A Journey In and Outside of Stem Cells --- Exosomes
Transcript of A Journey In and Outside of Stem Cells --- Exosomes
A Journey In and Outside of Stem Cells --- Exosomes
Hua-Jung Li
National Health Research Institutes
PGE2/EP4-mediated signaling is required to maintain breast cancer stem cells
Li HJ et al. Cancer Discovery 2012 9:840-55
early
endosome
EP4 antagonism
EVs/exosomes with
cargos for SC properties
SCs lose
stemness
via the
EVs/exosom
es
EP4 antagonist
sChemo
killed
CSCCSCChemo-
sensitive
Non-CSC
EP4
receptor
drug efflux
transporter
EP4
antagonis
t
chemotherapy
EP4 antagonist
MSCAnti-EP4 elicited MSC EVs
recovered neurons
Intact BBB
A2 astrocyte
microglia
Inhibiting
inflamma
tion
Suppressing
Astrogliosis
Recovery of neurons
Station 1CANCER
THERAPY
REGENERATIVE
MEDICINE
Station 2
Station 3
EVs in Mammary Stem Cell
Homeostasis
EVs in Tumor Chemosensitivity
EVs in Regenerative
Medicine
early
endosome
EP4 antagonism
EVs/exosomes with
cargos for SC properties
SCs lose
stemness
via the
EVs/exosom
es
EP4 antagonist
sChemo
killed
CSCCSCChemo-
sensitive
Non-CSC
EP4
receptor
drug efflux
transporter
EP4
antagonis
t
chemotherapy
EP4 antagonist
MSCAnti-EP4 elicited MSC EVs
recovered neurons
Intact BBB
A2 astrocyte
microglia
Inhibiting
inflamma
tion
Suppressing
Astrogliosis
Recovery of neurons
Station 1CANCER
THERAPY
REGENERATIVE
MEDICINE
Station 2
Station 3
EVs in Mammary Stem Cell
Homeostasis
EVs in Tumor Chemosensitivity
EVs in Regenerative
Medicine
COX-2/Cytokeratin 8/DAPI
NA
ME
CH
ML
E
Number/500 cells
0 50 100 150
Cell surface EP4
Negative 2nd Ab only EP4 Ab
NAMECHMLE
Cou
nt
EP4/Golgi/DAPIH
ML
E
N
AM
EC
***
NAMEC
HMLE
Cou
nt
HM
LE
NA
ME
C
COX-2
PGDH
EP4
GAPDH
mPGES-1
MRP4
EP4-mediated signaling is required to maintain mesenchymal/stem cells of the mammary epithelium
EpCAM
Lin MC et al. Stem Cells 2017 35(2):425-444
Crl GW
0 h
48
h
BC
rlG
W2nd
only
EP4
Cell surface EP4
PG
E2
A
Co
un
t
D
Num
be
r o
f ce
lls
(mig
ratio
n)
0
200
400
600
800
1000
1200
1400
Crl GW
* * *
Num
be
r/3
00
ce
lls
F
0
10
20
30
40
50
60
70
80
90
Crl GW
* * *
C not resistantresistant
Crl GW
Cell p
erc
en
t
0%
20%
40%
60%
80%
100%
*
Crl
GW
Integrin
β1
Integrin
α3CD90
ECD44 Integrin α6
2nd antibody
only
Specific antibody
EP4-mediated signaling is required to maintain mesenchymal/stem cells of the mammary epithelium
Lin MC et al. Stem Cells 2017 35(2):425-444
Exo
so
ma
lG
AP
DH
/
ce
ll
CM-P4 Particles/cell
Crl GW PGE2
0.49X104 5.43X104 0.86X104
0
2
4
6
8
10
12
Crl GW PGE2
*
* * *
Diameter (nm)
Rela
tive
nu
mb
er
100
50
050 5000
D RN
CD81
NAMEC
(1x)HMLE
(1X)
Crl GW Crl GW Crl GW
HMLE
(2X)
GAPDHCD44
Long-
exposure
CD81
GAPDHCD44
pull down Input
CD44
CD90
Integrin α6
CD81
Integrin β1
NB NB
CellCM-P4
N NB NB
CellCM-P4
NN N
CM-P4
Crl GW PGE2 Crl GW PGE2
Cell
CD44
CD81
HSP70
GAPDH
TSG101
Alix
CD9
Blocking EP4-mediated signaling in MaSCs causes release of stem cell surface markers and integrin via extracellular
vesicle (EV) release
Lin MC et al. Stem Cells 2017 5(2):425-444
Lin MC et al. Stem Cells 2017 5(2):425-444
