Articles
Conversion of human fibroblasts to angioblast-like
progenitor cells
© 2012 Nature America, Inc. All rights reserved.
Leo Kurian1,8, Ignacio Sancho-Martinez1,8, Emmanuel Nivet1,8, Aitor Aguirre1, Krystal Moon1, Caroline Pendaries2,
Cecile Volle-Challier2, Francoise Bono2, Jean-Marc Herbert2, Julian Pulecio3, Yun Xia1, Mo Li1, Nuria Montserrat3,
Sergio Ruiz1, Ilir Dubova1, Concepcion Rodriguez1, Ahmet M Denli4, Francesca S Boscolo5–7, Rathi D Thiagarajan5–7,
Fred H Gage4, Jeanne F Loring5,6, Louise C Laurent5–7 & Juan Carlos Izpisua Belmonte1,3
Lineage conversion of one somatic cell type to another is
an attractive approach for generating specific human cell
types. Lineage conversion can be direct, in the absence
of proliferation and multipotent progenitor generation,
or indirect, by the generation of expandable multipotent
progenitor states. We report the development of a
reprogramming methodology in which cells transition through
a plastic intermediate state, induced by brief exposure to
reprogramming factors, followed by differentiation. We use
this approach to convert human fibroblasts to mesodermal
progenitor cells, including by non-integrative approaches.
These progenitor cells demonstrated bipotent differentiation
potential and could generate endothelial and smooth muscle
lineages. Differentiated endothelial cells exhibited
neo-angiogenesis and anastomosis in vivo. This methodology
for indirect lineage conversion to angioblast-like cells adds to
the armamentarium of reprogramming approaches aimed at the
study and treatment of ischemic pathologies.
Somatic cell reprogramming has highlighted the plasticity of adult
somatic cells as well as the possibility of generating any desired
cell type in unlimited amounts. Three approaches for somatic
cell reprogramming based on the forced expression of transcription factors (TFs) have been described1. First, somatic cells can
be reprogrammed into induced pluripotent stem cells (iPSCs),
embryonic-like cells with the potential to generate any adult cell
type2. Second, TFs defining or specifying target-cell identity have
proven successful for the direct lineage conversion of mouse and
human cells into several cell types3–6. Finally, the fact that reprogramming, or de-differentiation to iPSCs, proceeds in a stepwise manner suggests that the process can be stopped before the
acquisition of an embryonic-like signature. Indeed, coupling of a
partially de-differentiated state to specific differentiation conditions has demonstrated a feasible alternative method to generate
murine cardiac and neuronal cells7–11.
Here we present a method for the simple and efficient conversion
of human fibroblasts to CD34+ progenitor cells with bipotent differentiation potential. We use a reprogramming strategy in which
complete reprogramming to pluripotency is shortened or bypassed
and the cells transition through a plastic intermediate state. This
allows redifferentiation into CD34+ progenitor cells and subsequently to functional endothelial and smooth muscle cells. We
thus demonstrate for the first time, to our knowledge, that a reprogramming strategy involving partial de-differentiation is feasible in
human cells for the generation of multipotent progenitors.
RESULTS
Differentiation to angioblast-like cells
Prior to establishing our lineage-conversion conditions, we
developed a robust medium suitable for the differentiation of
pluripotent stem cells (PSCs) to mesodermal progenitor cells.
We systematically analyzed well-known mediators of mesodermal development in different human PSC (hPSC) lines12. We
established a mesodermal induction medium (MIM) for efficient differentiation of hPSCs to a mesodermal fate (Fig. 1a and
Supplementary Fig. 1).
We tested MIM-mediated differentiation by assessing the
expression of CD34, an early marker for mesoderm-derived progenitor cells with hematopoietic and/or endothelial and smooth
muscle differentiation potential, in multiple hPSC lines (Fig. 1b–f
and Supplementary Figs. 1 and 2). We observed a peak of CD34+
cells by day 8 in every analyzed cell line. In parallel, we observed
upregulation of the vascular marker CD31 and mesodermal progenitor markers (Fig. 1b–g and Supplementary Figs. 1 and 2)
accompanied by rapid downregulation of pluripotency-related
markers (Fig. 1g and Supplementary Fig. 2). Additionally, we
observed upregulation of several early markers related to hematopoiesis, including those encoded by RUNX1 and TAL1 (SCL)
(Supplementary Fig. 2). Although this initially suggested that
differentiation in MIM may lead to the generation of a tripotent
1Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, California, USA. 2Sanofi-Aventis R&D, Toulouse, France. 3Center of Regenerative Medicine
in Barcelona, Barcelona, Spain. 4Laboratory of Genetics, Salk Institute for Biological Studies, La Jolla, California, USA. 5Department of Chemical Physiology, The Scripps
Research Institute, La Jolla, California, USA. 6Center for Regenerative Medicine, The Scripps Research Institute, La Jolla, California, USA. 7Department of Reproductive
Medicine, University of California, San Diego, La Jolla, California, USA. 8These authors contributed equally to this work. Correspondence should be addressed to
J.C.I.B. ([email protected]).
Received 31 July; accepted 1 November; published online 2 December 2012; DOI:10.1038/NMETH.2255
nature methods | ADVANCE ONLINE PUBLICATION | Articles
b
60
20
20
10
0
10
0
0
d0
d2
d4
d6
d8 d14
5.0
0.5
P < 0.0001
0
d0
d2
d4
d6
d0
d8 d14
d2
f
103
103
2
102
10
2
10
h
3
10
4
10
5
+
CD34 /CD31
CD34+/CD31–
2
10
+
10
3
10
4
10
Endoglin
Hoechst
5.0
2.5
5
0
Merge
0
VE-cad
VWF
Hoechst
Merge
30
20
0
P < 0.0001
d0 d2 d4 d6 d8 d14
ANGPT2
750
100
250
d0 d2 d4 d6 d8 d14
j
0
P < 0.0001
d0 d2
d4 d6 d8 d14
Primary EC
100
50
40
500
P < 0.0001
1,500
1,000
CXCR4
P < 0.0001
50
GATA2
0
d8 d14
10
d0 d2 d4 d6 d8 d14
200
d0 d2 d4 d6 d8 d14
i
KiPSEndo
VWF
7.5
10
CD34
TIE2
P < 0.0001
10.0
0
d8 d14
P = 0.0059
104
Day 8
d6
CD34 P = 0.023
CD31
KDR
ENG
CDH5
VWF
ANGPT1
ANGPT2
TIE2
HOXB4
ACTA2
SMMHC
CALD1
SM22-alpha
104
CD34–/
CD31+
Gene expression
level
CD31
© 2012 Nature America, Inc. All rights reserved.
105
Gene expression
level
Isotype
5
10
d4
2.5
d6
0
–2.0 down
20
7.5
1.0
d4
iEC
PSC
k
0 hypo
20
T (brachyury)
10.0
d2
0.5
30
40
1.5
d0
1.0 hyper
CD34+
CD31+
CD34+/CD31+
40
NANOG
SOX2
KiPS
OCT4
d0 d2 d4 d6 d8 d14
P < 0.0001
60
g
KiPS
Gene expression
level
CD34+
+
CD31
CD34+/CD31+
80
Percentage of
positive cells
e
CBiPS
30
40
0
d
40
2.0 up
Day 8
P = 0.0058
Day 6
H1
CD34+
+
CD31
CD34+/CD31+
50
P = 0.0011
Day 4
P = 0.0006
Day 2
Percentage of
positive cells
Day 0
c
HuES9
CD34+
+
CD31
CD34+/CD31+
80
P = 0.0009
Progenitor generation
P = 0.0157
Mesodermal commitment
a
Primary EC
Figure 1 | Differentiation of hPSCs into mesodermal progenitor and endothelial cells. (a) Scheme and
iEC
representative bright-field micrographs during differentiation of hPSCs toward CD34 + progenitor cells.
