Induction of Endothelial Cell

from Fibroblast by 5 Defined Factors

Hyo-Soo Kim, MD, PhD, FAHA Director, National Research Laboratory for Cardiovascular Stem Cell,

Seoul National University Hospital (SNUH),

Seoul, Republic of Korea

Presenter Disclosure

• Nothing to disclose

• New vessel formation using endothelial cells

holds great therapeutic promise.

• ESC- or iPSC-derived ECs

– Ethical hurdles

– No standardized protocol

– Necessity for complex manipulation of EB

– Low differentiation efficiency

– Risk of contamination by feeder cells

Background

Waddington’s Epigenetic Landscape

Ladewig et al., Nat. Rev. Mol. Cell Biol. 2013

Conventional concept De-differentiation Trans-differentiation

Adapted from Ladewig et al., Nat. Rev. Mol. Cell Biol. 2013

Fibroblasts

(mouse dermal or cardiac)

Exocrine pancreatic cells

(mouse)

Fibroblasts

(mouse embryonic or dermal

or tail tip)

Fibroblasts

(mouse skin or tail)

Fibroblasts

(human skin)

Gata4, Hnf1α, Foxa3

Gata4, Mef2C. Tbx5, Hand2

miR-1

Immature

cardiomyocytes

miR-133

miR-208, miR-499

NGN3( or NeuroD1), PDX1, MAFA

Hnf4α, Foxa1 / Foxa2 / Foxa3

Gata4, Hnf1α, Foxa3

Brn2, Myt1l Ascl1

mir-9/9*

mir-124

Ascl1, Brn2,

Myt1l

Ascl1

Immature

neurons

Acsl1, Myt1l,

NeuroD1

NeuroD1

Ngn2

small molecules

Cardiomyocytes

Endocrine (insulin-producing)

pancreatic β-cells

Hepatocytes

Functional

neurons

No ethical hurdles

No necessity for complex manipulation of EB

No risk of contamination by feeder cells…

Aim:

• To develop a novel methodology providing

endothelial cells from fibroblasts via direct

conversion.

mesodermal progenitor cell

hemangioblast

hematopoietic

stem cell angioblast

Tal1, Gata2, Fli1

Ets family

(ER71=Etv2,

ETS-1, ELF-1, Erg)

endothelium

Ets family,

Klf proteins

Key T.F.s in EC development

+ Co-workers of endothelial transcription

– FOXO1, FOXC, LMO2 (co-factor)

+ Regulator of endothelial functions

– Klf2

• Target cells: adult fibroblast

• Species: mouse

• Read-out: tie2-GFP

Adapted from De Val et al., Cell 2009

11 Candidate Factors

Gene symbol Accession Number

Gata2 NM_008090

Foxc2 NM_013519

Elf1 NM_007920

Erg NM_133659

Fli1 NM_008026

Tal1 NM_011527

Foxo1 NM_019739

Lmo2 BC057880

Ets1 BC010588

Er71(Etv2) NM_007959

Klf2 NM_008452

JK Han,,, HS Kim. Circulation 2014

PE

Tie2-GFP

100%

0%

DMEM+FBS10% EBM2+FBS10% EBM2+FBS10%

SFB iEC

Tie-2 promoter GFP Tie-2 promoter GFP

Tie2-GFP Tg

Lentiviruses

FACS

D0 D2 D12

11 factors

PE

Tie2-GFP

0.9% 0%

Mock

JK Han,,, HS Kim. Circulation 2014

Gata2 Foxc2 Elf1

Erg Foxo1 Fli1 Tal1

Er71 Lmo2 Ets1 Klf2

Mock P

E

Tie2-GFP

0% 0% 0% 0%

0% 0% 0%

0% 0% 0%

0.2%

0.4%

Series of ‘Single Factor’ Transduction Experiments

Mo

ck

5 f

ac

tors

Ga

ta2

Fo

xc

2

Elf

1

Erg Fli

Ets

1

0

1

2

3

4

5

Tie

2-G

FP

+ c

ell

s (

%)