Blocking EP4-mediated signaling in MaSCs causes release of stem cell surface markers and integrin via EV release
Lin MC et al. Stem Cells 2017 5(2):425-444
Blocking EP4-mediated signaling in MaSCs causes release of stem cell surface markers and integrin via EVrelease
Lin MC et al. Stem Cells 2017 5(2):425-444
Blocking EP4-mediated signaling in MaSCs causes release of stem cell surface markers and integrin via EV release
0
10
20
30
40
50
60
0.5 0.75 1 1-
Num
ber
of m
am
mosphere
s/3
00
ce
lls
--
***
**
***
***
0
200
400
600
800
1000
1200
1400
Num
ber
of m
igra
ting c
ells/c
ham
ber
0.5 0.75 1 1Dy (μM)
GW
-
-+ ++ +
-
-
*** **
***
**
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
not resistant resistant
Cell P
erc
en
t
***
***
***
0.75 1Dy (μM)
GW
-
+ + +
-
-
GAPDH
E-cad
+ + + - GW
0.75 1 Dy (μM)-- Crl
GW
Integrin β1Integrin α3 CD90 CD44 Integrin α6
Dy+
GW
2nd antibody only Specific antibody
EP4 antagonist-induced EVs are responsible for the loss of basal SC properties by MaSCs
Dy (μM)
GW -+ ++ +
Lin MC et al. Stem Cells 2017 35(2):425-444
Crl
GW/GW GW/Exo
GW/PBS
0
10
20
30
40
50
60
70
80
90
100
Crl GW/PBS GW/EV GW/GW
**
**
Cell P
erc
en
t (%
)
not resistant resistant
0
500
1000
1500
2000
2500
3000
3500
4000
*
*
**
****
0
10
20
30
40
50
60
70
80
*
*
***
***
Nu
mb
er
of
mig
ratin
g c
ells/c
ham
be
r
Nu
mb
er
of
ma
mm
osp
he
res/3
00
ce
lls
EP4 antagonist-induced EVs are responsible for the loss of basal SC properties by MaSCs
Lin MC et al. Stem Cells 2017 35(2):425-444
0
1
2
3
4
5
6
Crl Exo
Crl EV
HMLE NAMEC
Crl
GW
CFSEnegative controlCFSE-labeled EV
EV/DAPI/Plasma membrane
Crl GW
0
200
400
600
800
1000
1200
Nu
mb
er
of ce
lls
*
***
0
10
20
30
40
50
60
70
80
Crl Non-migrating
EV-HMLE
Migrating
EV-HMLE
Nu
mb
er/
20
00
ce
lls**
* * ** * *
Crl HMLE+EV EV-HMLE
Nu
mb
er/
20
00
ce
lls
Crl EV
Crl EV400
y axi
s [
μm
]
200
0
-200
-400
400 400200200-200-400 0 -200-400 0y axis [μm] y axis [μm]
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Crl Exo
cell
mig
ration d
ista
nce (
m/d
ay)
***
EV
EP4 antagonist-induced basal MaSC EVs contain molecules that can transfer basal SC properties
Lin MC et al. Stem Cells 2017 35(2):425-444
K
Collecting EP4
agonist/antagonist
induced EVs from
primary mammary
epithelial cells
Sorting out primary
mammary luminal cells
Isolating primary
mammary epithelial
cells
Evaluating the
transfer of mammary
gland-forming ability
by EVs in cleared fat
pads
EP4 agonist
/antagonist
EVs
Primary luminal cells
Ep
CA
M
integrin α6
EpCAMhi/integrin α6lo
luminal cell
EpCAMlo/integrin α6hi
basal cell
EP4 antagonist-induced basal MaSC EVs contain molecules that can transfer mammary gland-forming ability
Lin MC et al. Stem Cells 2017 35(2):425-444
EP4 antagonist-induced EVs contain proteins required to maintain basal mammary epithelial cell properties
Lin MC et al. Stem Cells 2017 35(2):425-444
A2 3 4 5 7 8 961
Caveolin
-1G
ang
liosid
e
GM
1G
AP
DH
p-A
kt
Akt
TG
Fβ
-R1
TG
Fβ
-R2
crl
GW
PG
E2
crl
GW
PG
E2
crl
GW
PG
E2
crl
GW
PG
E2
crl
GW
PG
E2
crl
GW
PG
E2
crl
GW
PG
E2
LRF Non-LRF
B
Crl
GW
Surface GM1
negative
CTB-Alexa 488
