Whartons Jelly Stem Cells: A Novel

Cell Source for

Oral Mucosa and Skin Epithelia
Regeneration
-Bhagyesha Patil
- Bhagyashree Bachhav
- Harini Krishnan
- Snehal Kolhekar
1
Scientists used the stem cells taken from the umbilical cord.
These stem cells can be stored in tissue banks, -> used
instantly when injuries are caused.
The scientists used Wharton jelly mesenschymal stem
cells from the human umbilical cord and combined with a
biomaterial made of fibrin - a protein found in the clotting of
blood - and agarose - a polymer usually extracted from
seaweed.
The combination of the Wharton jelly mesenschymal stem
cells and biomaterial led to the growth of artificial skin and
oral mucosa - a mucous membrane lining the inside of the
mouth.

THE ARTICLE
2
Anatomy of skin
and oral mucosa
Both have two things
in common, epethilia
and the connective
tissue.
Epithilia consists of
keratinocytes and are
avascular.
The roles of the
connective
tissue are related to the
sustenance of the
differentiation
status of the overlying
epithelium

3
Embryonic
Stem Cells
Adult Stem
cells
Perinatal
Stem Cells
Ethical issues, human
rejection, tertoma
formation
Limited proliferation
potential and
differentiation capability
Obtained from tissue
that is normally
discarded.
low risk to the mother
and the newborn and
with few ethical
concerns.
Human
Whartons
jelly stem
cells
(HWJSCs) are
considered
immunoprivil
eged
perinatal
cells that
have high
differentiatio
n capability
and are
easily
available for
therapeutic
applications.

As mesenchymal
stem cells, HWJSCs
retain the ability
to differentiate to
mesodermic
tissues (cartilage,
tendon, bone, and
ligament)
4
A mixture of human fibrin obtained from frozen human
plasma and 0.1% agarose.
An average of 250,000 cultured oral mucosa and skin
fibroblasts were added to 5 ml of the mixture immediately
before inducing the polymerization of the artificial stroma.
Once the stromas jellified, HWJSCs were seeded on top of the
oral mucosa and skin artificial stromas and cultured for 7
days. H-hOM (heterotypical human oral mucosa) and H-hS.
For in vivo evaluation a segment of skin of 2.5 x 2.5 cm was
excised from the back of the animals. Then the H-hOM and H-
hS was engrafted on the surgical wounds.
DEVELOPMENT OF 3D BIOACTIVE SYSTEMS
TO INDUCE EPITHELIAL DIFFERENTIATION OF HWJSCs

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Protein-based polymers have the advantage of mimicking many features of
extracellular matrix and thus have the potential to direct the migration, growth
and organization of cells during tissue regeneration and wound healing and for
stabilization of encapsulated and transplanted cells.
Properties of fibrin:
Fibrin is a protein matrix produced from fibrinogen, which can be autologously
harvested from the patient providing an immunocompatible carrier for delivery
of active biomolecules.
In addition, fibrin naturally contains sites for cell binding, and therefore has been
investigated as a substrate for cell adhesion, spreading, migration and
proliferation.
Fibrin provides a material that can be rapidly invaded, remodeled and replaced by
cell-associated proteolytic activity.
Moreover, due to its biomimetic and physical properties it is also widely used as a
cell carrier to many cell types, such as keratinocytes, tracheal epithelial cells ,
murine embryonic stem cells, mesenchymal progenitor cells, etc.
But, problems of instability and degradation invivo, so combination with agarose
provides improved properties resembling natural skin


FIBRIN-AGAROSE SCAFFOLD
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These biomaterials possessed "added
resistance, firmness and elasticity to the
skin" compared to other biomaterials like
collagen, chitosan, polyglycolic acid, etc.

FIBRIN-AGAROSE SCAFFOLD
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ANALYSIS OF THE MESENCHYMAL NATURE OF HWJSCs
Aim: To confirm the differential capability of cells

Method Used: Flow Cytometry

Principle: Flow cytometry is a technology that simultaneously measures and
then analyzes multiple physical characteristics of single particles, usually
cells, as they ow in a uid stream through a beam of light. The properties
measured include a particles relative size, relative granularity or internal
complexity, and relative uorescence intensity.

Staining: 1. osteogenic alizarin red S
2. adipogenic Oil red O
3. chondrogenic Alcian blue


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IN VIVO EVALUATION OF THE EPITHELIAL
DIFFERENTIATION POTENTIAL
AIM: In vivo evaluation of the Epithelial differentiation potential of HWJSCs

BRIEF PROCEDURE:
-a segment of skin 2.5x2.5cm was excised from the backs of the
immunodeficient athymic mice
- grafting of H-hOM and H-hS using absorbable suture material
- grafted tissue harvested for histological analysis
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HISTOLOGICAL ANALYSIS
AIM: Histological analysis of in vitro and in vivo samples using light
microscopy, SEM and TEM

Histology is the study of the cellular organization of body tissues and organs

OUTLINE:
-Staining using hematoxylin and eosin
- for immunofluoresence labelling with epithelial markers eg. CK1, CK4,
CK8, CK13, involucrin, filaggrin, plakoglobin
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HWJSCs :
1.
Can be easily harvested at
low cost
2.
Can be easily expanded and
cryopreserved
3.
Shorter doubling time, rapid
propagation and expansion
DISCUSSIONS AND CONCLUSIONS
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In vitro
1.
Differentiation level of HWJSCs in vitro
is limited
2.
Lack of well defined strata, rete ridges,
chorial papillae and surface patterns
3.
Lack of well defined epithelial
markers.
DISCUSSIONS AND CONCLUSIONS
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In vivo
1.
Differentiation level of HWJSCs is
efficient with high maturation level
2.
Well defined basal, spinosum,
granulosm, and corneum cell layers
3.
Specific differentiation patterns on
cell surface in 20 days
DISCUSSIONS AND CONCLUSIONS
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In vivo
1.
Differentiation process requires
longer time in vivo grafting
2.
Cell cell contacts in H-hom and
Hhs after 30 days
3.
Cells tend to join and form tight
epithelial barrier.
DISCUSSIONS AND CONCLUSIONS
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General
1.
Heterotypical tissues have lower
differentiation rate in vivo
2.
Typically seen in H-hom
3.
Potential to differentiate in to
ectodermal layers
DISCUSSIONS AND CONCLUSIONS
15