c-Kit+ Progenitors Generate Vascular Cells for Tissue-Engineered Grafts
Through Modulation of the Wnt/Klf4 Pathway
Campagnolo P PhD1*, Tsung-Neng T PhD2, Hong X MD2, Kirton JP PhD2, So P-W
PhD3, Margariti A PhD2, Di Bernardini E PhD2, Wong MM PhD2, Hu Y MD2,
Stevens MM PhD1 and Xu Q MD PhD2*
1
Department of Materials, Royal School of Mines, Imperial College, London, United
Kingdom
2
Cardiovascular Division, British Heart Foundation Centre, King’s College, London,
United Kingdom
3
Institute of Psychiatry, Psychology & Neuroscience, King’s College, London,
United Kingdom
Short Title: c-Kit+ progenitors and vessel graft
Word count: 5041
*
Corresponding authors:
Paola Campagnolo
Department of Materials, Royal School of Mines, Imperial College, Prince Consort
Road, London, United Kingdom, Email: p.campagnolo@imperial.ac.uk, Tel: +44
(0)2075946761, Fax: +44 (0)20 75946757
Qingbo Xu
Department of Cardiology, King’s College London, 125 Coldharbour lane, London,
United Kingdom, Email: qingbo.xu@kcl.ac.uk, Tel: +44 (0)20 7848 5295, Fax: +44
(0)20 7848 5296
Keywords: vascular graft; stem cells; endothelialization; cell signalling
1
Abstract
The development of decellularised vessel scaffolds for small diameter grafts is
hampered by their limited long-term patency, mostly due to the lack of luminal cell
coverage by endothelial cells (EC) and to the low tone of the vessel due to absence of
a contractile smooth muscle cell (SMC) layer. In this study, we identify a novel
population of vascular progenitor c-Kit+/Sca-1- cells readily available in large
numbers and derived from an immuno-privileged source of embryonic stem cells
(ESCs). We also define an efficient and controlled differentiation protocol yielding
fully differentiated ECs and SMCs in sufficient numbers to allow the repopulation of
a tissue engineered vascular graft, improving the long-term performance and
mechanical resistance of the graft. We showed that Krüppel-like transcription factor 4
(Klf4) could regulate the choice of differentiation pathway of these cells.
Interestingly, Klf4 was regulated by the canonical Wnt pathway activator lithium
chloride and was able to modulate the downstream pathway by acting on β-catenin.
When seeded ex vivo on a decellularised vessel, cells derived from c-Kit+/Sca-1- cells
recapitulated the native vessel structure and upon in vivo implantation in the mouse,
markedly reduced neointima formation and mortality, restoring functional
vascularisation. Our data shows that ESC-derived c-Kit+/Sca-1- cells differentiated
through a Klf4/β-catenin dependent pathway are a suitable source of vascular
progenitors for the creation of superior tissue-engineered vessels from decellularised
scaffolds.
2
Introduction
Therapies for coronary and peripheral vascular diseases often involve vascular grafts
to restore blood perfusion. In clinical practice, the transplantation of an autologous
vessel is the primary choice; however, almost 40% of patients lack autologous vessels
of an adequate quality. To overcome this limitation, artificial alternatives have been
devised, including synthetic expanded polytetrafluoroethylene (ePTFE) and Dacron
grafts and decellularisation of natural vessels [1]. In particular, the development of
decellularised vessel scaffolds allows for the creation of an ‘off-the-shelf’ material
combining the characteristics of the natural vessels and as a substrate for cell adhesion
and growth with low immunogenicity. Despite their promise, the application of
decellularised vessel scaffolds in the clinic has been hampered by inadequate longterm patency due to thrombosis and neointimal hyperplasia1. We previously showed
in an animal model that the patency of decellularised vessels is limited due to the lack
of adequate endothelialisation [2]. The outcome of the graft was greatly improved by
the local application of vascular endothelial growth factor (VEGF), which induced the
local recruitment of endothelial progenitor cells to the scaffold [2]. For the same
reason, the seeding of the luminal side of the scaffold with endothelial cells has been
demonstrated to improve graft success rate by ensuring a non-thrombogenic surface
of contact with the blood. Similarly, the seeding of smooth muscle cells (SMCs) in the
media of the decellularised vessel was shown to improve the mechanical stability of
the graft and its compliance [3,4]. However, the use of mature endothelial cells (ECs)
and SMCs is hampered by their limited availability from an autologous source and
low proliferation rate. For this reason, the exploitation of the immuno-privileged and
plastic embryonic stem cells (ESCs) is highly advantageous. Stem cells are highly
proliferative and can give rise to a high number of differentiated cells including ECs
3
and SMCs provided the mechanisms regulating the differentiation process are well
understood and controlled.