*

* *

* * *

*

5F+1 M

oc

k

5 f

ac

tors

Fo

xo

1

Er7

1

Klf

2

Ta

l1

Lm

o2

0

1

2

3

4

5

Tie

2-G

FP

+ c

ell

s (

%)

*

* *

* *

*

5F-1

2F+1

Co

ntr

ol

2 f

ac

tors

Ga

ta2

Fo

xc

2

Elf

1

Erg Fli

Ta

l1

Lm

o2

Ets

1

Klf

2

0

1

2

3

4

5

Tie

2-G

FP

+ c

ell

s (

%)

*

Klf2

on Foxo1/Er71

3F+1

Co

ntr

ol

3 f

ac

tors

Ga

ta2

Fo

xc

2

Elf

1

Erg Fli

Ta

l1

Lm

o2

Ets

1

0

1

2

3

4

5

Tie

2-G

FP

+ c

ell

s (

%)

*

Tal1 out of 8

Mo

ck

4 f

ac

tors

Ga

ta2

Fo

xc

2

Elf

1

Erg Fli

Lm

o2

Ets

1

0

1

2

3

4

5

Tie

2-G

FP

+ c

ell

s (

%) *

4F+1

Lmo2 out of 7

JK Han,,, HS Kim. Circulation 2014

5 Key Factors for Endothelial Reprogramming

5F Mock

PE

Tie2-GFP

4.0% 0%

Mock 5F

CD31

mock

5F

D3

vWF

0.9%

11.9%

7.3%

Flk1

0.5%

1.8%

2.6%

VE-Cadherin

0.5%

4.1%

4.2%

0.5%

1.8%

2.3%

PE

Count

D7

D12

*

*

*

*

* *

*

EC markers

during the

Programming

Lentiviral Silencing

D0

SFB

D3

5F

D7

5F

D12

5F

iEC

0

500

1000

1500

Rela

tive m

RN

A e

xp

ressio

n

D0

SFB

D3

5F

D7

5F

D12

5F

iEC

0

1000

2000

3000

Rela

tive m

RN

A e

xp

ressio

n

D0

SFB

D3

5F

D7

5F

D12

5F

iEC

0

10

20

30

Rela

tive m

RN

A e

xp

ressio

n

D0

SFB

D3

5F

D7

5F

D12

5F

iEC

0

100

200

Rela

tive m

RN

A e

xp

ressio

n

D0

SFB

D3

5F

D7

5F

D12

5F

iEC

0

100

200

300

Rela

tive m

RN

A e

xp

ressio

n

Foxo1 Er71

Tal1 Klf2 Lmo2

* * *

* * *

*

*

*

EC Markers after Endothelial Reprogramming

Mock 5F

VE-cadherin/DAPI

Mock 5F

CD31/DAPI

Mock 5F

Flk1/DAPI

Before sorting by Tie2

JK Han,,, HS Kim. Circulation 2014

Induced Endothelial Cells:

after Tie2 sorting

Mock-SFB iEC

JK Han,,, HS Kim. Circulation 2014

iEC Characterization: RT-PCR

* *

*

* *

*

N.D.

*

* *

*

* *

Mock-SFB

iEC

Primary EC

MS1

Relative mRNA expression

VE-cadherin

0.0

0.5

1.0

1.5

CD31

0.0

0.5

1.0

1.5

ICAM2

0.00

0.02

0.04 0.5

1.0

1.5

Tie2

0.0

0.5

1.0

1.5

JK Han,,, HS Kim. Circulation 2014

Mock-SFB iEC

VE-cadherin/DAPI

Mock-SFB iEC

CD31/DAPI

Mock-SFB iEC

BS1 lectin/DAPI

Mock-SFB iEC

DiI-Ac-LDL/DAPI

iEC Characterization: IF, EC Fx.