Crl
GW
PG
E2
Surface GM1
C
Blocking PGE2/EP4 signaling causes the relocation of signaling receptors and mediators into lipid
Lin MC et al. Stem Cells 2017 35(2):425-444
A
LR
P6
HE
R2
Me
tE
GF
R
C
EInte
gri
n β
1In
teg
rin
α6
1 2 3 4 5 7 8 96
C
E
C
E
C
E
C
E
C
E
Re
ce
pto
rM
igra
tio
n
p-A
KT
Src
GA
PD
HC
av-1
GM
1
C
E
C
E
C
E
C
E
C
E
Sig
na
ling
LR
FN
on
-LR
F
1 2 3 4 5 7 8 96Integrin β1
Integrin α6
B
EGFR
HER2
LRP6
p-Akt
p-Src
GW PGE2
GM1
Cav-1
Met
TGFβ-R2
Src
AKT
CD44
CD90
TGFβ-R1
3 4 52 3 4 52
Blocking PGE2/EP4 signaling causes the relocation of signaling receptors and mediators into lipid rafts and then
the release of proteins via extracellular vesicles
Lin MC et al. Stem Cells 2017 35(2):425-444
- + + +
- - 0.5 1
EVs/cell
MβCD (mM)
GW
GAPDH
CD81
GAPDH
Caveolin-1
Akt
p-Akt
CD44
- 0.5 1 MβCD
(mM)
p-PDK
p-Src
TGFβ-R1
EGFR
HER2
LRP6
Frizzled5
Met
Caveolin-2
TGFβ-R2
β-Actin
CD71/Trf R
Integrin β1
Recepto
rM
igra
tion
Ste
mness
Src
Sig
naling
CD90
Integrin α6
LR
FN
on-L
RF
GAPDH
0
0.2
0.4
0.6
0.8
1
1.2
EV
-Cav-
1/E
V-G
AP
DH ***
***
Cav-1
MβCD (mM) - 0.5 1
1X
2X
5X
0
0.2
0.4
0.6
0.8
1
1.2
EV
-GM
1/E
V-G
AP
DH
MβCD (mM) - 0.5 1
******
CD44
p-Akt
Akt
GAPDH
Cav-1
Cav-2
TGFβ-R1
p-Src
TGFβ-R2
Met
β-Actin
EGFR
Src
Recepto
rM
igra
tion
Ste
mness
Sig
naling
CD90
Integrin α6
LR
FN
on-
LR
F
HER2
LRP6
Crl siCav1siCav1+2
Integrin β1
GAPDH
CD81
GAPDH
Cav-1Cav-2
Cell
EV
vehicle GW
B
GM
1
EV proteins are sorted in EP4 antagonist-induced extracellular vesicles in a caveolae/lipid-raft-dependent manner
Lin MC et al. Stem Cells 2017 5(2):425-444
EV EV EV0
200
400
600
800
1000
1200
1400
Crl GW PGE2 MβCD+GW
Nu
mb
er
of
ce
lls
***
***
***
*
EV-treated
Crl GW PGE2 MβCD+GWEV-treated
***
The lipid-raft-associated factors of EP4 antagonist-induced extracellular vesicles are required to transfer basal MaSC
properties
Lin MC et al. Stem Cells 2017 35(2):425-444
0
1
2
3
4
5
6
7
8
9
10
Nu
mb
er /
200
0 ce
lls
* **
EV-treated
Ra
b4
En
do
so
me
re
cyclin
g
Ra
b2
7b
/35
EV
re
lea
se
<
Ra
b4
En
do
so
me
re
cyclin
g
Ra
b2
7b
/35
EV
re
lea
se
<
EarlyEndosome
EarlyEndosome
EarlyEndosome
Multi-vesicular Endosome (MVE)
MVEs
EV
few “empty” EVs lots of EVs with
LRF-proteins/miRNAs
bMaSCs retainstemness
luminal cells remain
bMaSCs lose stemness
luminal cells acquire bMaSCproperties via the EVs with LRF-proteins/miRNAs
EP4 agonism EP4 antagonism
i i
iiii
iii
iviv
iii
Exosome release can be regulated by extracellular signals
Lin MC et al. Stem Cells 2017 Feb;35(2):425-444
early
endosome
EP4 antagonism
EVs/exosomes with
cargos for SC properties
SCs lose
stemness via the
EVs/exosomes
EP4 antagonist
sChemo
killed
CSCCSCChemo-
sensitive
Non-CSC
EP4
receptor
drug efflux
transporter
EP4
antagonis
t
chemotherapy
EP4 antagonist
MSCAnti-EP4 elicited MSC EVs
recovered neurons
Intact BBB
A2 astrocyte
microglia
Inhibiting
inflamma
tion
Suppressing
Astrogliosis
Recovery of neurons
Station 1CANCER
THERAPY
REGENERATIVE
MEDICINE
Station 2
Station 3
EVs in Tumor Chemosensitivity
EVs in Regenerative
Medicine
Non-SCs acquire SC
properties via
the induced EVs/exosomes