CD34+
(b–e) Flow cytometry analysis of the mesoderm markers CD34 and CD31 during differentiation of HuES9
PSC
embryonic stem cells (b), H1 embryonic stem cells (c), two-factor cord blood–derived iPS cells (CBiPS)
(d) and four-factor keratinocyte-derived iPS (KiPS) (e). (f) Representative flow cytometry plots after
8 d of PSC differentiation in the presence of MIM. Left, isotype controls. (g) mRNA fold change of pluripotency and mesodermal markers during
differentiation of KiPS cells. (h) Fluorescence micrographs show expression of indicated endothelial cell markers in KiPS-derived endothelial cells
(KiPSEndo). (i) mRNA expression profile showing specific upregulation of endothelial markers in KiPS Endo. (j,k) Heat map and representative clustering of
hPSCs compared to CD34+ cells differentiated from hPSCs (CD34+), endothelial cells differentiated from hPSCs (iECs) and primary human umbilical vein
endothelial cells (primary ECs). Genome-wide transcriptome analysis (j) and methylation profiling (k) are shown. For all figures, see Supplementary
Table 2 for specific gene expression changes. Error bars, s.d.; n > 3. Scale bars: 200 µm (a), 50 µm (h).
hemangioblast-like state (with hematopoietic, endothelial and
smooth muscle differentiation potential), MIM-differentiated
PSC-derived CD34+ cells (PSCCD34+) did not result in the expression of hematopoietic markers at the protein level or the formation of hematopoietic colonies in standard assays.
MIM-induced CD34+ cells may thus represent a developmental
stage similar to that of angioblast cells 13, and we consequently
investigated their potential to differentiate along endothelial
and smooth muscle lineages. Sorting of CD34+ cells after 8 d
of MIM differentiation for mesoderm commitment, followed by
subsequent differentiation toward the endothelial lineage, yielded
60–90% endoglin (encoded by ENG)- and vascular endothelial–
cadherin (encoded by CDH5)-positive endothelial cells for all
PSC lines analyzed (Fig. 1h and Supplementary Fig. 3). Notably,
we readily detected expression of von Willebrand factor (VWF),
a mature endothelial marker and pro-coagulant protein (Fig. 1h
and Supplementary Fig. 3). Quantitative PCR (qPCR) analysis
| ADVANCE ONLINE PUBLICATION | nature methods
demonstrated upregulation of endothelial markers, but not of
smooth muscle markers (Fig. 1i and Supplementary Fig. 3).
Similarly, we sorted PSCCD34+ cells and subjected them to
smooth muscle differentiation conditions, yielding a population
consisting of more than 50% smooth muscle cells as indicated by
immunofluorescence staining. Using qPCR, we found significant
upregulation of smooth muscle markers, including expression of
high–molecular weight caldesmon type 1 (CALD1), but not of
endothelial markers. With single-cell differentiation assays, we
demonstrated that the MIM-differentiated PSCCD34+ cells were
multipotent (Supplementary Fig. 4).
Our data thus indicate that MIM can be used for differentiation of hPSCs to CD34+ cells with the potential to generate both
endothelial and smooth muscle lineages. Moreover, CD34+ cells
were generated more efficiently (at least 30%, depending on the
PSC line) than in previously described protocols14–16. Genomewide DNA methylation and gene expression studies indicated
Articles
pMXs-OCT4/SOX2/KLF4/c-MYC
(± miRs 302 and 367)
+ MIM
HuES9
HFF
104
103
102
Q3 Q4
2
3
TRA 1-60
10 10 104 10
105
4
10
103
102
5
Q1 Q2
Q3 Q4
102 103 104 105
TRA 1-81
Q1
Q2
105
104
103
2
10
Q3
Q4
4
102 103 10 105
105
104
103
2
10
Q1 Q2
Q3 Q4
Q1
Q2
Q3
Q4
4
Q1
Q2
Q3
Q4
105
104
103
102
105
104
3
10
102
102 103 10 105
e
660
Q1 Q2
Q3 Q4
102 103 104 105
4
102 103 10 105
80
60
40
20
0
HFF +4F/miRs
102 103 104 105
105
4
10
103
102
CD34
CD31
ENG
CDH5
VWF
ACTA2 *
CNN1
CD45
FOXA1 *
*
Q1 Q2
f
Q3 Q4
2
3
10 10 104 10
5
*
HFF +4F
200
Endo
150
TEL
GATA1
GATA2
ETV2
ANGPT1
ANGPT2
*
HOXB4
KDR
T
CXCR4
RUNX1
TAL1
AC133
TIE2
*
*
*
HFF
*
**
HFF
+4F
Endo
HFF +4F/miRs
*
100
*
50
**
HFF
+4F/miRs
g
*
*
* * **
* * *
**
* **
*
*
* * **
** *
50
0
*
*
150
100
0
*
450
300
200
HFF
HFF
MIM
+4F
HFF
MIM
+4F/miRs
Primary EC
cEC
a clear distinction between all differentiated cells and undifferentiated PSCs at both the transcriptome and the methylome
levels (Fig. 1j,k and Supplementary Figs. 5 and 6), although
we observed some differences between PSC-endothelial differentiated cells and primary endothelial cells (see Discussion).
Conversion of human fibroblasts to angioblast-like cells
We next asked whether MIM could be coupled to partial de-differentiation or ‘plastic’ induction to convert human fibroblasts to
CD34+ angioblast-like progenitor cells (FibCD34+). We induced
plasticity by short-term exposure of fibroblasts to iPSC reprogramming conditions7,8 followed by MIM differentiation (Fig. 2a).
We first used retroviral approaches and the traditional four-factor combination of proteins (encoded by SOX2, OCT4 (POU5F1),
KLF4 and c-MYC (MYC)) in two neonatal human fibroblast lines
(HFF and BJ) and in adult human dermal fibroblasts. An 8-d exposure of cells to reprogramming factors and iPSC-like culture conditions resulted in a plastic state in the absence of pluripotent marker
expression that, upon MIM differentiation for an additional 8-d
period, led to the appearance of a prominent FibCD34+ population
(Fig. 2b,c and Supplementary Table 1). Of note, precise frequency
analysis was technically difficult because our procedure involves
nonclonal expanding populations of cells. We calculated angioblast
conversion efficiencies by estimating the ratio between the final
number of converted cells and the initial number of fibroblasts.
Given that 75,000 fibroblasts gave rise to ~2 × 106 cells by the end
of MIM commitment, of which 20%–60% were CD34+ (depending
on the cell line of origin and the method used for plasticity induction), conversion efficiencies ranged between 400% and 1,200%.
0.5
0 hypo
1.0 hyper
Figure 2 | Conversion of human
Fibroblasts
*
*
fibroblasts into mesodermal
160
progenitors and endothelial
150
*
h
cells by retroviral approaches.
*
(a) Schematic representation
50
Primary EC
40
*
*
*
of the conversion process.
**
**
*
0
cEC
(b) Representative flow cytoHFF
HFF
HUVEC
HFF
+4FEndo
+4F/
metry plots of pluripotency markers
Fibroblasts
Endo
miRs
upon plastic induction. 4F, four-factor
condition. (c) Flow cytometry analysis of CD34 expression after MIM induction in neonatal human fibroblasts in the presence of miR302–367 or scrambled
controls. (d) mRNA profiling of mesodermal genes upon plastic induction (left) and upon induction followed by MIM differentiation (right). (e) mRNA
expression profiling of endothelial cell (EC) markers upon differentiation of sorted FibCD34+ cells. (f) Fluorescence micrographs show expression of the indicated
endothelial markers in converted cells. Green, endoglin and VE-cadherin; magenta, VWF; blue, nuclear stain. (g,h) Heat map and representative clustering of
starting fibroblasts compared to endothelial cells (cECs) differentiated from the converted FibCD34+ cells and primary human umbilical vein endothelial cells
(primary ECs). Genome-wide transcriptome analysis (g) and methylation profiling (h) are shown. Scale bars, 50 µm; error bars, s.d.; n > 3; *P < 0.05.
Gene expression level
© 2012 Nature America, Inc. All rights reserved.