In this study, we focused on the isolation of an abundant and clinically relevant c-Kit
positive/Sca1- negative (c-Kit+/Sca-1-) vascular progenitor population from ESCs
and their differentiation to both ECs and SMCs. We then applied these cells to
repopulate the decellularised scaffold for vascular graft. The c-Kit antigen has
previously been used to define progenitor cells in the human and mouse heart where
they participate to repair after myocardial infarction [5] and form both ECs and SMCs
[6,7]. Regulating the choice of differentiation pathway in progenitor cells is
fundamental: (a) to obtain a highly differentiated and pure population for tissue
engineering; and (b) for the maintenance of the physiological state once the vessel is
grafted. For vascular progenitor cells, the mechanisms have been only partially
elucidated. In this paper, we focused on Krüppel-like factor 4 (Klf4), which is known
to play an atheroprotective function in the vessel [8-12] and participate to antiinflammatory [13-16] and shear stress response [17,18]. We also investigated the
interaction of Klf4 with Wnt/β-catenin signaling, which plays an important role in
vasculature development and endothelial function/remodeling [19-21] through the
shuffling of β-catenin and activation of T cell factor (TCF) target genes [13,14,22].
Understanding the differentiation mechanisms underpinning c-Kit+/Sca-1- cell fate is
crucial for their use as a cell source for vascular grafts.
Material and methods
For expanded Materials and Methods, refer to the Supplemental Material provided
4
Cell culture, isolation and differentiation
Mouse ESCs (ES-D3 cell line, American Type Culture Collection [ATCC]) and
isolated c-Kit+/Sca1- cells were cultured as previously reported [23]. Differentiation
was induced by plating c-Kit+/Sca1- cells on collagen IV-coated flasks in presence of
differentiation medium (DM; α-MEM containing 10% FBS, 0.2 mM 2mercaptoethanol, 100 U/ml penicillin, and 100 μg/ml streptomycin; Invitrogen)
containing either VEGF (50 ng/ml, Peprotech) or platelet-derived growth factor
(PDGF, 20 ng/ml, Sigma) for up to 21 days. The endothelial differentiation protocol
was improved with the application of shear stress between Day 3 and 5 using an
orbital shaker.
Decellularised vessel preparation and seeding
The preparation of the decellularised vessels was performed as previously
described[2] treating the descending aorta with 0.075% SDS for 2 h. c-Kit+ ECs were
seeded using a bioreactor where the grafts were fixed between two needles and the
bioreactor was set up in a standard incubator at 37°C. Scaffolds were preconditioned
for 2 h with the indicated coating. 2 x 106 c-Kit+ EC were then injected and allowed
to seed for 12 h at a continuous rotational movement before initial flow was set up.
For double seeded scaffolds, a second seeding step was performed with 1 x 106 c-Kit+
derived SMCs. After seeding, medium flow rate was adjusted stepwise to reach a
shear stress of 30 dynes/cm2. Vessels were harvested on Day 5 and then used for
further analysis ex vivo or immediately grafted to animals. Decellularised vessels were
used as a control.