early
endosome
EP4 antagonism
EVs/exosomes with
cargos for SC properties
SCs lose
stemness via the
EVs/exosomes
EP4 antagonist
sChemo
killed
CSCCSCChemo-
sensitive
Non-CSC
EP4
receptor
drug efflux
transporter
EP4
antagonis
t
chemotherapy
EP4 antagonist
MSCAnti-EP4 elicited MSC EVs
recovered neurons
Intact BBB
A2 astrocyte
microglia
Inhibiting
inflamma
tion
Suppressing
Astrogliosis
Recovery of neurons
Station 1CANCER
THERAPY
REGENERATIVE
MEDICINE
Station 2
Station 3
EVs in Tumor Chemosensitivity
EVs in Regenerative
Medicine
Non-SCs acquire SC
properties via
the induced EVs/exosomes
Ove
rall
Su
rviv
al (%
)100
80
60
40
20
00 50 100 150 200 250 300
P=0.00798
i
Breast Invasive Carcinoma
N=96
N=719
100
80
60
40
20
00 20 40 60 80 100 120 140
HER2+ Breast Tumor
P=4.3x10-4
Ove
rall
Su
rviv
al (%
)ii
N=16
N=104
COX-2 gene amplification
Months Survival
HER2+ Breast Tumor
Re
lap
se
Fre
e S
urv
iva
l (%
)
100
80
60
40
20
00 20 40 60 80 100 120 140
P=0.0263
iii
N=12
N=92
No COX-2 gene amplification
*
CO
X-2
mR
NA
4.5
0.80.6
0.4
0.20
1
5.5
EP
2m
RN
A
4
2
0
6
EP
1m
RN
A
0.4
0.2
0
0.6 ***
EP
4m
RN
A
0.4
0.2
0
0.6
0.8*
EP
3m
RN
A
4
2
0
6
8
Elevated COX-2 and EP4 expression in mesenchymal/basal breast cancer cells
Months SurvivalMonths Survival
Lin MC, Chen SY et al. Int J Cancer. 2018;143(6):1440-1455
Merged COX-2 Pan-Cytokeratinp
atie
nt 1
pa
tie
nt 2
pa
tie
nt 3
Elevated COX-2 and EP4 expression in mesenchymal/basal breast cancer cells
Lin MC, Chen SY et al. Int J Cancer. 2018;143(6):1440-1455
A C
E
0
1
2
3
4
5
6
PG
E2
(pg
/10
5ce
ll) *
0
20
40
60
80
Num
bers
/500 c
ells
***
B
Crl SQ120 SQ240 GW
D
0
400
800
1200
1600
Crl
SQ
12
0
SQ
24
0
GW
Nu
mb
er
of
ce
lls
***
Crl
SQ 120 SQ 240
GW
GAPDH
CD81
TSG101
Alix
G
H
CD44
GAPDH
CD81
N-cad
CD90
GAPDH(L)
CD81(L)
0
1000
2000
3000
4000
5000
6000
EV
s/c
ell
F
***
EP4
GAPDH
PGDH
COX-2
MRP4
EP4 antagonist induces EV/exosome release from Ras-transformed mammary epithelial stem cells
Lin MC, Chen SY et al. Int J Cancer. 2018;143(6):1440-1455
A HMLE
-R
NAMEC
-R
GW-
NAMEC
-R
1x106
2x105
5x104
0 ~
4x10 -7
8x10 -7 ~
4x10 -6
8x10 -8 ~
1x10 -6CSC/TICfrequency
0/6
0/6
0/6
4/6
3/6
1/6
2/6
0/6
0/6
injected cells not
forming tumor
injected cells
forming tumor
C NAMEC-Ras GW-NAMEC-RasMOCK
0
2 0
4 0
6 0
8 0
1 0 011 0 0 6
21 0 0 6
31 0 0 6
41 0 0 6
51 0 0 6
61 0 0 6
E not forming liver metastasis
forming liver metastasis
*
2x106
4x106
6x106
100
60
20
Num
ber
of tu
mor
cells/2
5 m
g
lung tis
sue
B
Lung
Liv
er
NAMEC-R GW-NAMEC-R
D ii iiii
v viiv
viii ixvii
xi xiix
*
* **
**
*
0
NA
ME
C-R
as
GW
-NA
ME
C-R
as
EP4 antagonist decreases CSC properties ofRas-transformed mammary epithelial stem cells
Lin MC, Chen SY et al. Int J Cancer. 2018 Sep 15;143(6):1440-1455
MCF-7
(CD44lo/Cd24hi
)
MDA-MB231
(CD44hi/Cd24lo)
Percentage 97.8 % +/-0.2% 96.6 %+/- 0.1%
MFI of CD44 22.3+/- 0.6 293+/- 9.5
MFI of CD24 817+/- 13.6 33+/- 0.6CD24
CD
44
GW (μg/ml)0
5
10
15
0 1 3 6
EV
pro
tein
(p
g)/
ce
ll MCF-7 MDA-MB231*
0
3
6
GW
(μ
g/m
l)
CD44 Integrin β1
MCF7 MDA-MB231
Cell n
um
ber
X 0 1 3 6 0 1 3 6
MCF7 231
0 1 3 6
