Authorship
Contribution: R.C. designed the research plan, performed
most experiments, analyzed results, and wrote the manuscript;
D.R. and L.J.A. helped with cytokine multipex experiments; E.K.W.
provided bone marrow from healthy individuals; S.N. performed
western blot analysis for IDO; K.M. performed b-galactosidase
assays; D.A. and G.G. helped with qPCR arrays; and J.G.
25 APRIL 2017 x VOLUME 1, NUMBER 11
designed the research plan, analyzed results, and wrote the
manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Jacques Galipeau, Department of Medicine,
University of Wisconsin Carbone Comprehensive Cancer Center,
University of Wisconsin–Madison, 1111 Highland Ave, Madison,
WI 53705; e-mail: [email protected].
References
1.
Phinney DG, Galipeau J, Krampera M, Martin I, Shi Y, Sensebe L. MSCs: science and trials. Nat Med. 2013;19(7):812.
2.
Kallekleiv M, Larun L, Bruserud Ø, Hatfield KJ. Co-transplantation of multipotent mesenchymal stromal cells in allogeneic hematopoietic stem cell
transplantation: a systematic review and meta-analysis. Cytotherapy. 2016;18(2):172-185.
3.
Galipeau J. The mesenchymal stromal cells dilemma–does a negative phase III trial of random donor mesenchymal stromal cells in steroid-resistant graftversus-host disease represent a death knell or a bump in the road? Cytotherapy. 2013;15(1):2-8.
4.
Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284(5411):143-147.
5.
von Bahr L, Sundberg B, Lönnies L, et al. Long-term complications, immunologic effects, and role of passage for outcome in mesenchymal stromal cell
therapy. Biol Blood Marrow Transplant. 2012;18(4):557-564.
6.
Hayflick L, Moorhead PS. The serial cultivation of human diploid cell strains. Exp Cell Res. 1961;25:585-621.
7.
Tarte K, Gaillard J, Lataillade JJ, et al; Société Française de Greffe de Moelle et Thérapie Cellulaire. Clinical-grade production of human mesenchymal
stromal cells: occurrence of aneuploidy without transformation. Blood. 2010;115(8):1549-1553.
8.
Sensebé L, Tarte K, Galipeau J, et al; MSC Committee of the International Society for Cellular Therapy. Limited acquisition of chromosomal aberrations in
human adult mesenchymal stromal cells. Cell Stem Cell. 2012;10(1):9-10, author reply 10-11.
9.
Bernardo ME, Zaffaroni N, Novara F, et al. Human bone marrow derived mesenchymal stem cells do not undergo transformation after long-term in vitro
culture and do not exhibit telomere maintenance mechanisms. Cancer Res. 2007;67(19):9142-9149.
10. Ksia˛ żek K. A comprehensive review on mesenchymal stem cell growth and senescence. Rejuvenation Res. 2009;12(2):105-116.
11. Wagner W, Bork S, Lepperdinger G, et al. How to track cellular aging of mesenchymal stromal cells? Aging (Albany NY). 2010;2(4):224-230.
12. Baxter MA, Wynn RF, Jowitt SN, Wraith JE, Fairbairn LJ, Bellantuono I. Study of telomere length reveals rapid aging of human marrow stromal cells
following in vitro expansion. Stem Cells. 2004;22(5):675-682.
13. Wagner W, Horn P, Castoldi M, et al. Replicative senescence of mesenchymal stem cells: a continuous and organized process. PLoS One. 2008;3(5):
e2213.
14. Katsube Y, Hirose M, Nakamura C, Ohgushi H. Correlation between proliferative activity and cellular thickness of human mesenchymal stem cells.
Biochem Biophys Res Commun. 2008;368(2):256-260.
15. Bork S, Pfister S, Witt H, et al. DNA methylation pattern changes upon long-term culture and aging of human mesenchymal stromal cells. Aging Cell.
2010;9(1):54-63.
16. Kasper G, Mao L, Geissler S, et al. Insights into mesenchymal stem cell aging: involvement of antioxidant defense and actin cytoskeleton. Stem Cells.
2009;27(6):1288-1297.
17. Shibata KR, Aoyama T, Shima Y, et al. Expression of the p16INK4A gene is associated closely with senescence of human mesenchymal stem cells and is
potentially silenced by DNA methylation during in vitro expansion. Stem Cells. 2007;25(9):2371-2382.
18. Izadpanah R, Kaushal D, Kriedt C, et al. Long-term in vitro expansion alters the biology of adult mesenchymal stem cells. Cancer Res. 2008;68(11):
4229-4238.
19. Griffiths S, Baraniak PR, Copland IB, Nerem RM, McDevitt TC. Human platelet lysate stimulates high-passage and senescent human multipotent
mesenchymal stromal cell growth and rejuvenation in vitro. Cytotherapy. 2013;15(12):1469-1483.
