1. -5
5
15
25
CD31 VECadherin vWF
RelativeGeneExpression
Endothelial Cell (EC) Markers
Human embryonic stem cells- a novel source for
in-vitro three dimensional vascularized oral mucosa.
Handral Harish K 1, Gopu Sriram4, Toh Wei Seong1,3 and Cao Tong1,2,3
1- Oral Sciences, Faculty of Dentistry, National University of Singapore, Singapore.
2- NUS Graduate School of Integrative Science and Engineering, National University of Singapore, Singapore.
3- Tissue Engineering Program, Life Science Institute, National University of Singapore, Singapore.
4- Experimental Dermatology, Institute of Medical Biology, A*Star, Singapore
Oral mucosa, a stratified epithelium which covers the inner lining of the mouth except
teeth part. Its main function is to act as a barrier and protecting deeper tissues from
external insults as well as preventing from the entry of microbial and other toxins into
the body. Oral mucosa has been studied since long years under both in-vivo (animal
models) and in-vitro (primary or immortalized cells) conditions. However, till date no
one has reported to establish a 3D vascularized oral mucosa from human embryonic
stem cells (hESCs). In current study, we developed a novel in-vitro 3D vascularized
oral mucosa from single source of hESCs by differentiating to keratinocytes,
endothelial cells, vascular smooth muscle cells, after which they were tri-cultured in a
PEG-Fibrin based scaffold under animal-component free medium conditions. 3D oral
mucosa carries great potential applications in high throughput screening of
pharmaceutical chemicals, dental cosmetics, drug discovery, etc.
INTRODUCTION
H1 hESC lines were purchased from WiCell Research Institute (Madison, WI) and
were propagated under feeder free conditions on Matrigel™ coated tissue culture
plates in complete mTeSR1™ culture medium. HESC cell lines were differentiated to
keratinocytes, endothelial cells and vascular smooth muscle cells by modifying
previously published protocols1,2 &3 . A PEG-Fibrin scaffold4 was considered as dermal
substitute in which hESC differentiated cells were tri-cultured. Cultured 3D
organotypic tissues were analysed by H & E staining and immuno-fluorescence
staining.
MATERIALS AND METHODS
Mesoderm
Ectoderm
A
Keratinocytes
Vascular smooth muscle cells
Endothelial cells
Blood vessel
formation
Addition of
Keratinocytes
Formation of
Stratified and
vascularized epithelia
B
Figure 1. A) Represents the hESC differentiation to ectodermal and mesodermal lineage
cells. B) Represents tri-culture of hESC differentiated cells to develop organotypic in-vitro
vascularized 3D stratified epithelia.
RESULTS-1. Differentiation
Day0 Day2 Day5Day4
Day0 Day3 Day9Day6
hESC differentiation
to Keratinocytes
hESCdifferentiation
to Vascular
progenitors
0
10
20
30
40
50
Cytokeratin-14 α6Integrin ΔNp63
RelativeGeneExpression
Keratinocyte Markers
α6Integrin Hgh/CD71Lw
2.Characterization:
2.1. mRNA level by q-PCR.
-10
-5
0
5
10
15
hESC-vSMCs hESC-EC
RelativeGeneExpression
Vascular Smooth Muscle (SMC) Markers
αSMA
SM22α
Calponin
PDGFβ
Sorted for α6H and CD 71L
Keratinocytes (α6H / CD 71L)
α6INTEGRINAPC
CD71 FITC
Vascular smooth muscle cells
Sorted for 1). CD34+&CD31+ (ECs)
2). CD34- CD31- PDGFb+ (vSMCs)
Endothelial cells
CD34-PE
CD31-APC
2.2. Flow-cytometry analysis.
ΔNP63K19
a b
2.3. Immuno-fluorescence staining.
VE-CadherinVWF α-SMA
Immuno-fluorescence staining showing the expression of K19 (a) & ΔNp63 (b) in hESC-Keratinocytes;
Von Willebrand Factor (VWF) (c) & VE-Cadherin (d) in hESC-endothelial cells; α-SMA (e) & Calponin (f) in hESC-
vascular smooth muscle cells.
Formation of network of blood vessels from GFP-
hESC-endothelial cells was confirmed by Matrigel
tube formation (g&h).
4X
Contraction of hESC-vascular smooth muscle cells was
confirmed by culturing in presence of muscarinic antagonist for
30 minutes. Arrows represents contracted cells (i & j).
k l
Epidermis
Dermis
Blood
vessels
m
(k) Z-stack images of 3D blood vessels formed by GFP-hESC-endothelial cells has been confirmed by confocal
microscopy. (l) 4x & (m) 20x images of H & E staining of 3D tissue; Immunofluorescence staining of 3D tissues
expressing K19 (n), fibronectin(o) and Collagen-type4 (p). Arrows represents presence of blood vessels.
DISCUSSION AND CONCLUSION
Testing of drugs is always as important as formulating them. Various existing in-vitro platforms used for
testing drugs, drug discovery have their own list of drawbacks such as, lack of reliable results, poor in
reflecting human in-vivo milieu, which gave opportunities to scientists to look forward for alternative in-vitro
models. Discovery of hESCs by James Thomson, has created the demand of the hESC-derived in-vitro
models. Three dimensional architecture of tissues are more promising and more reflective to human in-vivo
conditions. In current stem cell research area, we are the first to report differentiation of hESCs into various
lineages which can establish a 3D in-vitro vascularized platform from a single cell source. These models have
great industrial, pre-clinical as well as academic research value, especially in pharmaceuticals and drug
discovery.
* NOTE- Scale bar of all images 200µm.
1.Kidwai, F.K et al. 2013. Differentiation of human embryonic stem cells to clinically amenable
keratinocytes in an autogenic environment.
2.Tan, J.Y et al. 2013. Efficient differentiation of lateral plate and paraxial mesoderm subtypes from human
embryonic stem cells through GSKi-mediated differentiation.
3.Sriram, G. 2014. In-vitro vascularised tissue equivalents from human embryonic stem cell derived
endothelial and smooth muscle cells.
4.Natesan, S. et al. 2012. Engineering a bi-layered hydrogel to control ASC differentiation.
5..B.Calenic. et al. 2010. Characterization of oral keratinocyte stem cells and prospects of its differentiation
to oral epithelial equivalents.
REFERENCES
hESCs
3D Culture media
n l
c
c e
g
Calponin
Cytokeratin-19 Alpha 6 Integrin ΔNP63
3. Functional studies
C d e f
10Xh
o p
AfterBefore i j