HFF +4F
105
Q1 Q2
Endothelial
differentiation
(8 d)
M
Mesodermal
induction
(8 d)
Gene expression level
Plastic
induction
(8 d)
H H
Percentage of CD34+
FF F
cells
+4 F +
F/ 4F M
BJ B miR IM
+4 J + s MIM
F/ 4F M
m
iR IM
s MI
105
104
103
102
+ EGM-2
VE-cad/VWF/Hoechst Endoglin/VWF/Hoechst
Isotypes
b
d
2.0 up
0
–2.0 down
c
a
We additionally asked whether the miR302 and miR367
(miR302–367) microRNA clusters, which have been demonstrated to play a role during reprogramming to iPSCs17,18, could
increase the efficiency of the process. We observed that inclusion
of miR302–367 improved the efficiency of FibCD34+ cell generation in some but not all lines (Fig. 2c and Supplementary
Table 1). We next sought to determine the minimal requirements for the conversion of human fibroblasts to FibCD34+ by
systematic single-factor removal. We observed marginal levels of
CD34+ cells, with low fluorescence intensities, when SOX2 was
used alone (~5% CD34low), and subsequent differentiation of
sorted CD34+ cells did not yield endothelial or smooth muscle
cells. Other single-factor combinations did not give rise to CD34+
cells. All together, combination of the four Yamanaka factors,
alongside the use of iPSC-like culture conditions, was necessary
for the conversion to CD34+ cells resembling an angioblastlike state.
Similarly to PSC differentiation results, MIM differentiation
led to a significant upregulation of angioblast-related markers in
all conditions analyzed (Fig. 2d). Sorting of MIM-differentiated
+
FibCD34 cells and subsequent culture in medium promoting
endothelial or smooth muscle cell differentiation resulted in the
upregulation of lineage-specific markers at both the RNA and
protein levels (Fig. 2e,f and Supplementary Figs. 7–9). Lineage
conversion of human fibroblasts toward the endothelial lineage
resulted in the mixed expression of markers for different endo­
thelial subtypes, including expression of arterial, venous and
lymphatic endothelial genes19 (Supplementary Fig. 8). Similarly,
analysis of smooth muscle cell populations derived from human
nature methods | ADVANCE ONLINE PUBLICATION | Articles
5
10
4
103
103
Q1 Q2
2
10 Q3 Q4
Q1 Q2
102 Q3 Q4
TRA1-60
10
103
103
Q1 Q2
102 Q3 Q4
Q1 Q2
10 Q3 Q4
2
102 103 104 105
BJ +6FEndo
250
200
150
100
50
P3
BJ +6F/miRsMIM
250
200
150
100
50
P3
P3
102 103 104 105
102 103 104 105
250
200
150
100
50
102 103 104 105
250
200
150
100
50
102 103 104 105
P3
102 103 104 105
CD34
BJ +6F/miRsEndo
h
BJ +6FEndo
Endoglin/VWF/
Hoechst
Isotype control
Mean fluorescence
intensity
1,000
VE-cad/VWF/
Hoechst
*
C
BJ
F
+6
LDL
P < 0.0001 P < 0.0001
800
600
400
200
0
do
m
BJ
*
*
2,000
1,000
PROX1
VEGFR3
VWF
CDH5
i
500
BJ
iR
s En
m
+6
F/
En
F
+6
BJ
do
do
2
1
0
BJ
do
F/ BJ
m
iR
s En
do
2
1
0
Lymphatic/pan-endothelial markers
Gene expression level
*
En
do
BJ
iR
s En
m
F/
+6
BJ
+6
F
En
BJ
do
2
1
0
F
100
+6
400
900
Venous endothelial markers
COUP-TFII
EPHB4
*
NRP2
+6
*
ALK1
BMX
CXCR4
EPHB2
CX40
JAG1
NRP1
UNC5B
BJ
*
Gene expression level
Arterial endothelial markers
800
BJ
g
Gene expression level
*
*
*
0
Endo
BJ +6FEndo BJ +6F/miRs
© 2012 Nature America, Inc. All rights reserved.
200
150
*
*
*
*
*
VE
0
*
do
20
f
*
*
*
4
U
40
*
105
4
10
BJ +6FMIM
TRA1-81
CNN1
CD45
FOXA1
*
55,000
45,000
35,000
25,000
15,000
14,000
105
H
60
VWF
ACTA2
BJ
iR +6F
s En /
80
e
ENG
CDH5
do
Mean % of positive
cells
100
CD34
CD31
Endothelial differentiation
(8 d)
En
VE-cadherin+
Endoglin+
VE-cadherin+/endoglin+
Gene expression level
d
Mesodermal induction
(8 d)
c
102 103 104 105
102 103 104 105
Plastic induction
(8 d)
BJ +6F/miRs
Isotypes
+ EGM-2
10
BJ
(is +6
ot F En
yp do
BJ e)
BJ (L +6F E
+6 DL ndo
(is F/m )
o
BJ typ iRs E
+6 e) ndo
F
(L /m
D iR
L) s En
+ MIM
Isotypes
OCT4/SOX2/KLF4/LMYC/LIN28/shP53
(± miRs 302 and 367) episomes
BJ +6F
104
105
SSC
b
a
Figure 3 | Conversion of human fibroblasts to mesodermal progenitors and endothelial cells by non-integrative
approaches. (a) Schematic representation of the conversion process. (b) Representative flow cytometry plots of
pluripotency-associated markers upon plastic induction with non-integrative plasmids followed by MIM differentiation. 6F, six-factor condition.
(c) Representative flow cytometry plots of CD34 expression before and after MIM differentiation in neonatal human fibroblasts (BJ) induced to a plastic
state in the presence of miR302–367 or scrambled controls. Upper panel shows isotype controls. SSC, side scatter. (d) Flow cytometry analysis of BJderived VE-cadherin+ and endoglin+ endothelial cells. (e) mRNA expression profile of endothelial markers upon differentiation of sorted BJ FibCD34+ cells.
(f) Fluorescence micrographs show expression of the indicated endothelial markers in converted endothelial cells. Green, endoglin and VE-cadherin;
magenta, VWF; blue, nuclear stain. For all gene expression plots, the levels of expression were normalized to corresponding GAPDH values and are
shown as fold change relative to the value of the control sample. (g) Characterization of endothelial subtypes in BJ converted endothelial cells.
(h) Representative images of endothelial cells upon fluorescent LDL uptake (upper panels). The plot shows mean fluorescence intensities of LDL taken up by the
indicated converted endothelial cells. Controls are cells incubated in the presence of Alexa Fluor 488 alone to monitor background fluorescence. (i) Capillary-like
structures spontaneously formed by BJ-derived endothelial cells in vitro. Scale bars: 50 µm (f); 100 µm (h); 200 µm (i). Error bars, s.d.; *P < 0.05.
fibroblasts demonstrated mixed expression of smooth muscle
markers20, including expression of the pericyte marker NG2
(Supplementary Fig. 9).
Converted endothelial cells lost many features of fibro­blast
gene expression and DNA methylation profiles, and they
acquired characteristics of primary endothelial cells (Fig. 2g,h
and Supplementary Fig. 5). When all samples, regardless of their
method of derivation, were compared by unsupervised hierarchical
clustering of the mRNA and methylation array data, two major
groups were observed: pluripotent cells and differentiated cells.
As expected, fibroblasts clustered more closely to differentiated
cells than to PSCs (Supplementary Fig. 5 and 6). Both mRNA
expression and DNA methylation results were similar in terms
of describing the relationships among the different cell types
(Supplementary Figs. 5 and 6).
All together, our results demonstrate that 8-d exposure of
human fibroblasts to iPSC reprogramming factors and iPSC
| ADVANCE ONLINE PUBLICATION | nature methods
culture conditions induced an intermediate plastic state.
Subsequent mesodermal induction by 8-d exposure to MIM yielded
intermediate CD34+ bipotent progenitor populations, which
could be further differentiated to endothelial and smooth muscle
cell populations.