After sorting by Tie2

Mock-SFB iEC

iEC Characterization: Matrigel Tube Formation

Primary EC MS1

JK Han,,, HS Kim. Circulation 2014

iEC Characterization: NO Production

0

2

4

6

NO

(u

M)

VEGF

Ach

L-NAME

A23187

-

+

- -

-

- +

- -

- - +

+

+

- -

+

- +

- +

- - +

*

-

+

- -

-

- +

- -

- - +

+

+

- -

+

- +

- +

- - +

-

+

- -

-

- +

- -

- - +

+

+

- -

+

- +

- +

- - +

-

+

- -

-

- +

- -

- - +

+

+

- -

+

- +

- +

- - +

* * * * *

*

* *

# # # # # # #

#

Mock-SFB iEC Primary EC MS1

#

After overnight incubation in 2 ng/ml VEGF, culture supernatants were harvested,

and NO was assayed using a NO Detection Kit.

JK Han,,, HS Kim. Circulation 2014

Endothelial Epigenetics / Genetics +

2.0

0

-2.0

SFB-1

SFB-2

Mock-SFB-1

Mock-SFB-2

Mock-SFB-3

iEC-1

iEC-2

Primary EC-1

Primary EC-2

MS1-1

MS1-2

iEC-3

●●●○○●●●

●●●●○●●●

●●●●●●●●

●●●○○●●●

●●●●●●●●

●●●●●●●●

●●●●○●●●

●●●●●●●●

Mock-

SFB

iEC

Primary

EC

MS1

VE-

cadherin

○●○○○○○○

○●○○○○●○ ○○●○○○●○

○●●○○○○○

○○○○○○●○ ○○○○○○○○

○○○○○○○○ ○○●○○○○●

○●○○○○●○

○●●○○○○○ ○○○○○○●○ ○●○○●○○○

●●○○○○●○

●○○○○○○● ○○○○○○○○ ○●○○●○○○

○○○○○○○○

○○○○○○○○

○○○○○○○○

○○○○○○○○

○○○○○○○○

○○○○○○●●

○○○○○○○○ ○○○○○○○○

Tie2

○○○○○

○○●○○ ○○○○○

○○○○○

○○○○○

○○○○○ ○○○○○

●○○○○

●●●●●

○○●●○ ●●●●●

○●●○●

●●●●●

●●●●● ○●●○●

○●●○●

●○●○○

○○●○○ ○○●●○

●○●○○

○○●○○

○○●○○ ○○○○○

○○●○○

●○○○○

○○○●● ○○○○○

○○○○○

○○○○○

○○○○○

○○○○○

○○○●○

Affymetrix GeneChip Mouse Gene 1.0 ST Array Bisulfite sequencing

EC specific

genes

SF

B-1

S

FB

-2

Mock-S

FB

-1

Mock-S

FB

-2

Mock-S

FB

-3

iEC

-1

iEC

-2

Prim

ary

EC

-1

Prim

ary

EC

-2

MS

1-1

M

S1-2

iEC

-3

H-cadherin

Sox7

Flt1

E-selectin

Emcn

P-selectin

VE-cadherin

Ecscr

Jag1

Pear1

Tie1

Egfl7

Pecam1

Notch1

S1pr1

Gpr116

Procr

Lyve1

Flt4

Sox18

Icam2

Thbd

Esm1

Cxcr4

SF

B-1

S

FB

-2

Mock-S

FB

-1

Mock-S

FB

-2

Mock-S

FB

-3

iEC

-1

iEC

-2

Prim

ary

EC

-1

Prim

ary

EC

-2

MS

1-1

M

S1-2

iEC

-3

Tnmd Agt Plod3 Acvr1 Cul7 Etv2 F2rl1 Tgfbr1 Xdh Bmpr1a Smad4 Prox1 Dmd Bmp6 Hey2 Ccm2 Ctnnb1 Vezf1 Btg1 Bmpr2 Gdf2 Wnt7b Acvt2b Rbpj Mesp1 Lect1 Ren1 Hoxa13 Tmem100 Gpx1 Id1 Clic4 Wnt7a Heg1 Nr2f2 Hoxb5 Eng Bmp4 Abcb1b Csnk2b Pde2a Kdr Alox12 Acvrl1 Hey1 Notch1 Dll4 Col18a1 Notch4 Sox17 Sox18 Slc40a1

Endothelium

development

(GO003158)