MCF7 231
Exo Cell
EP4
GAPDH
COX-2
PGDH
mPGES-1
MCF-7 MDA-MB-231
0 1 3 6
CD44
CD81GAPDH
E-cadherin
Integrin α6
Integrin β1
β-catenin
Fibronectin
Vimentin
GW (μg/ml)
0
5000
10000
15000
20000
25000
30000
0 1 3 6
EV
x 1
03/c
ell
MCF7 MDA-MB231
***
***
*
GW (μg/ml)
5
35
20
25
15
10
0
100 101 102 103 104
102
103
104
101
100
100 101 102 103 104
MRP4
101 102 103 100 101
119
123
126
595
533
474
4.26
4.00
3.86
2.65
2.59
2.56
EP4 antagonist decreases CSC properties by inducing exosome release
Lin MC, Chen SY et al. Int J Cancer. 2018;143(6):1440-1455
Num
ber
of cells/f
ield
0
100
200
300
400
500 ***
Dy+GWCrl GW
***
Crl GW Dy+GWN
um
ber/
300 c
ells
Crl GW Dy+GW
*****
0
20
40
60
80
Crl GW Dy+GW
Dy+GWCrl GW
injected cells not
forming tumor injected cells
forming tumor
*
injected cells not
forming tumor
injected cells
forming tumor
p/s
ec/c
m2/s
r
l
Crl
GW
Dy+
GW
2x109
Tota
l F
luore
centF
lux (photo
ns/s
ec)
1.5x109
1x109
0.5x109
0
EP4 antagonist decreases CSC properties by inducing exosome release
Crl GW Dy+GW
1x105
1x104
1.4x10-4 4.6x10-5 2.1x10-4CSC/TIC
frequency
0/6
0/6 3/9
2/6
0/6
injected cells not
forming tumor
injected cells
forming tumor
8/8 0/69/9 0/68/8
6/8 7/8
Lin MC, Chen SY et al. Int J Cancer. 2018;143(6):1440-1455
Crl 231-EVGW-in-
231-EV
GW-in-
NA-EV
E
H1 2 3 4 5 6
Lung metastasis
Crl
GW-in-
NA-EV
1 2 3 4 5 6Liver metastasis
Crl
GW-in-
NA-EV
D
Crl 231-EV GW-in-
231-EV
GW-in-
NA-EV
5x105
1x105
5x104
1x104
CSC/TIC
frequency9 x 10-6 6 x 10-6 3 x 10-5 3 x 10-5
injected cells not
forming tumor injected cells
forming tumor
6/6
4/9
4/9
2/9
7/7
4/9
1/9
1/9
6/6
7/7
6/9
2/9
8/8
9/9
5/9
4/9
A
02468
10121416
Crl
Crl EV
Num
ber/
2000 c
ells
EV E-cad/DAPI
BCrl 231 EV
GW-in-231 EV GW-in-NA EV
C
0
10
20
30
40
50
Num
ber/
2000 c
ells
*********
*
F Crl GW-in-NA-EV
H&
EV
IM/D
AP
Iα
-SM
A/K
i67/D
AP
I
***
Crl EV
0
0.05
0.1
0.15
Tum
or
Weig
ht (g
)
**
G
0
20
40
60
80
Ki6
7 Index (
%)
****
EP4 antagonist-induced CSC exosomestransfer CSC properties to non-CSCs
Lin MC, Chen SY et al. Int J Cancer. 2018 Sep 15;143(6):1440-1455
Blocking EP4-mediated signaling reduces CSC numbers in tumors
Lin MC, Chen SY et al. Int J Cancer. 2018;143(6):1440-1455
05
101520253035
***
% C
ell
Via
bili
ty
Paclitaxel (nM)
HMLE-RNAMEC-R
10-4 10-2 100 102 104
100
50
0
GW-NAMEC-R
- + - - + - - + - + - + - +231 231 NAMEC
NAMEC
-R NAMEC
NAMEC
-R
BCRP
MRP2
MRP3
MRP4
GAPDH
-
cell cell EVEV
CD81
TSG101
Alix
% C
ell
Via
bili
ty
Tumor chemoresistance is decreased via EP4 antagonist-induced exosome release
Lin MC, Chen SY et al. Int J Cancer. 2018;143(6):1440-1455
ns**
***
* nsns
***
Lo
Vo
tum
or
we
igh
t (g
)
iii iv
01020304050607080
Lo
Vo
tum
or
gro
wth
ra
te
(mm
3/d
ay)
***
***
**
**
ii
***
5 8 11 15days of treatment
i
Lo
Vo
tum
or
vo
lum
e (
mm
3)
Lo
Vo
tum
or
vo
lum
e (
mm
3)
1000
800
600
400
200
0
DMSO GW Pac GW+Pac
1500
1000
500
0
0.8
0.6
0.4
0.2
1.72
1.8ns
ns*
HC
C1
80
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mo
r w
eig
ht (g
)
0.5
1.0
0
ns
****
***
MD
A-M
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31
(fL
uc)