Conversion to angioblasts by non-integrative approaches
We observed residual expression of the Yamanaka factors transgenes upon differentiation to CD34+ cells as well as their endothelial and smooth muscle derivatives (Supplementary Fig. 10). We
thus pursued the establishment of non-integrative approaches for
conversion of human fibroblasts to other cell types.
We chose a six-factor combination (OCT4, SOX2, KLF4, nontransforming LMYC (MYCL1), LIN28 and short hairpin RNA
against p53) proven to generate human iPSCs in the presence of
murine feeder layers when delivered episomally21 (Fig. 3). We
obtained plastic reprogramming intermediates by electroporation
a
BJ
BJ +6F/
HUVEC HuES9Endo KiPSEndo
Endo
Endo
+6F
miRs
bFGF
17 d
c
IF Ulex lectin
BJ +6F
DAPI
HuNu
Endo
ISH hDNA
d
KiPS
IHC hCD31
IF Ulex lectin
Endo
IF Ulex lectin
HuES9
IF Ulex lectin
b
ISH hDNA
Endo
CD31
Merge
Endo
BJ +6F/miRs
DAPI
HuNu
CD31
Merge
Figure 4 | Converted endothelial cells are functional in vivo.
e
(a) Images show Matrigel plugs extracted from mice 17 d
after the implantation of endothelial cells derived from the
indicated experimental groups as well as a negative control
in the presence of bFGF. (b,c) Representative micrographs of
extracted Matrigel plugs with HuES9- (b) and KiPS- (c) derived
endothelial cells showing the identification of human cells
by in situ hybridization (ISH) on ALU+ sequences (dark blue dot),
anti-human CD31 immunohistochemistry (IHC) staining (brown)
and Ulex lectin rhodamine immunofluorescence (IF) staining (red). Note the presence of circulating
red blood cells through the vessel-graft. (d) Endothelial cells derived by non-integrative–mediated
conversion of human fibroblasts demonstrate anastomosis in vivo. Upon Matrigel plug extraction
and processing, human specific CD31 antibody demonstrates the presence of converted endothelial
cells (green). Colocalization with specific human nuclear antigen (HuNu) staining demonstrates
that the generated vessels are derived from the injected converted human endothelial cells. In the
rightmost images, all fluorescence channels have been merged over bright-field pictures to allow
for morphological identification of circulating erythrocytes. (e) Representative high-magnification
micrograph of the extracted Matrigel plugs demonstrating connection to the pre-existing vasculature
upon injection of converted endothelial cells generated by non-integrative approaches. Arrows,
circulating red blood cells. Scale bars: 5 µm (b,c); 50 µm (d,e). Green, CD31; magenta, HuNu; blue,
nuclear staining.
Functionality of converted cells in vitro
and in vivo
Two well-characterized physiological
hallmarks of smooth muscle cell function are calcium response and contractility20,23,24. Contraction of FibCD34+
cell–derived smooth muscle cells occurred
both spontaneously as well as upon drug
stimulation (Supplementary Fig. 11 and
Supplementary Video 1). Exposure to carbachol resulted in
rapid calcium transients. Of note, HEK293T human embryonic
kidney cells also show calcium transients in response to carbachol, but they do not physically contract as demonstrated by their
unchanged cell surface area (Supplementary Fig. 11).
We investigated the function of FibCD34+ cell–derived endo­
thelial cells by measuring acetylated low-density lipoprotein
(LDL) uptake, a characteristic of mature endothelial cells. The
cells showed significantly higher rates of LDL uptake as compared to differentiated endothelial cells in the presence of control
Alexa 488 used to measure nonspecific fluorescence background
(Fig. 3h and Supplementary Fig. 12). The converted endo­thelial
cells also aggregated into vessel-like structures in vitro (Fig. 3i
HuNu/CD31/DAPI
of each vector followed by a 6-d resting
phase before a switch to iPSC culture
medium (Fig. 3a). After 8 d of plastic
induction, expression of the pluripotency
markers TRA1-60 and TRA1-81 was
undetectable (Fig. 3b). Then, the medium
was changed to MIM for an additional 8 d,
yielding CD34+ cells (Fig. 3c). Sorting of
+
FibCD34 cells and subsequent differentiation into endothelial and smooth muscle
lineages resulted in the upregulation of cell
type–specific markers at both the RNA
and protein level (Fig. 3d–g). As observed
previously, the generated endothelial
and smooth muscle cells represented
mixed populations of different subtypes
(Fig. 3g). Notably, we observed the rapid
clearing of episomal vectors and did not
detect random integration of exo­genous
genes in the differentiated endothelial cells
(Supplementary Fig. 10).
TRA1-60 has recently been described
as the most reliable early marker for iPSC
generation, with a success prediction rate
of up to 90% (ref. 22). We did not observe
detectable expression of TRA1-60 or
TRA1-81 with either the non-integrative
approach or the retroviral approach for
plastic induction of fibroblasts (Figs. 2b
and 3b,c and Supplementary Fig. 10).
Furthermore, testis injection in mice of
1 million differentiated endothelial cells,
including cells generated by differentiation
of PSCs, did not result in teratoma formation in any of the groups analyzed after
10 weeks (Supplementary Fig. 10).
IHC hCD31
© 2012 Nature America, Inc. All rights reserved.
Articles
and Supplementary Fig. 12). Upon subcutaneous implantation in
a Matrigel plug, the cells formed functional vessels, allowing for
blood circulation, after 17 d in vivo (Fig. 4a), demonstrating the
connection of newly formed vessels to the pre-existing vasculature. Cells in the Matrigel plugs extracted from the animals at day
17 were endo­thelial cells as verified by Ulex europaeus lectin binding and were of human origin as determined by in situ hybridization (Fig. 4b,c) as well as by staining with antibodies specific for
human CD31 and human nuclear antigen (Fig. 4d,e).
DISCUSSION
We have established an efficient method for the conversion of
neonatal and adult human fibroblasts to CD34 + angioblast-like
nature methods | ADVANCE ONLINE PUBLICATION | © 2012 Nature America, Inc. All rights reserved.
Articles
progenitor cells (Supplementary Table 1). Our approach couples
the generation of plastic reprogramming intermediates with subsequent induction of an angioblast fate with chemically defined
MIM. These angioblast-like cells could be further differentiated
into functional endothelial and smooth muscle cells.
We observed differences at both the transcriptome and methy­
lome level when comparing our converted endothelial and smooth
muscle cells to the primary cells used as positive controls. One
reason could be that our generated cell populations include different subtypes of cells as compared to the primary cells (human
umbilical vein endothelial cells and arterial smooth muscle cells).
Alternatively, these differences could be reminiscent of experimental
variation as seen between and within iPSC and embryonic stem
cell lines25. Residual epigenetic marks from the initial fibroblasts
could also account for some observed differences6. Furthermore,
the fact that induction of ‘plasticity’ relies on a first phase of
epigenetic erasure, which by similarity with iPSC reprogramming might imply a stochastic process, strongly suggests that the
heterogeneity observed at the molecular level during the conversion process might be due to the presence of cells with varying
degrees of epigenomic plasticity. Nevertheless, all the cells generated (whether differentiated from PSCs or derived by conversion
of human fibroblasts) demonstrated functional properties, thus
highlighting the potential of these novel conversion methodologies as well as the importance of analyzing functional parameters
in reprogramming paradigms6,25,26.
Conversion to angioblast-like progenitor cells occurred in the
absence of detectable iPSC colony formation, surface-marker
expression and re-activation of the endogenous pluripotency
transcription network, therefore considerably shortening the
time required for generation of the desired cell types. Although
we cannot rule out that converted cells transitioned through a
pluripotent-like state, the lack of pluripotent marker expression
and teratoma formation indicates that the conversion process
does not result in typical iPSC features.
In contrast to a recent report describing the conversion of amniotic cells to endothelial cells27, this study demonstrates that shortterm induction by iPSC reprogramming conditions, followed by
exposure to a chemically defined differentiation medium, is sufficient for the conversion of neonatal and adult human fibroblasts
into angioblast-like progenitor cells with multipotent differentiation potential. Of note, not only the autonomous effects of the
reprogramming factors but also the overall combination of stem
cell culture conditions promoting cell proliferation, as exemplified by the requirement of basic fibroblast growth factor (bFGF),
are crucial. Interestingly, culture conditions have also been highlighted as a critical component during the conversion process in
similar studies performed in the murine system7,8.
