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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
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