Ets1

Lmo2

Foxo1

Tal1

Fli1

Erg

Elf1

Foxc2

Gata2

Er71

Klf2

11 factors

SF

B-1

S

FB

-2

Mock-S

FB

-1

Mock-S

FB

-2

Mock-S

FB

-3

iEC

-1

iEC

-2

Prim

ary

EC

-1

Prim

ary

EC

-2

MS

1-1

M

S1-2

iEC

-3

SF

B-1

SF

B-2

Mock-S

FB

-1

Mock-S

FB

-2

Mock-S

FB

-3

iEC

-1

iEC

-2

Prim

ary

EC

-1

Prim

ary

EC

-2

MS

1-1

MS

1-2

iEC

-3

Amot Bmper Hspb1 Fgf2 Snai2 Thbs1

Wnt5a Vegfc Pdcd6 Slit2 Gpld1 Srpx2 Ptk2b Agt Angpt4 Rgcc Apoe Bmp10 Sema4a Apoh Mir218-1 Prl7d1 Ager Nr2e1 Bmpr2 Ppap2b Prkca Mir218-2 Pdpk1 Angpt1 Hdac5 Nf1 Ccbe1 Tdgf1 Dab2ip Gdf2 Akt1 Itgb1bp1 Prkd1 Map2k5 Gpr124 Plcg1 Klf4 Hdac7 Ets1 Dll4 Prkd2 Efna1 Notch1 Pdgfb Kdr Alox12 Ptprm Krit1 Acvrl1 Ccdc23 Amotl1 Bcar1 Nr2f2 Csnk2b Angpt2 Anxa3 Epha2 Gata3 Bmp4 Mmrn2 Foxc2 Tgfb1 Itgb3 Flt4 Ptgs2 Cxcl13 Tek Vash1 Prox1 Hrg

Vegfa

Regulation of EC migration

(GO0010594)

Proliferation & Apoptosis of iECs

D0

D7

D14

D21

D28

D35

0

5

10

15

20Primary EC

iEC

To

tal cell n

um

ber

(X10

5)

Annexin V

7-A

AD

3.5±0.3%

2.1±0.9%

3.6±1.0% 4.2±0.2%

1.6±0.3% 2.9±1.8%

Growth Curve

Serum Starvation Mock-SFB iEC Primary EC

JK Han,,, HS Kim. Circulation 2014

Universal Effect of iEC-5 Factors: TTF

Mock 5F

0%

Mock

5.4%

5F

PE

Tie2-GFP

Tail-tip FB (TTF)

*

TTF

Effect of iEC-5 Factors on Monocytes

Mock 5F

BM MNC

0%

Mock

0.1%

5F

PE

*

Tie2-GFP

Unique Effect of iEC-5 Factors:

Compared with Rafii’s 3 Factors

Mock Er71/Erg/Fli1

0%

Mock

0.3%

Er71/Erg/Fli1

PE

Tie2-GFP

SFB

Not Through Pluripotency Induction:

Oct4-GFP SFBs

0% P

E

Oct4-GFP

Unstained

Mock 5F

0% P

E

Oct4-GFP

Tie2-PE ab.

stained

Mock 5F 4.9% 0%

Not Through Pluripotency Induction:

Nanog-GFP SFBs

0%

PE

Nanog-GFP

Unstained

Mock 5F

0%

PE

Nanog-GFP

Tie2-PE ab.

stained

Mock 5F 3.8% 0%

Not Through Pluripotency Induction: Oct4/Nanog stay silent during trans-differentiation

D0

MOCK 5F

D3 D7 D12 D3 D7 D12 mES

Oct4

Nanog

α-tubulin

Tie2

JK Han,,, HS Kim. Circulation 2014

In Vivo Functionality of iECs

Mock-

SFB

iEC

Primary

EC

MS1

Gross Pictures LDPI

Mouse Hindlimb

Ischemia Model: D14

JK Han,,, HS Kim. Circulation 2014

Pre D

0D3

D7

D14

0

50

100

Mock-SFB

Primary EC

MS1

iEC

Ra

tio

of

isc

he

mic

/ n

on

isc

he

mic

lim

b p

erf

us

ion

(%

)