Picrosirius collagen and Miller’s elastin staining was performed to assess the collagen
content in the tissue-engineered vessel.
5
Decellularised and tissue-engineered vessel grafting to the right carotid artery
Mice were anesthetised and the middle portion of the carotid artery was ligated and
dissected between the two ties. The graft was interposed as previously described using
a two-cuffs based system [2].
A total of 56, 28 and 13 mice were transplanted respectively with non-seeded, c-Kit+
EC seeded, and c-Kit+ ECs + SMCs seeded grafts, respectively. Survival was
monitored for up to 56 days and tissues for patency monitoring were collected at 2, 4
and 8 weeks.
Mice underwent magnetic resonance imaging (MRI) on a 7T Agilent MRI scanner
tuned to 400 MHz 1H frequency.
Lesion measurement
For en face staining, the tissue-engineered vessels were cut longitudinally and
mounted with the lumen facing up before incubation with Alexa Fluor 488 phalloidin
and/or 4'-6-Diamidino-2-phenylindole (DAPI). Lesion measurement was performed
by staining cross-sections obtained at the centre of the graft with hematoxylin and
eosin (H&E stain).
Statistical analysis
Data were analysed with GraphPad Prism 5.02 program using ANOVA and two-tailed
student’s t-test for two-groups or pair-wise comparisons. The results were expressed
as the mean ± standard error of the mean (SEM).
Results
6
Isolated c-Kit+/Sca-1- cells represent a novel population of ESC-derived vascular
progenitor cells
ESCs cultured on collagen IV became adherent and elongated and contained high
numbers of c-Kit+/Sca-1- cells (about 50% of the total). c-Kit+/Sca-1- cells were
isolated by flow cytometry (FACS) and stimulated with VEGF in combination with
shear stress or with PDGF-BB (Figure 1a). In the presence of VEGF, the cells
assumed cobblestone-like features (Figure 1aiii) and expressed EC markers CD31,
Flk-1 and VE-cadherin at both mRNA (Figure 1bi) and protein level (Figure 1bii-iii).
Functional differentiation was demonstrated by Matrigel assay and Ac-LDL uptake
(Figure 1Biv and Supplemental Figure 1). When cultured with PDGF-BB, the cells
grew in size, assuming a polygonal shape (Figure 1aiv), and started overexpressing
SMC specific genes (Figure 1ci) and proteins (Figure 1cii-iii).
c-Kit+ derived vascular cells repopulated the decellularised graft and improved in
vivo outcome
When suitable patient-derived vessels are unavailable for vessel graft procedures,
decellularised vessels offer an attractive alternative. Our lab has developed an
efficient mouse model mimicking the clinical situation whereby the aorta is harvested
and decellularised ex vivo before being grafted in the mouse carotid artery, thus
simulating the patient situation during vessel surgery [2]. In this study, we aimed to
exploit the potential of c-Kit+ derived cells for pre-clinical application by seeding
them on the decellularised scaffold and implanting it in the mouse model of vessel
graft. We tested a set of conditions to determine the most efficient seeding protocol
leading to a homogenous luminal attachment of ECs on the graft. Application of shear
force was initiated at a rate of 3 dyne/cm2 and then gradually increased to reach the
7
physiological shear stress of 30 dyne/cm2. The protocol proved crucial for the cell
attachment to the lumen; the best coverage was obtained when the flow was increased
hourly over the first 24 h (Figure 2a and b). Next, the decellularised vessel was coated
with gelatin or fibronectin and results showed that cell attachment was vastly
improved after coating, especially following the application of the shear (Figure 2c).
Under these conditions, c-Kit+ ECs efficiently attached to the luminal surface of the
vessel, producing a continuous monolayer of CD31+ ECs (Figure 2d) that responded
to the application of physiological shear stress by aligning to the direction of the flow
and becoming elongated (Figure 2e). Collagen and elastin staining showed that cell
seeding improved the extracellular matrix content (Figure 2f).