20. Chinnadurai R, Copland IB, Ng S, et al. Mesenchymal stromal cells derived from Crohn’s patients deploy indoleamine 2,3-dioxygenase-mediated immune
suppression, independent of autophagy. Mol Ther. 2015;23(7):1248-1261.
21. Debacq-Chainiaux F, Erusalimsky JD, Campisi J, Toussaint O. Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity,
a biomarker of senescent cells in culture and in vivo. Nat Protoc. 2009;4(12):1798-1806.
22. Chinnadurai R, Copland IB, Patel SR, Galipeau J. IDO-independent suppression of T cell effector function by IFN-g-licensed human mesenchymal
stromal cells. J Immunol. 2014;192(4):1491-1501.
23. Chinnadurai R, Garcia MA, Sakurai Y, et al. Actin cytoskeletal disruption following cryopreservation alters the biodistribution of human mesenchymal
stromal cells in vivo. Stem Cell Rep. 2014;3(1):60-72.
24. Dimri GP, Lee X, Basile G, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA. 1995;
92(20):9363-9367.
25. Krampera M, Galipeau J, Shi Y, Tarte K, Sensebe L; MSC Committee of the International Society for Cellular Therapy (ISCT). Immunological
characterization of multipotent mesenchymal stromal cells–The International Society for Cellular Therapy (ISCT) working proposal. Cytotherapy. 2013;
15(9):1054-1061.
25 APRIL 2017 x VOLUME 1, NUMBER 11
SENESCENT MSCS DISPLAY IMPAIRED IMMUNE SUPPRESSION
641
26. Chinnadurai R, Copland IB, Garcia MA, et al. Cryopreserved mesenchymal stromal cells are susceptible to T-cell mediated apoptosis which is partly
rescued by IFNg licensing. Stem Cells. 2016;34(9):2429-2442.
27. Ren G, Zhang L, Zhao X, et al. Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide. Cell
Stem Cell. 2008;2(2):141-150.
28. Romieu-Mourez R, François M, Boivin MN, Stagg J, Galipeau J. Regulation of MHC class II expression and antigen processing in murine and human
mesenchymal stromal cells by IFN-gamma, TGF-beta, and cell density. J Immunol. 2007;179(3):1549-1558.
29. Sepúlveda JC, Tomé M, Fernández ME, et al. Cell senescence abrogates the therapeutic potential of human mesenchymal stem cells in the lethal
endotoxemia model. Stem Cells. 2014;32(7):1865-1877.
30. Asp L, Johansson AS, Mann A, et al. Effects of pro-inflammatory cytokines on expression of kynurenine pathway enzymes in human dermal fibroblasts.
J Inflamm (Lond). 2011;8:25.
31. Duijvestein M, Wildenberg ME, Welling MM, et al. Pretreatment with interferon-g enhances the therapeutic activity of mesenchymal stromal cells in animal
models of colitis. Stem Cells. 2011;29(10):1549-1558.
32. Rafei M, Birman E, Forner K, Galipeau J. Allogeneic mesenchymal stem cells for treatment of experimental autoimmune encephalomyelitis. Mol Ther.
2009;17(10):1799-1803.
33. Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy
position statement. Cytotherapy. 2006;8(4):315-317.
34. Cheng H, Qiu L, Ma J, et al. Replicative senescence of human bone marrow and umbilical cord derived mesenchymal stem cells and their differentiation to
adipocytes and osteoblasts. Mol Biol Rep. 2011;38(8):5161-5168.
35. Zhong L, Huang X, Karperien M, Post JN. The regulatory role of signaling crosstalk in hypertrophy of MSCs and human articular chondrocytes. Int J Mol
Sci. 2015;16(8):19225-19247.
36. Chen S, Fu P, Cong R, Wu H, Pei M. Strategies to minimize hypertrophy in cartilage engineering and regeneration. Genes Dis. 2015;2(1):76-95.
37. Studer D, Millan C, Öztürk E, Maniura-Weber K, Zenobi-Wong M. Molecular and biophysical mechanisms regulating hypertrophic differentiation in
chondrocytes and mesenchymal stem cells. Eur Cell Mater. 2012;24:118-135, discussion 135.
38. Sivanathan KN, Gronthos S, Rojas-Canales D, Thierry B, Coates PT. Interferon-gamma modification of mesenchymal stem cells: implications of
autologous and allogeneic mesenchymal stem cell therapy in allotransplantation. Stem Cell Rev. 2014;10(3):351-375.
39. Krampera M. Mesenchymal stromal cell ‘licensing’: a multistep process. Leukemia. 2011;25(9):1408-1414.
40. Galipeau J, Krampera M. The challenge of defining mesenchymal stromal cell potency assays and their potential use as release criteria. Cytotherapy.
