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


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


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 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


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



Blocking EP4-mediated signaling in MaSCs causes release of stem cell surface markers and integrin via EV release


Blocking EP4-mediated signaling in MaSCs causes release of stem cell surface markers and integrin via EVrelease


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 -+ ++ +


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


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


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


EP4 antagonist-induced EVs contain proteins required to maintain basal mammary epithelial cell properties


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


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


- + + +

- - 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


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


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


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


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


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


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


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


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


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


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


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


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


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

6tu

mo

r w

eig

ht (g

)

0.5

1.0

0

ns

****

***

MD

A-M

B-2

31

(fL

uc)

tum

or

gro

wth

(Re

lative

fL

uc

activity)

0

20

15

10

5



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


Lin MC, Chen SY et al. Int J Cancer. 2018

early

endosome

EP4 antagonism

EVs/exosomes with


SCs lose

stemness via the

EVs/exosomes

EP4 antagonists Chemo

killed

CSC

CSC

Non-CSC

EP4 receptor

EP4

antagonist

chemotherapy

EP4 antagonist


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


early

endosome

EP4 antagonism

EVs/exosomes with


SCs lose

stemness via the

EVs/exosomes


killed

CSC

CSC

Non-CSC

EP4 receptor

EP4

antagonist

chemotherapy

EP4 antagonist


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


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

*** ***


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)

Camk2a/DTA mice (DC)

-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

)

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

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

SignalingBehavior

Reproductive

process

Rhythmic

process-l

og

10(p

-valu

e)

10

5

TS

C2

IFN

GT

NF

CR

EM

TG

FB

1

HT

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10

CR

EB

1

TG

FB

R2

GR

TC

F7L2

IL3

IL4

ZN

F106

Cdkn1a

Apold

1

Dusp1

Cyr6

1

Mfs

d2a

Zfp

36

Nfk

bia

Osm

r

Fos

GWEV/DC

EV/DC

GWEV/EV100

160

140

120

0

60

40

20

Fold

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)

2

18

16

14

12

10

8

6

4

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)


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

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ii

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+ a

rea

(μ

m2)

(5d

)

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ber

of G

FA

P+

/S100

β+

astr

ocyte

(5d)

0

20

40

60***

*******

iv

0

10

20

30

40 *****

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p

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a

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qc

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qa

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ql1

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qb

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s

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A2 marker A1 marker A1 inducerC

A

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GAPDH

CD44

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Induce

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DC

UC

DC


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


EP4 antagonist-elicited MSC EVs increase integrity of BBB in damage hippocampi


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


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

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10

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10

20

30

40

50

60

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-tu

bu

lin

po

lym

eri

za

tio

n

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*** ***

***


The CNP contributes to neurogenesis and neuritogenesisin the damaged hippocampi

C

0

5

10

15

20

25

***

***

MA

P2 p

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e a

rea (

%)

i

0

2

4

6

8

10

12

14

16

18

ii

***

***

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UB

positiv

e a

rea (

%)

0

10

20

30

40

50

60***

iii

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1 n

eu

ron

DA

PI

Th

ickn

ess (

μm

)

***


A

B


A B

40

45

50

55

60

65

70

Tim

e s

pe

nt o

n n

ew

lo

ca

tio

n (

%)

****

* *

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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)

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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


early

endosome

EP4 antagonism

EVs/exosomes with


SCs lose

stemness via the

EVs/exosomes


killed

CSC

CSC

Non-CSC

EP4 receptor

EP4

antagonist

chemotherapy

EP4 antagonist


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


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

Documents

A Journey In and Outside of Stem Cells --- Exosomes