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or
gro
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lative
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0
20
15
10
5
Tumor chemoresistance is decreased via EP4 antagonist-induced exosome release
Lin MC, Chen SY et al. Int J Cancer. 2018;143(6):1440-1455
CSCs are
resistant to
chemotherapy
EP4 antagonists
EP4 antagonists induce EV
release from CSCs
CSCs convert to non-CSCs after
releasing EVs
Non-CSCs convert to CSC-like
cells after taking up EVs
CSCs convert to non-CSCs after
releasing EVs
Non-CSCs and converted non-
CSCs are both killed by
chemotherapy
i
ii
iii
Tumor chemoresistance is decreased via EP4 antagonist-induced exosome release
Lin MC, Chen SY et al. Int J Cancer. 2018
early
endosome
EP4 antagonism
EVs/exosomes with
cargos for SC properties
SCs lose
stemness via the
EVs/exosomes
EP4 antagonists Chemo
killed
CSC
CSC
Non-CSC
EP4 receptor
EP4
antagonist
chemotherapy
EP4 antagonist
MSCAnti-EP4 elicited MSC EVs
recovered neurons
Intact BBB
A2 astrocyte
microglia
Inhibiting
inflamma
tion
Suppressing
Astrogliosis
Recovery of neurons
Station 1CANCER
THERAPY
REGENERATIVE
MEDICINE
Station 2
Station 3
EVs in Regenerative
Medicine
Non-SCs acquire SC
properties via
the induced EVs/exosomes
early
endosome
EP4 antagonism
EVs/exosomes with
cargos for SC properties
SCs lose
stemness via the
EVs/exosomes
EP4 antagonists Chemo
killed
CSC
CSC
Non-CSC
EP4 receptor
EP4
antagonist
chemotherapy
EP4 antagonist
MSCAnti-EP4 elicited MSC EVs
recovered neurons
Intact BBB
A2 astrocyte
microglia
Inhibiting
inflamma
tion
Suppressing
Astrogliosis
Recovery of neurons
Station 1CANCER
THERAPY
REGENERATIVE
MEDICINE
Station 2
Station 3
EVs in Regenerative
Medicine
Non-SCs acquire SC
properties via
the induced EVs/exosomes
from http://www.ahajournals.org/
Proposed therapies for brain damage and degeneration disease
Blocking PGE2/EP4 signaling of mesenchymal stem cells elicits the release of EVs and exosomal protein sorting
Chen SY et al. Stem Cells Transl Med, 2019 8:707-723
EP4 antagonist-elicited MSC EVs rescue memory and learning deficiencies caused by hippocampal damage
DTA
(UC)Camk2a/DTA
(DC)
H&
EN
euN
Camk2α
DTA DTATRE TRE
+Dox
Damaging neural cells in
CA1 region of hippocampus
tTA
0
1
2
3
4
5
6
7
PK
H2
6 p
ositiv
e a
rea
in
CA
1 (
%)
PKH26/DAPI
PKH26-iMSCEVPKH26-PBS PKH26-MSCEV
*** ***
Chen SY et al. Stem Cells Transl Med, 2019 8:707-723
40
50
60
70
80
*** ***
***
NS
E
Tim
e s
pent
on n
ew
location (%
)
40
50
60
70
80*** ***
NS
*
F
Tim
e s
pent
on n
ew
obje
ct (%
)
Birth 6 wk
PBSEV Inj.GWEV
DTA (UC)
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-Dox 25d 10d 10d
Behavior test
024
48
72
96
120
2 0
4 0
6 0
D a y s o f tra in in g
La
te
nc
y (s
)
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G
NS20
40
60
La
ten
cy (
s)
DCUC EV GWEV
***
**NS
**
*** ***
******
NS
024
48
72
96
120
2 0
4 0
6 0
D a y s o f tra in in g
La
te
nc
y (s
)
N o rm a l
In d u c e
E x o tre a te d in d u c e
G W E x o tre a te d in d u c e
Chen SY et al. Stem Cells Transl Med. 2019;8(7):707–723