When decellularised vessels seeded with c-Kit+ ECs were implanted in a model of
vascular graft, we observed increased residual lumen area (Figure 2g) and reduced
neointimal formation (Figure 2h). As reported in Supplemental Table 1, the major
complication observed in mice grafted with decellularised vessel was thrombus
formation, which was completely abolished by graft seeding (Supplemental Table 1).
Finally, decellularised vessels were seeded with a combination with c-Kit+ derived
SMCs to improve the stability of the vessel. Seeding of the graft with ECs only or
ECs + SMCs significantly increased the survival of the mice (Figure 2i) by reducing
graft occlusion (Figure 2l). MRI confirmed the superior performance of ECs + SMCs
seeded vessels (Figure 2m).
Together, these data indicate that the application of progenitor-derived vascular cells
for repopulation of vascular grafts is feasible and that seeding dramatically reduces
lumen occlusion and ultimately mortality.
Klf4 induces EC differentiation and inhibits SMC differentiation
8
Data presented so far demonstrated that c-Kit-derived vascular progenitor cells can
give rise to both ECs and SMCs and that their application to tissue-engineered
vascular grafts can significantly improve in vivo performance and survival. Precise
understanding and regulation of the molecular mechanisms determining the choice of
differentiation pathway of vascular progenitor cells is fundamental for the rational
design of repopulated decellularised vascular grafts.
We next tested whether the expression of the Krüppel-like transcription factor Klf4
was regulated upon EC and SMC differentiation. We found that Klf4 was strongly
induced during EC differentiation and downregulated during SMC differentiation
(Figure 3a). Upon adenoviral overexpression of Klf4 (Supplemental Figure 2), we
observed increased EC commitment of the c-Kit+ derived vascular progenitors as
shown by increased tube-formation capacity (Figure 3b). Klf4 overexpression led to
increased expression of EC markers CD31, VE-cadherin and eNOS at both RNA and
protein level, and a concomitant reduction in SMC markers (Figure 3c and d).
Additionally, lentiviral knockdown of Klf4 (Supplemental Figure 2) resulted in
increased expression of SMC and the inhibition of EC markers at both mRNA and
protein levels (Figure 3e and f). These results highlight the central role of Klf4 in the
determination of the differentiation fate in c-Kit-derived vascular progenitor cells.
Klf4 and canonical Wnt pathway interact to determine c-Kit+ cell differentiation
fate
The canonical Wnt pathway has been implicated in the fate decision of stem cellderived vascular cells [17,18,21,24]. Klf4 has also been shown to interact with βcatenin in the nucleus in the context of tumour growth [25,26]. In our study, we found
that Klf4 overexpression led to a nuclear accumulation of β-catenin as shown by
9
confocal imaging and western blot analysis (Figure 4a and b); this effect was reversed
when Klf4 expression was inhibited (Figure 4c). The reported downstream effectors
of the Wnt pathway Axin2, ID2 and Cyclin D1 were altered by Klf4 overexpression
and knockdown (Supplemental Figure 3).
Treatment of cells with lithium chloride (LiCl), a Wnt pathway and β-catenin
activator, resulted in an increase of Klf4 expression and consequently an increase in
EC markers and a reduction of SMC markers expression (Figure 4c), mimicking the
effects of the exogenous upregulation of Klf4 described above. Conversely, when βcatenin expression was knocked down, EC differentiation was impaired: EC marker
expression was reduced alongside increased levels of SMC markers (Figure 4d and e).
To confirm the importance of Klf4 in the activation of the Wnt pathway, we
established that the activity of the β-catenin-dependent TCF promoter was ablated by
knockdown of Klf4, as shown by TOP-flash luciferase assay (Supplemental Figure 4).