Unlike direct lineage conversion, which requires precise knowledge and screening of molecules defining target-cell identity,
induction of plastic or de-differentiation states coupled to specific
differentiation protocols might provide a general, more readily
accessible platform toward the broader generation of clinically
relevant cell types. Furthermore, whereas direct lineage conversion might be viewed as an ‘unnatural’ process6 occurring in
the absence of progenitor cell generation, our results and those
reported for the murine system7,8 show the formation of intermediate progenitor states, such as seen during normal embryogenesis. This may have two major practical implications. First, the
| ADVANCE ONLINE PUBLICATION | nature methods
generation of progenitor cells with multilineage differentiation
capacity strongly diversifies the spectra of applications as opposed
to direct lineage conversion. Second, the inability to generate proliferative populations by direct lineage conversion could represent
a major limitation for applications in which large numbers of
cells are required6,9. In the case shown here, the conversion of
human fibroblasts into vascular smooth muscle and endothelial
cells proceeds through the generation of an expandable population of vascular progenitors with multilineage differentiation
capacity. All together, our results describe a novel methodology
for the reprogramming of somatic cells, and they support a complementary approach to direct lineage conversion as well as to
full reprogramming to induced pluripotency for the generation
of human cell types with clinical implications.
Methods
Methods and any associated references are available in the online
version of the paper.
Accession codes. Data sets for DNA methylation analysis and
gene expression microarray analysis are available on the Gene
Expression Omnibus (GSE40927).
Note: Supplementary information is available in the online version of the paper.
Acknowledgments
We are thankful to Y. Zheng for his expertise and assistance with sorting
procedures, C. Maiza for his expertise and assistance with in vivo procedures
and M. Schwarz for administrative support. L.K. was partially supported by the
California Institute for Regenerative Medicine. E.N. was partially supported
by an F.M. Kirby Foundation postdoctoral fellowship. A.M.D. was supported
by the Helmsley Foundation. L.C.L., R.D.T., F.S.B. and J.F.L. are supported by
the California Institute for Regenerative Medicine (CL1-00502, RT1-01108,
TR1-01250, RN2-00931), US National Institutes of Health (R33MH087925),
US National Institutes of Health/National Institute Child Health and Human
Development K12 Career Development Award (L.C.L.), Hartwell Foundation
(L.C.L., R.D.T., F.S.B.), Millipore Foundation (J.F.L.) and Esther O’Keefe
Foundation (J.F.L.). Work in the laboratory of F.H.G. was supported by the JPB
Foundation, G. Harold and Leila Y. Mathers Charitable Foundation and Ellison
Medical Foundation. Work in the laboratory of J.C.I.B. was supported by grants
from Fundacion Cellex, the G. Harold and Leila Y. Mathers Charitable Foundation,
the Leona M. and Harry B. Helmsley Charitable Trust, Sanofi, Ministerio de
Economia y Competitividad (PLE2009-0100), Instituto de Salud Carlos III
(ISCIII), Terapia Celular (TerCel) (RD06/0010/0016) and Fondo Europeo de
Desarrollo Regional (FEDER).
AUTHOR CONTRIBUTIONS
L.K., I.S.-M., E.N. and J.C.I.B. designed all experiments. I.S.-M., E.N. and J.C.I.B.
wrote the manuscript. L.K., I.S.-M., K.M., E.N. and A.A. performed and analyzed
all experiments. C.P., C.V.-C., F.B., E.N., I.D. and J.-M.H. performed in vivo
experiments. K.M. was responsible for all cell culture–related work. M.L., A.M.D.
and F.H.G. provided microRNA constructs and reagents. J.P., Y.X., S.R., I.D.,
N.M., C.R., A.M.D. and F.H.G. contributed to the overall design of the project.
F.S.B., R.D.T., J.F.L. and L.C.L. performed and analyzed genome-wide array DNA
methylation and gene expression studies.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Published online at http://www.nature.com/doifinder/10.1038/nmeth.2255.
Reprints and permissions information is available online at http://www.nature.
com/reprints/index.html.
1. Sancho-Martinez, I., Nivet, E. & Izpisua Belmonte, J.C. The labyrinth of
nuclear reprogramming. J. Mol. Cell Biol. 3, 327–329 (2011).
2. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from
mouse embryonic and adult fibroblast cultures by defined factors.
Cell 126, 663–676 (2006).
© 2012 Nature America, Inc. All rights reserved.
Articles
3. Laiosa, C.V., Stadtfeld, M., Xie, H., de Andres-Aguayo, L. & Graf, T.
Reprogramming of committed T cell progenitors to macrophages and
dendritic cells by C/EBPα and PU.1 transcription factors. Immunity 25,
731–744 (2006).
4. Ieda, M. et al. Direct reprogramming of fibroblasts into functional
cardiomyocytes by defined factors. Cell 142, 375–386 (2010).
5. Vierbuchen, T. et al. Direct conversion of fibroblasts to functional neurons
by defined factors. Nature 463, 1035–1041 (2010).
6. Vierbuchen, T. & Wernig, M. Direct lineage conversions: unnatural but
useful? Nat. Biotechnol. 29, 892–907 (2011).
7. Efe, J.A. et al. Conversion of mouse fibroblasts into cardiomyocytes
using a direct reprogramming strategy. Nat. Cell Biol. 13, 215–222
(2011).
8. Kim, J. et al. Direct reprogramming of mouse fibroblasts to neural
progenitors. Proc. Natl. Acad. Sci. USA 108, 7838–7843 (2011).
9. Sancho-Martinez, I., Baek, S.H. & Izpisua Belmonte, J.C. Lineage
conversion methodologies meet the reprogramming toolbox. Nat. Cell Biol.
14, 892–899 (2012).
10. Papp, B. & Plath, K. Reprogramming to pluripotency: stepwise resetting of
the epigenetic landscape. Cell Res. 21, 486–501 (2011).
11. Plath, K. & Lowry, W.E. Progress in understanding reprogramming to the
induced pluripotent state. Nat. Rev. Genet. 12, 253–265 (2011).
12. Goldman, O. et al. A boost of BMP4 accelerates the commitment of human
embryonic stem cells to the endothelial lineage. Stem Cells 27,
1750–1759 (2009).
13. Dzierzak, E. & Speck, N.A. Of lineage and legacy: the development
of mammalian hematopoietic stem cells. Nat. Immunol. 9, 129–136
(2008).
14. Choi, K.D. et al. Hematopoietic and endothelial differentiation of human
induced pluripotent stem cells. Stem Cells 27, 559–567 (2009).
15. James, D. et al. Expansion and maintenance of human embryonic stem
cell–derived endothelial cells by TGFβ inhibition is Id1 dependent. Nat.
Biotechnol. 28, 161–166 (2010).
16. Levenberg, S., Ferreira, L.S., Chen-Konak, L., Kraehenbuehl, T.P. & Langer, R.
Isolation, differentiation and characterization of vascular cells derived
from human embryonic stem cells. Nat. Protoc. 5, 1115–1126 (2010).
17. Anokye-Danso, F. et al. Highly efficient miRNA-mediated reprogramming of
mouse and human somatic cells to pluripotency. Cell Stem Cell 8, 376–388
(2011).
18. Subramanyam, D. et al. Multiple targets of miR-302 and miR-372 promote
reprogramming of human fibroblasts to induced pluripotent stem cells.
Nat. Biotechnol. 29, 443–448 (2011).
19. Narazaki, G. et al. Directed and systematic differentiation of cardiovascular
cells from mouse induced pluripotent stem cells. Circulation 118, 498–506
(2008).
20. Cheung, C., Bernardo, A.S., Trotter, M.W., Pedersen, R.A. & Sinha, S.
Generation of human vascular smooth muscle subtypes provides insight
into embryological origin-dependent disease susceptibility. Nat.