9.98 s 56.23 s 36.75 s 13.51 s
EP4 antagonist-elicited MSC EVs rescue memory and learning deficiencies caused by hippocampal damage
EP4 antagonist-elicited MSC EVs increase the expression of genes involved in anti-inflammation in damaged
hippocampus
Immune system
Process (18.26%)
Biological processCellular process
Response to
stimulus
Single-organism
process
Metabolic
process
Multi-organism
process
Cellular component
organization or biogenesisBiological
adhesion
Locomotion
Developmental
process
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process
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process-l
og
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r
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160
140
120
0
60
40
20
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s
C/E
BP
δ
Birth 6 wk
PBS
EV Inj.
GWEV
-Dox 25d 10d 10d
DTA
Camk2a/DTA
5d
30d
Analyzing
Hippocampus
(short) (long)
gene expression in PBS-treated hippo DC (log2)
gene
exp
ress
ion
in
MS
CE
V-t
reat
ed h
ippo
c(lo
g2)
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18
16
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2 1816141210864gene expression in
PBS-treated hippo DC (log2)
2
18
16
14
12
10
8
6
4
2 1816141210864
gene
exp
ress
ion
in
MS
CG
WE
V-t
reat
ed h
ippo
(lo
g2)
Chen SY et al. Stem Cells Transl Med, 2019 8:707-723
EP4 antagonist-elicited MSC EVs suppress reactive astrogliosis
Num
ber
of G
FA
P+
astr
ocyte
(30d)
0
10
20
30
40
NC
ind
NC
EV
GW
EV
*****
***iii
010203040506070 ***
***
Ei
Num
ber
of G
FA
P+
astr
ocyte
(5d)
0
1000
2000
3000
4000
* ****
ii
C3
+ a
rea
(μ
m2)
(5d
)
Num
ber
of G
FA
P+
/S100
β+
astr
ocyte
(5d)
0
20
40
60***
*******
iv
0
10
20
30
40 *****
***v
Num
ber
of G
FA
P+
/S100β+
astr
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(30d)
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qa
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s
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EV
NC
D
UC
DC
EV
GW
EV
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PI
S100β/DAPI GFAP/DAPI S100β/DAPI
5 Day 30 Day
Induce
UC
DC
UC
DC
Chen SY et al. Stem Cells Transl Med, 2019 8:707-723
EP4 antagonist-elicited MSC EVs suppress extensive inflammation in damage hippocampi
DC
EV
GW
EV
A
Iba1/DAPI
UC
EV
GW
EV
UC
B
Iba1/DAPI
DC
0
10
20
30
40
50
60**
**
Nu
mb
er
of m
icro
glia
in C
A1
0
10
20
30
40
50
******
***
****
Nu
mb
er
of m
icro
glia
in C
A1
Chen SY et al. Stem Cells Transl Med, 2019 8:707-723
EP4 antagonist-elicited MSC EVs increase integrity of BBB in damage hippocampi
Chen SY et al. Stem Cells Transl Med, 2019 8:707-723
EP4 antagonist-elicited MSC exosomes increase neuroregeneration
Chen SY et al. Stem Cells Transl Med. 2020;9:499–517 FEATURED ARTICLE
EP4 antagonist-induced MSC exosomes promote neurogenesis and neuritogenesis
Chen SY et al. Stem Cells Transl Med. 2020;9:499–517 FEATURED ARTICLE
The elevated CNP in EP4 antagonist-induced MSC exosomes contributes to β3-tubulin polymerization
0
20
40
60
80
100
120
140 ******
****
adenosin
e (
pm
ole
)
D
A
CNP