Interestingly, LiCl-dependent EC marker upregulation was reduced in Klf4-deficient
cells (Figure 4f). Conversely, β-catenin knockdown blunted the Klf4-induced
upregulation of CD31 expression and downregulation of SMA (Figure 4g). Together,
these data corroborate our findings that the mechanism of LiCl induction of EC
differentiation is mediated by Klf4, and that Klf4 in turn requires β-catenin to
promote the process of differentiation.
Discussion
Acute coronary obstruction requires artery bypass surgery where an autologous vessel
is grafted to the narrowed artery [15,16,27]. When autologous grafts are unavailable,
the decellularisation of native vessels can potentially provide alternative material
endowed with comparable characteristics [28-32]. However, the delayed
10
endothelialisation dramatically increases the potential for neointimal formation [2,812], which creates the need for a suitable source of transplantable ECs. Stem cellderived vascular cells allow the generation of a greater number of ECs compared to
mature sources that could be exploited in the clinical practice owing to their
privileged immunogenic profile [19-21,33]. In this study, we provide evidence that
ESC-derived c-Kit+/Sca-1- cells are a suitable source of ECs and SMCs and can
therefore be considered bona fide vascular progenitor cells. In particular, we
demonstrated the clinical relevance of ESC-derived cells in tissue engineering by
seeding them on a decellularised scaffold using a bioreactor and applying it to a
model of vascular graft. The optimised graft preparation and seeding described
allowed the creation of an improved cellularised graft displaying a continuous luminal
layer of endothelial cells and a robust SMC layer. This later improved survival and
patency and reduced graft failure by avoiding thrombus, neointima development, and
graft rupture [32,34,35]. The use of an abundant population of mouse progenitor cells
characterised by the expression of the c-Kit antigen makes our findings easily
translatable to their human counterpart. The c-Kit antigen is expressed in several
progenitor populations in adult human tissues including the heart where they act as a
reservoir of cardiomyocytes, ECs and SMCs [5,36].
A thorough understanding of the molecular mechanisms underlying the differentiation
of stem cells into the vascular cell lines used for the creation of the grafts is of great
importance to ameliorate graft coverage and performance. In this study, we present a
novel mechanism involving the transcription factor Klf4 and the canonical Wnt
pathway that could be exploited in future to improve the levels of endothelialisation
of vascular grafts and control SMC overgrowth leading to neointimal growth. In
particular, we described the central role of the transcription factor Klf4 in driving
11
differentiation preferentially towards the EC lineage and therefore reducing SMC
differentiation. This was in line with previous findings showing that Klf4 is
upregulated by shear stress [18], a typical EC differentiation stimulus, and that Klf4
regulates eNOS [10,14,31,37] and VE-cadherin expression [16] and inhibits SMC
maturation [38]. Furthermore, a recent publication showed that Klf4 played a
fundamental role in the maintenance of the vascular phenotype of endothelial
progenitor cells [39]. Interestingly, the Klf4-induced canonical Wnt pathway
activation and effect on differentiation was mimicked by LiCl, a Wnt activator. These
results are in line with previous studies describing the importance of Wnt in the
development of the embryonic vasculature [20,21] and could therefore lead to the
supplementation of LiCl in the medium used for graft luminal seeding.
The data presented described for the first time the vasculogenic potential of ESCderived c-Kit+ cells and their suitability as a source of vascular cells for the creation
of alternative grafts. We provided a novel insight into the mechanism regulating their
differentiation, thus providing the translational potential for this stem cell therapy.
Acknowledgement
This work was supported by British Heart Foundation grant RG/09/004.
We would like to thank Mr Zhongyi Zhang for the exceptional technical work.