Biotechnol. 30, 165–173 (2012).
21. Okita, K. et al. A more efficient method to generate integration-free
human iPS cells. Nat. Methods 8, 409–412 (2011).
22. Chan, E.M. et al. Live cell imaging distinguishes bona fide human iPS cells
from partially reprogrammed cells. Nat. Biotechnol. 27, 1033–1037 (2009).
23. Ohta, T., Ito, S. & Nakazato, Y. All-or-nothing responses to carbachol in
single intestinal smooth muscle cells of rat. Br. J. Pharmacol. 112,
972–976 (1994).
24. Kohda, M., Komori, S., Unno, T. & Ohashi, H. Carbachol-induced [Ca2+]i
oscillations in single smooth muscle cells of guinea-pig ileum. J. Physiol.
(Lond.) 492, 315–328 (1996).
25. Yamanaka, S. Induced pluripotent stem cells: past, present, and future.
Cell Stem Cell 10, 678–684 (2012).
26. Yusa, K. et al. Targeted gene correction of α1-antitrypsin deficiency in
induced pluripotent stem cells. Nature 478, 391–394 (2011).
27. Ginsberg, M. et al. Efficient direct reprogramming of mature amniotic cells
into endothelial cells by ETS factors and TGFβ suppression. Cell 151,
559–575 (2012).
nature methods | ADVANCE ONLINE PUBLICATION | © 2012 Nature America, Inc. All rights reserved.
ONLINE METHODS
Reagents and antibodies. The following antibodies were used at
the specified concentrations: mouse anti-human CD34-APC 1:10
(130-046-703, Miltenyi), mouse anti-human CD133/2 (293C3)PE 1:10 (130-090-853, Miltenyi), mouse anti-human CD144-PE
1:10 (VE-cadherin; 560410, BD Biosciences), mouse anti-human
CD144-APC 1:10 (VE-cadherin; 348507, BioLegend), CD105-PE
1:10 (endoglin; ab60902, Abcam), mouse anti-human CD105PE 1:10 (endoglin; 560839, BD), CD31-FITC 1:10 (555445,
BD), CD117-PeCy7 1:10 (c-Kit; 339195, BD), VEGFR2-PE 1:10
(KDR; 560494, BD), mouse anti-human CD45-FITC 1:10 (130080-202, Miltenyi), anti-human CD235a-PE 1:10 (340947, BD),
mouse APC isotype control 1:10 (555751, BD), mouse FITC
isotype control 1:10 (555748, BD), PeCy7 isotype control 1:10
(557872, BD), PE isotype control 1:10 (555749, BD), VE-cadherin
1:500 (555661, BD), endoglin 1:500 (M3527, DAKO), anti–von
Willebrand Factor 1:200 (VWF; 7356, Millipore), calponin 1:500
(Dako, M3556), α-SMA 1:500 (AB56994, Abcam), α-SMA 1:1,000
(A5228, Sigma), PECAM-1 (M-20) 1:100 (CD31; sc1506, Santa
Cruz Biotechnology) anti-human nuclei 1:100 (MAB1281,
Millipore), DAPI (5 mg ml−1) 1:2,000 (D1306, Invitrogen),
Hoechst 33342 (5 mg ml−1) 1:2,000 (B2261, Sigma), Alexa Fluor
488 goat anti-mouse (A11001, Invitrogen), Alexa Fluor 488
donkey anti-goat (A11055, Invitrogen), Alexa Fluor 568 donkey
anti-mouse (A10037, Invitrogen) and Alexa Fluor 568 donkey
anti-rabbit (A10042, Invitrogen).
Directed differentiation of hES/hiPS cells in chemically defined
conditions. Human ES/iPS cells cultured as described above were
freshly split on Matrigel-coated plates, and we made sure the subcolonies were of small size (~300–500 cells per colony). After 24 h
of recovery in mTeSR, the cells were gently washed using DMEM:
F12 (Invitrogen) and allowed to grow in chemically defined MIM
differentiation medium. Medium was changed every second day
with addition of half the volume of medium every other day.
Cell culture. Human ES cells, H1 (WA1, WiCell), HuES 9
(http://grants.nih.gov/stem_cells/registry/current.htm?id=40)
and human iPS cells CBiPS28 and KiPS29 (KIPS 4F2, CBiPS
2F4) (passage 25–45) were cultured in chemically defined
hES/hiPS growth medium (mTeSR30 on growth factor–reduced
Matrigel (356230, BD)-coated plates). Briefly, 70–80% confluent
hES/iPS cells were treated with dispase (Invitrogen) for 7 min
at 37 °C, and colonies were dispersed to small clusters and
lifted carefully using a 5-ml glass pipette at a splitting ratio of
~1:4. Neonatal human fibroblasts (HFF-1, BJ; ATCC) and adult
human ­dermal fibroblasts (HDF-693) were cultured in DMEM
containing 10% FBS, 2 mM GlutaMAX (Invitrogen), 50 U ml−1
penicillin and 50 mg ml−1 streptomycin (Invitrogen). Human
umbilical vein endothelial cells (HUVECs) were purchased from
PromoCell and cultured in EBM medium supplemented with
EGM-2 SingleQuots (cc-3162, Lonza), 2% FBS, hEGF 10 µg
ml−1 and heparin 100 µg ml−1 (Sigma). Mesodermal induction medium (MIM) consists of DMEM:F12, 15 mg ml−1 stem
cell–grade BSA (MP Biomedicals), 17.5 µg ml−1 human insulin
(SAFC Biosciences), 275 µg ml−1 human holo-transferrin
(Sigma), 20 ng ml−1 bFGF (Stemgent), 50 ng ml−1 human VEGF165 aa (Humanzyme), 25 ng ml−1 human BMP4 (Stemgent),
450 µM monothioglycerol (Sigma) and 2.25 mM each
l-glutamine and non-essential amino acids (Invitrogen). iPS/
ES-derived endothelial cells were cultivated in EBM-2 medium
supplemented with EGM-2 SingleQuot kit (cc-3162, Lonza).
iPS/ES-derived smooth muscle cells were cultured in SmBM
medium supplemented with SmGM-2 SingleQuot kit (cc-3182,
Lonza). All the cells were grown in collagen I–coated plates
(BD). All cell lines were maintained in an incubator (37 °C, 5%
CO2) with medium changes every day (hES/iPS) or every second
day (HUVEC/fibroblasts).
Conversion of human fibroblasts into angioblast-like CD34+
progenitor cells. For retroviral infection, human fibroblast cells
(HFF-1, BJ, HFF-693) were plated on Matrigel-coated six-well
plates at 75,000 cells per well. The next day, cells were infected
with an equal ratio of a combination of either four pMX-derived
retroviruses encoding OCT4, SOX2, KLF4 and c-MYC (4F) or
five pMX-derived retroviruses encoding OCT4, SOX2, KLF4,
c-MYC and miR302–367 (4F/miRs). Scramble miRNA control
(PMIRH000PA-1, SBI) was used whenever appropriate. The plates
were infected by spinfection of the cells at 1,850 r.p.m. for 1 h
at room temperature in the presence of polybrene (4 µg ml−1)
and put back in the incubator without medium change. 24 h
later, the medium was switched to WiCell medium composed
of DMEM/F12 (Invitrogen), 20% KnockOut serum replacement,
10 ng ml−1 bFGF, 1 mM GlutaMAX, 0.1 mM non-essential amino
acids and 55 µM β-mercaptoethanol; with medium changes every
day. After 6 d, cells were split at a ratio of 1:3 on to Matrigelcoated six-well plates supplemented with WiCell medium for
another 2 d. The cells were then washed once with DMEM/F12
and induced for differentiation for 8 d in the presence of MIM.
Medium was changed every second day with addition of half the
volume of medium every other day.