GAPDH
shG
FP
shcontr
ol
shC
NP
-1
shC
NP
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B C
shC
NP
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shG
FP
shcontr
ol
shC
NP
-1
CNP
GAPDH
0
10
20
30
40
50
60
Eβ
3-t
ub
ulin
po
lym
eri
za
tio
n
* ****
0
10
20
30
40
50
60
β3
-tu
bu
lin
po
lym
eri
za
tio
n
F
*** ***
***
Chen SY et al. Stem Cells Transl Med. 2020;9:499–517 FEATURED ARTICLE
The CNP contributes to neurogenesis and neuritogenesisin the damaged hippocampi
C
0
5
10
15
20
25
***
***
MA
P2 p
ositiv
e a
rea (
%)
i
0
2
4
6
8
10
12
14
16
18
ii
***
***
β3-T
UB
positiv
e a
rea (
%)
0
10
20
30
40
50
60***
iii
CA
1 n
eu
ron
DA
PI
Th
ickn
ess (
μm
)
***
Chen SY et al. Stem Cells Transl Med. 2020;9:499–517 FEATURED ARTICLE
A
B
Chen SY et al. Stem Cells Transl Med. 2020;9:499–517 FEATURED ARTICLE
A B
40
45
50
55
60
65
70
Tim
e s
pe
nt o
n n
ew
lo
ca
tio
n (
%)
****
* *
UC
40
45
50
55
60
65
70T
ime
sp
en
t o
n n
ew
ob
ject (%
)
**
***
**
**
10
30
50
Day 1 Day 2 Day 3 Day 4 Day 5
NC shGFP shcontrol
shCNP-1 shCNP-2
La
ten
cy (
s)
C
The elevated CNP contributes to restoring brain functions
CNS therapy by EP4 antagonist-elicited MSC extracellular vesicles
EP4 antagonist
MSC Anti-EP4 elicited MSC EVs(IL-2↑, IL-10↑, TIMP1↑,
VEGF-a↑, BDNF↑)
recovered neurons
Intact BBB(CLDN5+)
A2 astrocyte (CD109+/GFAPlo)
microglia
A1 reactive
Astrocyte(C3+/GFAPhi/S100β+)
activated
Microglia(Iba1+)
damagedneuron
broken BBB
Cq1
C3
C3, S100β
Cq1
Activation Damage
Damage
Activation
Damage
ActivationActivation
Chen SY et al. Stem Cells Transl Med, 2019 8:707-723 FEATURED ARTICLE
Chen SY et al. Stem Cells Transl Med. 2020;9:499–517 FEATURED ARTICLE
early
endosome
EP4 antagonism
EVs/exosomes with
cargos for SC properties
SCs lose
stemness via the
EVs/exosomes
EP4 antagonists Chemo
killed
CSC
CSC
Non-CSC
EP4 receptor
EP4
antagonist
chemotherapy
EP4 antagonist
MSCAnti-EP4 elicited MSC EVs
recovered neurons
Intact BBB
A2 astrocyte
microglia
Inhibiting
inflamma
tion
Suppressing
Astrogliosis
Recovery of neurons
Station 1CANCER
THERAPY
REGENERATIVE
MEDICINE
Station 2
Station 3
Non-SCs acquire SC
properties via
the induced EVs/exosomes
Acknowledgement LI LAB (NHRI)
Meng-Chieh LinShih-Yin ChenPei-Lin HeHo-Min TsaiJia-Shiuan TsaiWinty Lo
FORMOR COLLABORATOR
Dr. Harvey Herschman (UCLA)Sarah Dry (UCLA)Dr. Robert Weinberg (MIT/WI)Wai Leong Tam (MIT/WI)Xin Ye (MIT/WI)
CURRENT COLLABORATOR
Immunomodulation
Dr. Li-Tzong Chen (NHRI/NCI)Dr. Wen-Chun Hung (KMU)Dr. Yan-Shen Shan (NCKU/NCKUH)
Drug Development
Dr. Chiung-Tong Chen (NHRI/IBPR)
Brain Technology
Dr. Ing-Ming Chiu (NHRI/ICSM)Dr. Gin-Shin Chen (NHRI/IBEN)
Protein Interaction
Dr. Wen-Hung Kuo (NTUH)
Small Molecules for Exosome Induction
Dr. Lun Kelvin Tsou (NHRI/IBPR)GRANT SUPPORT
NHRIMOSTIndustry-Academy Collaboration Grant
Acknowledgement
LI LAB (NHRI)
Meng-Chieh LinShih-Yin ChenPei-Lin HeHo-Min TsaiJia-Shiuan TsaiWinty Lo
HERSCHMAN LAB (UCLA)
Harvey HerschmanSarah Dry
WEINBERG LAB (MIT/Whitehead Institute)
Robert WeinbergWai Leong TamXin Ye
GRANT SUPPORT
NHRIMoSTSusan G. Komen foundation