*

Mock-SFB iEC

Primary EC MS1

Mock-SFB iEC

Primary EC MS1

JK Han,,, HS Kim. Circulation 2014

PBS

Mock

-SFB

iEC

Prim

ary

EC

MS1

0.5

1.0

1.5

2.0

Rati

o o

f cap

illa

ries /

myo

tub

e * * *

Mouse Hindlimb Ischemia Model:

Capillary Density

iEC

MS1 Primary EC

Mock-SFB

BS1-lectin / DAPI

In Vivo Engraftment Ratio = 10%

500,000

GFP+ cells

21,000

GFP+ cells

Engraftment ratio =

9.4%, 9.8%, 12.0%

in iEC, primary EC, and MS1

In Vivo Participation as EC in Capillary: 0.3%

JK Han,,, HS Kim. Circulation 2014

In Vivo Participation as EC : 0.3%

JK Han,,, HS Kim. Circulation 2014

Fibroblasts

(mouse dermal or cardiac)

Exocrine pancreatic cells

(mouse)

Fibroblasts

(mouse embryonic or dermal

or tail tip)

Fibroblasts

(mouse skin or tail)

Fibroblasts

(human skin)

Gata4, Hnf1α, Foxa3

Gata4, Mef2C. Tbx5, Hand2

miR-1

Immature

cardiomyocytes

miR-133

miR-208, miR-499

NGN3( or NeuroD1), PDX1, MAFA

Hnf4α, Foxa1 / Foxa2 / Foxa3

Gata4, Hnf1α, Foxa3

Brn2, Myt1l Ascl1

mir-9/9*

mir-124

Ascl1, Brn2,

Myt1l

Ascl1

Immature

neurons

Acsl1, Myt1l,

NeuroD1

NeuroD1

Ngn2

small molecules

Cardiomyocytes

Hepatocytes

Functional

neurons

Endocrine (insulin-producing)

pancreatic β-cells

Fibroblasts

(mouse skin or tail) Endothelial cell

Foxo1, Er71, Klf2, Tal1, Lmo2

Endothelial

differentiation

iEC vs. ESC/iPSC-derived ECs

Trans-differentiation

Induced

pluripotency

Endothelial

differentiation

iPS cell

Endothelial cell ES cell

▪ Ethical problem

▪ Tumorigenesis risk

▪ Feeder cell contam.

▪ Low efficiency

▪ Sophisticated culture condition

▪ No standardized protocol

▪ Easily obtainable adult skin cell

▪ Direct conversion into EC

▪ No need for feeder cell

Therapeutic Strategy

Transplantation

▪ > 50% cells in heart:

fibroblasts! iEC

Transduction

of iEC 5 factors

Direct

Conversion

• Cell, 2012

• Human amniotic cells

→ ECs

• 3 ETS factors:

ER71/ERG1/FLI1

• Only amniotic cells

– Not readily available

– Immunogenicity / allograft

rejection

• Not terminally

differentiated FBs

• Nat. Med, 2012

• PNAS, 2012

• Human FBs → ECs

• Y’ iPS 4 factors:

OCT4/SOX2/KLF4/MYC

• Via partial iPSC or

progenitor cells status

– Concerns over tumorigenic

potential

Summary

• The first study demonstrating that adult

fibroblasts can be directly converted to ECs by

defined factors.

• These iEC 5 factors are Foxo1, Er71, Klf2,

Tal1 and Lmo2.

• iECs exhibit endothelial features and functions

in vitro and in vivo.

Conclusions

• Our study provides further evidence that cell fate determination is not eternal, but plastic by the formation of new transcriptional network.

• Our findings identify the molecular background of endothelial differentiation and trans-differentiation.

• This study makes significant progress towards future clinical application.

iEC Project

Jung-Kyu Han, MD

Sung-Hwan Chang,

Hyun-Ju Cho, PhD

Saet-Byeol Choi,

Hyo-Suk Ahn, MS

Jung-Soo Lee

Fund

• Innovative Research

Institute for Cell Therapy

(IRICT), Korea

• National Research

Foundation (NRF), Korea