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CD31 VECadherin vWF

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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

AKeratinocytes

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 lineagecells. B) Represents tri-culture of hESC differentiated cells to develop organotypic in-vitrovascularized 3D stratified epithelia.

RESULTS-1. Differentiation

Day0 Day2 Day5Day4

Day0 Day3 Day9Day6

hESC differentiationto Keratinocytes

hESC differentiationto Vascularprogenitors

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Cytokeratin-14 α6Integrin ΔNp63Rela

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Keratinocyte Markers

α6Integrin Hgh/CD71Lw

2.Characterization:

2.1. mRNA level by q-PCR.

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hESC-vSMCs hESC-EC

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Vascular Smooth Muscle (SMC) Markers

αSMA

SM22α

Calponin

PDGFβ

Sorted for α6H and CD 71L

Keratinocytes (α6H / CD 71L)

α6 I

NT

EG

RIN

AP

C

CD71 FITC

Vascular smooth muscle cells

Sorted for 1). CD34+&CD31+ (ECs)

2). CD34- CD31- PDGFb+ (vSMCs)

Endothelial cells

CD

34

-PE

CD31-APC

2.2. Flow-cytometry analysis.

ΔNP63K19a 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