For episomal transfection, 2 × 106 cells were transfected with
1.5 µg each of pCXLE-episomal vectors encoding OCT4, SOX2,
KLF4, LMYC, LIN28 and shRNA-p53 (ref. 22) (27077, 27078 and
27080, Addgene) with and without addition of pcDNA3.1 encoding for miR302–367 (6F or 6F/miRs). Fibroblasts were transfected
by nucleofection (Amaxa NHDF Nucleofector Kit, VPD-1001)
according to the manufacturer’s instruction and plated back
on to Matrigel-coated wells. Cells were then rested for 6 d in
DMEM/F12 supplemented with 10% FBS, 0.1 mM non-essential
amino acids and 2 mM GlutaMAX, and then the medium was
nature methods
Single-cell differentiation assays. Upon MIM differentiation for
8 d, CD34+ angioblast-like cells were sorted and plated in collagen
I–coated 48-well plates at a density of one cell per well in either
EBM-2 (for endothelial differentiation) or SmBM (for smooth
muscle differentiation) supplemented as described above. After
7 d in the respective differentiation conditions, cells were washed
once with PBS and fixed with 4% paraformaldehyde (PFA) in 1×
PBS. Following fixation, cells were blocked and permeabilized for
1 h at 37 °C with 5% BSA/5% appropriate serum/1× PBS in the
presence of 0.1% Triton X-100. Subsequently, cells were incubated
overnight at 4 °C with an anti-endoglin antibody in case of cells
in EBM-2/EGM-2 or with an anti-calponin antibody in the case
of cells in SmBM/SmGM-2. Cells were then washed thrice with
1× PBS, incubated for 1 h at 37 °C with the respective secondary
antibodies and 20 min with DAPI for nuclear staining. Following
incubation, cells were washed thrice with 1× PBS before micro­
scopy analysis and scoring.
doi:10.1038/nmeth.2255
© 2012 Nature America, Inc. All rights reserved.
switched to WiCell medium, with medium changes every day.
After 6 d, cells were split at a ratio of 1:3 on to Matrigel-coated
six-well plates with WiCell medium for another 2 d. The cells
were then washed once with DMEM:F12 and induced for differentiation for 8 d in the presence of MIM. Medium was changed
every second day with addition of half the volume of medium
every other day.
RNA isolation and real-time PCR analysis. Total cellular RNA
was isolated using Trizol reagent (Invitrogen) according to the
manufacturer’s recommendations. 2 µg of DNase 1– (Invitrogen)
treated total RNA was used for cDNA synthesis using the
SuperScript II Reverse Transcriptase kit for RT-PCR (Invitrogen).
Real-time PCR was performed using the SYBR Green PCR Master
Mix (Applied Biosystems). The levels of expression of respective
genes were normalized to corresponding GAPDH values and are
shown as fold change relative to the value of the control sample.
All the samples were done in triplicate. A list including precise
mRNA fold-change quantifications of the qPCR data summarized in Figures 1–3 is provided in Supplementary Table 2.
The primers used for real-time PCR experiments are listed in
Supplementary Table 3.
Flow cytometry analysis. Human ES/iPS cells undergoing
directed differentiation, lineage-converted CD34 + cells or their
respectively derived endothelial cells were harvested using TrypLE
(Invitrogen), washed once with PBS and further incubated with
the corresponding antibodies in the presence of FACS blocking
buffer (1× PBS/10% FCS) for 1 h on ice in the absence of light.
After incubation, cells were washed thrice with 1 ml of FACS
blocking buffer and resuspended in a total volume of 200 µl
before analysis. A minimum of 10,000 cells in the living population were analyzed by using a BD LSRII flow cytometry machine
equipped with five different lasers and the BD FACSDiva software.
Percentages are presented after the subtraction of isotype background and refer to the total living population analyzed. Results
are representative of at least three independent experiments with
a minimum of two technical replicates per experiment.
Cell sorting. After 8 d of differentiation, CD34+ cells were stained
as described above and sorted by using a BDAria II FACS sorter
(BD Biosystems). Alternatively, CD34+ cells were enriched using
anti-CD34–conjugated magnetic beads (Miltenyi) according to
the manufacturer’s instructions with slight modifications. Briefly,
up to 109 cells were incubated with constant mixing at 4 °C with
100 µl of the corresponding magnetic beads in the presence of
100 µl of Fc-blocking solution in a total volume of 500 µl FACS
blocking buffer. After 1 h, cells were sorted by two consecutive
rounds of column separation to increase purity by applying
MACS separation magnets. Shortly, cells were passed through
the first MS separation column, which allowed the binding of
labeled cells. Nonlabeled cells were washed thoroughly with
3 ml FACS blocking buffer before elution of the labeled fraction.
Eluted labeled cells were then subjected to a second purification
step as described above.
Differentiation of CD34+ cells to endothelial cells. PSC and
lineage-converted CD34+ cells, isolated by MACS or by FACS
sorting after 8 d of differentiation in MIM, were plated in
doi:10.1038/nmeth.2255
collagen I–coated plates (50,000 cells per well in a 12-well plate)
and cultured in EBM-2/EGM-2 (Lonza) with medium changes
every day. After 5–8 d in culture, upon reaching 90% confluence, cells were split 1:4 with TrypLE (Invitrogen). The cells were
cultured for at least eight passages.
Differentiation of CD34+ cells to smooth muscle cells. PSC and
lineage-converted CD34+ cells, isolated by MACS or by FACS
sorting after 8 d of differentiation in MIM, were plated in collagen I–coated plates (50,000 cells per well in a 12-well plate) and
cultured in SmBM/SmGM-2 (Lonza) with medium changes every
day. After 5–8 d in culture, upon reaching 90% confluence, cells
were split 1:4 with TrypLE (Invitrogen). The cells were cultured
for at least eight passages.
DNA methylation analysis. Illumina 450K Infinium Methylation
Arrays were normalized and preprocessed in Genome Studio.
Probes with missing values were removed. A filter for an average β-value difference between groups (PSCs, fibroblasts, primary human arterial smooth muscle cells (PriSMCs), primary
human umbilical vein endothelial cells (PriECs), PSC→CD34+
progenitor cells, PSC→endothelial cells (iECs), converted
endothelial cells (cECs), PSC→smooth muscle cells (iSMCs),
converted smooth muscle cells (cSMCs)) of ≥0.3 was applied.
The resulting probes were used for ANOVA analysis using R
scripts with a P-value filter of <0.0001 (at this point, 30,000
probes remained). Probes with a β-value difference of ≥0.3
((max - min) > = 0.3) were used. ANOVA (P < 0.05; variance =
0.58) was applied to obtain statistically significant differentially methylated probes among the five groups in both SMC
and EC sample groups. The resulting probes (Supplementary
Data) were used for hierarchical clustering using Cluster 3.0
with complete linkage. Venn Diagram Plotter (http://omics.pnl.
gov/software/VennDiagramPlotter.php) was used to generate
area-proportional Venn diagrams.
Gene expression microarray analysis. The following groups
were analyzed: PSCs, fibroblasts, PriSMCs, PriECs, iECs, cECs,
iSMCs and cSMCs. Briefly, total RNA was extracted from
collected sample pellets (Ambion mirVana; Applied Biosystems)
according to the manufacturer’s protocol. RNA quantity (Qubit
RNA BR Assay Kits; Invitrogen) and quality (RNA6000 Nano Kit;
Agilent) were determined to be optimal for each sample before
further processing. 200 ng RNA per sample was amplified using
the Illumina Total Prep RNA Amplification Kit according to the
manufacturer’s protocol and quantified as above. 750 ng RNA
per sample was hybridized to Illumina HT-12v3 Expression
BeadChips, scanned with an Illumina iScan Bead Array Scanner
and checked for quality control in GenomeStudio and the lumi
bioconductor package. All RNA processing and microarray
hybridizations were performed in house according to the manufacturer protocols. Differential expression was defined as a minimum 2× fold change and multiple testing–corrected P < 0.05 by
ANOVA. The resulting probes (Supplementary Data) were used
for hierarchical clustering using Cluster 3.0 with complete linkage. Probes with minimum gene expression differences between
groups of 2× fold change were obtained (Supplementary Data).
Venn Diagram Plotter was used to generate area-proportional
Venn diagrams.
nature methods
© 2012 Nature America, Inc. All rights reserved.