2015;17(2):125-127.
41. Galipeau J, Krampera M, Barrett J, et al. International Society for Cellular Therapy perspective on immune functional assays for mesenchymal stromal cells
as potency release criterion for advanced phase clinical trials. Cytotherapy. 2016;18(2):151-159.
42. Sebastián C, Herrero C, Serra M, Lloberas J, Blasco MA, Celada A. Telomere shortening and oxidative stress in aged macrophages results in impaired
STAT5a phosphorylation. J Immunol. 2009;183(4):2356-2364.
43. Herrero C, Sebastián C, Marqués L, et al. Immunosenescence of macrophages: reduced MHC class II gene expression. Exp Gerontol. 2002;37(2-3):
389-394.
44. Herrero C, Marqués L, Lloberas J, Celada A. IFN-gamma-dependent transcription of MHC class II IA is impaired in macrophages from aged mice. J Clin
Invest. 2001;107(4):485-493.
45. Xaus J, Comalada M, Barrachina M, et al. The expression of MHC class II genes in macrophages is cell cycle dependent. J Immunol. 2000;165(11):
6364-6371.
46. Tchkonia T, Zhu Y, van Deursen J, Campisi J, Kirkland JL. Cellular senescence and the senescent secretory phenotype: therapeutic opportunities. J Clin
Invest. 2013;123(3):966-972.
47. Baraibar MA, Hyzewicz J, Rogowska-Wrzesinska A, et al. Impaired energy metabolism of senescent muscle satellite cells is associated with oxidative
modifications of glycolytic enzymes. Aging (Albany NY). 2016;8(12):3375-3389.
48. Capasso S, Alessio N, Squillaro T, et al. Changes in autophagy, proteasome activity and metabolism to determine a specific signature for acute and
chronic senescent mesenchymal stromal cells. Oncotarget. 2015;6(37):39457-39468.
49. Baraibar M, Hyzewicz J, Rogowska-Wrzesinska A, et al. Impaired metabolism of senescent muscle satellite cells is associated with oxidative modifications
of glycolytic enzymes. Free Radic Biol Med. 2014;75(Suppl 1):S23.
50. Milan AM, D’Souza RF, Pundir S, et al. Older adults have delayed amino acid absorption after a high protein mixed breakfast meal. J Nutr Health Aging.
2015;19(8):839-845.
51. Tang JP, Melethil S. Effect of aging on the kinetics of blood-brain barrier uptake of tryptophan in rats. Pharm Res. 1995;12(7):1085-1091.
52. Newton RB, Sullivan JL, Debusk AG. Neutral amino acid transport and in vitro aging. Mech Ageing Dev. 1984;27(1):63-72.
53. Loisel S, Dulong J, Ménard C, et al. Brief report–proteasomal indoleamine 2,3-dioxygenase degradation reduces the immunosuppressive potential of
clinical grade-mesenchymal stromal cells undergoing replicative senescence. Stem Cells. 2017.
54. François M, Romieu-Mourez R, Li M, Galipeau J. Human MSC suppression correlates with cytokine induction of indoleamine 2,3-dioxygenase and
bystander M2 macrophage differentiation. Mol Ther. 2012;20(1):187-195.
642
CHINNADURAI et al
25 APRIL 2017 x VOLUME 1, NUMBER 11
55. Chiossone L, Conte R, Spaggiari GM, et al. Mesenchymal stromal cells induce peculiar alternatively activated macrophages capable of dampening both
innate and adaptive immune responses. Stem Cells. 2016;34(7):1909-1921.
56. Kim KS, Kang KW, Seu YB, Baek SH, Kim JR. Interferon-gamma induces cellular senescence through p53-dependent DNA damage signaling in human
endothelial cells. Mech Ageing Dev. 2009;130(3):179-188.
57. Croitoru-Lamoury J, Lamoury FM, Caristo M, et al. Interferon-g regulates the proliferation and differentiation of mesenchymal stem cells via activation of
indoleamine 2,3 dioxygenase (IDO). PLoS One. 2011;6(2):e14698.
58. Pollock K, Sumstad D, Kadidlo D, McKenna DH, Hubel A. Clinical mesenchymal stromal cell products undergo functional changes in response to
freezing. Cytotherapy. 2015;17(1):38-45.
59. Lalu MM, McIntyre L, Pugliese C, et al; Canadian Critical Care Trials Group. Safety of cell therapy with mesenchymal stromal cells (SafeCell):
a systematic review and meta-analysis of clinical trials. PLoS One. 2012;7(10):e47559.
25 APRIL 2017 x VOLUME 1, NUMBER 11
SENESCENT MSCS DISPLAY IMPAIRED IMMUNE SUPPRESSION
643