Conflicts of interest
None
12
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Figure legends
Figure 1. Isolation and differentiation of c-Kit+/Sca-1- cells. a: Differentiated ESC acquire adherent
phenotype (ai) and c-Kit+/Sca-1- were isolated by flow cytometry (red box, aii). Addition of VEGF and
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application of shear induced endothelial phenotype (EC, aiii) while PDGF induced smooth muscle cells
phenotype (SMC, aiv). b: VEGF treated cells over expressed EC markers as shown by real time-PCR
(bi), immunocytochemistry (bii) and flow cytometry (biii). Functional differentiation was assessed by in
vitro tube formation assay (biv). c: PDGF treatment induced expression of SMC markers, as shown at
mRNA level (ci) and by immunocytochemistry (cii) and western blot analysis (ciii). Data are expressed
as fold-change over Day 0 (dotted lines). Scale bars: 200 μm (a), 25 μm (bii), 100 μm (biv), 50 μm (cii) *
P<0.05; ** P<0.01; *** P<0.001 vs. Day 0.
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Figure 2 Ex vivo seeding of decellularised vessel scaffolds and in vivo grafting. Two protocols
were followed to increase the shear from 3 to 30 dyne/cm2 in the tissue engineered grafts after luminal
seeding of ECs: ‘daily step-wise’ (protocol A) or ‘hourly step-wise’ (protocol B) (a); cell retention was
quantified in (b). Effect of PBS or gelatin or fibronectin coating on cell retention in static (empty bars)
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and shear (grey bars) was quantified in (c). c-Kit+ ECs formed a homogeneous CD31+ luminal
monolayer on the decellularised vessel (d) and elongate in response to shear (e). Picosirius staining for
collagen and Miler’s elastin staining of decellularised vessels showed improved matrix deposition after
cell seeding (f). In vivo implantation of vessels seeded with c-Kit+ ECs showed improved lumen patency
(g) and reduced neointima formation (h). Both the seeding with ECs and with the combination of ECs
and SMCs (ECs + SMCs) increased the survival of grafted animals (i) and reduced vessel obstruction
(l). MRI detection of the blood flow in the normal carotid (m, i), an occluded carotid grafted with a
decellularised vessel (m, ii) and a patent carotid grafted with ECs + SMCs seeded scaffolds at Day 2 (m,
iii), and 2 weeks (m, iv). Red arrows indicate the position of the grafts. Scale bars: 50 μm (d, f, l) and
100 μm (e). * P<0.05; ** P<0.01; *** P<0.001.
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Figure 3. Effect of Klf4 expression in differentiating c-Kit+ cells. VEGF treatment induced, while
PDGF decreased Klf4 expression (a, vs. Day 0, dotted line). Overexpression of Klf4 (AdKlf4) increased
EC differentiation and reduced SMC markers as shown by tube-formation (b), PCR (c) and western blot
(d), as compared to control virus (AdCTL). Klf4 knockdown (ShKlf4) reduced endothelial marker
expression and increased levels of SMC markers at RNA (e) and protein level (f) *P<0.05, **P<0.001,
***P<0.0001 vs. control.
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Figure 4. Klf4 and canonical Wnt pathway are interconnected in the determination of c-Kit+ cells
differentiation. Confocal imaging showing the membrane localisation of β-catenin in control cells
(AdCTL) and nuclear accumulation in Klf4 overexpressing cells (AdKlf4, a). Western blot confirmed βcatenin activation by AdKlf4 (b) and inhibition by ShKlf4 (c). LiCl (β-catenin activator) induced Klf4
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expression, induced EC marker and reduced the SMC marker expression in a concentration-dependent
manner (c: vs. NaCl, dotted line). Knockdown of β-catenin (Shβcat) impaired VEGF-dependent EC
differentiation and increased SMC markers (d, vs. control virus: dotted line and e). LiCl-induced increase
of EC markers was inhibited by Klf4 knockdown (f, ShKlf4). Klf4-induced EC marker upregulation and
SMC marker downregulation is ablated by β-catenin knockdown (g). * P<0.05; ** P<0.01. Scale bars:
10μm (a). * P<0.05; ** P<0.01 and *** P<0.001.
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