Determination of copy number by quantitative PCR. Briefly,
total DNA was extracted using the Qiagen DNeasy Blood & Tissue
kit (QIAGEN). The purity and quantity of DNA were measured
using a NanoDrop 8000 spectrophotometer (Thermo Scientific)
and then used as templates for absolute quantitation by qPCR
assay22. The primers used are listed in Supplementary Table 3,
and their amplification efficiencies as well as specificity were
checked by performing standard curve and melting curve analyses.
Immunocytochemistry and fluorescence microscopy. Briefly,
cells were washed thrice with PBS and fixed using 4% PFA in 1×
PBS. After fixation, cells were blocked and permeabilized for 1 h
at 37 °C with 5% BSA/5% appropriate serum/1× PBS in the presence of 0.1% Triton X-100. Subsequently, cells were incubated
with the indicated primary antibody either for 1 h at room temperature or overnight at 4 °C. The cells were then washed thrice
with 1× PBS and incubated for 1 h at 37 °C with the respective
secondary antibodies and 20 min with DAPI or Hoechst 33342.
Cells were washed thrice with 1× PBS before analysis. Sections
were analyzed by using an Olympus 1X51 upright microscope
equipped with epifluorescence and TRITC, FITC and DAPI
filters. Confocal image acquisition was performed using a Zeiss
LSM 780 laser scanning microscope (Carl Zeiss Jena) with 20×,
40× or 63× immersion objectives.
were conducted with approval of the Salk Institute Institutional
Animal Care and Use Committee (IACUC).
Matrigel plug assay. Anesthesia was induced using a mixture of
xylazine (Rompun 2%, Bayer) at 10 mg per kilogram body weight
and ketamine (Imalgene1000, Merial) at 100 mg per kilogram
body weight in NaCl at 0.9%, i.p. injected at a dose of 10 ml
per kilogram body weight. The animals’ backs were shaved and
swabbed with hexomedine. Prior to injection, HUVECs, HUES9-,
KiPS-, BJ 6F- and BJ 6F/miRs-derived endothelial cells were
harvested using TrypLE (Invitrogen). A total of 1 × 106 cells
were resuspended in 500 µl of cold Matrigel (Matrigel basementmembrane matrix from BD adjusted to 9.8 mg ml−1 with PBS)
supplemented with 150 ng of bFGF. We then injected Matrigel
solutions containing or lacking cells subcutaneously in the backs of
mice, carefully positioning the needle between the epidermis and
the muscle layer. Seventeen days later, mice were sacrificed, and the
Matrigel plugs were removed by a wide excision of the back skin,
including the connective tissues (skin and all muscle layers).
Hematopoietic colony-forming assays. Hematopoietic clonogenic assays were performed in 35-mm low-adherent plastic
dishes (Stem Cell Technologies) using 1.1 ml per dish of methyl­
cellulose semisolid medium (MethoCult H4434 classic, Stem
Cell Technologies) according to the manufacturer’s instructions.
Briefly, enriched CD34+ cells were sorted and immediately plated
at various densities: 1.5 × 103, 3 × 103 and 6 × 103 cells per ml.
All assays were performed in duplicate. After 21 d of incubation,
plates were analyzed for the presence of both colony-forming
units (CFU) and burst-forming units (BFU).
Tissue processing and analyses. For immunohistochemistry
(IHC), in situ hybridization (ISH) or immunofluorescence (IF)
analysis, cell-containing implants with associated connective tissues were fixed with Accustain (SIGMA) for 24 h, dehydrated
through an ethanol series and then processed for paraffin embedding before being sliced with a microtome. Slices from paraffinembedded samples were stained with appropriate antibodies or
probes. Alternatively, plugs were harvested and fixed with a 4%
paraformaldehyde solution overnight at 4 °C, washed thrice in
PBS and then incubated in a glucose solution (30%) for another
48 h before being sliced (45 µm thickness) with a cryostat (Leica).
Both methodologies were equally successful in identifying neovasculature derived from human cells.
For IHC, slides were stained with an anti-human CD31 monoclonal
antibody and then incubated with biotin-labeled secondary antibody
followed by incubation with streptavidin-HRP (Ventana Roche).
For ISH, slides were hybridized according to the manufacturer’s protocol with an Alu probe (780-2845, Ventana Roche) and
then labeled with the ISH iView Blue Detection Kit (760-092,
Ventana Roche).
For IHC and ISH, images were then captured with a camera
mounted on a light microscope (Nikon E-800).
For immunofluorescence assays, slides were stained with either
rhodamine-labeled Ulex europaeus agglutinin I (UEA I, a marker for
human endothelial cells, Vector Laboratories) or PECAM-1 (M-20)
(CD31; sc1506, Santa Cruz Biotechnology) and anti-human nuclei
1:100 (MAB1281, Millipore) counterstained with DAPI. Images were
captured with confocal microscopes (Zeiss, LSM 510 or LSM780).
Animals. Mice were housed in an AAALAC-accredited
facility and in compliance with European Directive related
to Laboratory animal protection. All murine experiments
were conducted with approval of the local laboratory animal
Ethics Committee of Toulouse Sanofi Research Center. NOD.
Cg-PrkdcscidIl2rgtm1Wjl /SzJ mice (or NOD-Scid IL2rγnull
abbreviated as NSG; age, 7 weeks; weight, 20 g) were purchased
from Charles River Laboratories, housed in air-flow racks on a
restricted-access area and maintained on a 12-h light/dark cycle
at a constant temperature (22 ± 1 °C). Teratoma experiments
Calcium live-cell imaging. Subconfluent cells were washed
with DMEM:F12 and incubated for 45 min with 1 µM Fluo4/AM (Molecular Probes) in 0.5% BSA, DMEM:F12 in an
incubator at 37 °C, 95% CO2. After a washing step to remove
unloaded dye, cells responses to 100 µM carbachol or vehicle
(water) were imaged in HEPES-buffered, phenol red–free
DMEM:F12 in a wide-field fluorescence microscope
(Olympus BX61WI) equipped for fast fluorescence imaging. Image capture was performed with Metamorph and an
EM-CCD camera (Hamamatsu). Image analysis was carried
Acetylated LDL uptake assay and vascular tube-like structure
formation assay. In short, 80% confluent endothelial cells
derived from human ES/iPS cells were incubated with 10 µg ml−1
Dil-Ac-LDL (L23380, Molecular Probes) for 3 h in DMEM:F12.
The cells were washed three times with PBS, dissociated using
TrypLE and analyzed by flow cytometry.
Briefly, to assess the formation of capillary structures, a suspension of 4 ×105 endothelial cells per ml in the presence EBM-2/
EGM-2 was prepared. Subsequently, 100 µl per well was dispensed
on flat-bottom 96-well plates coated with Matrigel (BD). Tube
formation was observed after 24 h of incubation, and a minimum
of three replicates per experiment was analyzed.
nature methods
doi:10.1038/nmeth.2255
out with Metamorph and Fiji software. To determine
functional SMC contraction after stimulations, the cell surface
area was determined before and after carbachol exposure.
28. Giorgetti, A. et al. Generation of induced pluripotent stem cells from
human cord blood using OCT4 and SOX2. Cell Stem Cell 5, 353–357 (2009).
29. Aasen, T. et al. Efficient and rapid generation of induced pluripotent stem
cells from human keratinocytes. Nat. Biotechnol. 26, 1276–1284 (2008).
30. Ludwig, T.E. et al. Derivation of human embryonic stem cells in defined
conditions. Nat. Biotechnol. 24, 185–187 (2006).
© 2012 Nature America, Inc. All rights reserved.
Statistical evaluation. Statistical analyses of all endpoints were
performed by using standard unpaired Student t-test (one-tailed,
95% confidence intervals) using the SPSS/PC + statistics 11.0 software (SPSS). All data are presented as mean ± s.d. or s.e.m. where
indicated and represent a minimum of two independent experiments with at least two technical duplicates.
doi:10.1038/nmeth.2255
nature methods