Effects of umbilical cord exctracts on proliferation of human keratinocyte and expression of tyrosine kinase gene of melaninocyte in vitro

Journal of military pharmaco-medicine 7-2013

EFFECTS OF UMBILICAL CORD EXCTRACTS ON PROLIFERATION OF HUMAN
KERATINOCYTE AND EXPRESSION OF TYROSINE KINASE GENE OF
MELANINOCYTE IN VITRO
Phan Kim Ngoc*; Pham Van Phuc*; Dang Thi Tung Loan*
Dinh Thanh Uyen**; Le Van Dong***
summary
Skin aging is a contuinuous process influenced by many factors such as free redicals, sun
exposure etc. This process is characterized with changes in many skin components, primarily
affected by three main cell types: fibroblast, keratinocyte and melaninocyte. Decreased number of
keratinocytes leads to less soften skin; while increase or decrease melanine production will lead to
imbalance of skin color leading to either backhead or vitiligo. This suty evaluates the effects of umbilical
cord exctracts on proliferation of human keratinocyte and melaline production of melaninocyte in
vitro. 25 preparations made of 4 types of extracts included: extracellular exctract of cord tissue;
intracellular extract of cord cells; mixtures of these two extracts; intracellular extract of umbilical cord
stem cells, were supplemented to culture media of human skin keratinocyte and melaninocyte.
Keratinocyte cell proliferation was evalutaetd by MTT technique; melanine production was indirectly
evaluated via tyrosine kinase gene expression by Realtime RT-PCR technique. The results show that
supplementation of 7.5% of preparation 3-5 containing a combination of both extra- and intra-cellular
extracts of umbilical cord led to the highest cell proliferation of k eratinocyte. However, at this
concentration, the 3-5 preparation did not inhibite the expression of tyrosine kinase gene in
melaninocyte. These data suggests that umbilical cord extract would be a potential source of material
for skin softening product but not brightening one.
* Key words: Skin aging; Skin softening; Skin brightening; Stem cell; Umbilical cord extract.

INTRODUCTION
Skin aging is known to be caused mainly
by exposure to light, mostly important is
sunlight UV. Aging affects three main skin
cell types: fibroblast, keratinocyte and

melaninocyte. Among them, skin softness
and brightness are mainly determined by
keratinocyte.
Keratinocyte is the main type of dermis,
accounting for about 90% of dermis cells,
works as barrier to protect skin from harmful
factors from environment such as pathologic

microbes (bacteria, fungi, parasites, and
viruses), heat, UV light or dehydration [5].
Once a pathogen comes to contact to skin,
keratinocyte may react by secreting inflamation
mediators such as chemokines CXCL10,
CCL2 to attract white blood cells to the
invasion places. Keratinocyte plays an
important role in filling up the defects of
injured skin. Once a wound happened,
keratinocytes from hair bulb will migrate in
to fix the wound temporally the keratincytes
from epithelial will replace them [3, 4].

* Hochiminh City Nationnal University
** Mekostem Stem Cell Bank
*** Vietnam Military Medical University
Address correspondence to Le Van Dong: Vietnam Military Medical University

56


Journal of military pharmaco-medicine 7-2013

E.mail:

Melaninocytes are cells which help produce
skin pigment - melanin, to form skin color.
By melanogenesis process, melaninocyte
produces melanin for skin, eye and hair.
In human, melanogenesis is classified into
two types: basal melanogenesis and activated
melanogenesis. In general, people who have
bright skin have low basal melanogenesis.
Exposure to UV-B radiation will increase
activated melanogenesis. The purpose of
melanogenesis is to protect dermis layers
as UV can damage DNA. Melanin absorbs
UV very well and prevents it from penetration
to dermis [2]. Melanogenesis require amino
acid tyrosine as material and enzyme
tyrosinase, by which tyrosinase convert
tyrosine to melanin.

been standardized in our previous study.
Human skin samples, which were voluntarily
donated after plastic surgery, were obtained
from Cho Ray Hospital. Skin samples were
kept in PBS supplemented with penicillinstreptomycin and anti-mycotic and transferred
to the laboratory. In the lab, skin samples
were washed with 1X antibiotic-mycotic two
times followed two times with D-PBS, and
then placed on petri dish to remove fat tissue
with blade and scissors. Cleaned skin was cut

into small pieces of 0.5x0.5 cm then incubated
in dispase II solution 0.5% (Sigma-Aldrich,
St Louis, CA) at 370C for 2 hours. Finally,
separate dermis and epidermis layers out
of each others and used them for the next
experiments.

Recently, stem cells and its extract have
been extensively studied to demonstrate
that they have stimulating effects on skin
regeneration and rejuvenation, especially
skin softening and brightening. It is postulated
that stem cells and stem cell-derived factors
directly and/or indirectly affect on keratinocyte
proliferation and inhibit melanogenesis
process in melaninocyte. Since then, this
research was carried out to: Test the effects
of umbilical cord extracts on proliferation of
human keratinocyte and activity of enzyme
responsible for melaline production of human
melaninocyte in vitro.

In order to isolate keratinocyte, the dermis
layer was cut into small pieces of 2 - 3 mm2
and then placed in flask T-25 (Nunc, Denmark)
with 5 - 10 pieces per flask. 2 mL medium
Stemline Keratinocyte medium (Sigma-Aldrich,
St Louis, CA) supplemented with HKGS
(Life Technologies, USA) was then added to
the flask for culturing at 37oC, 5% CO2 in an

incubator. The medium was replaced every
four days. Sub-culture was done when the
cells reached 70 - 80% confluence using
trypsin/EDTA 0.25% (GeneWorld, Hochiminh
City, Vietnam). During subculturing, protease
inhibitor (Sigma-Aldrich, St Louis, CA) was
used for neutralization of eccess trypsin.

MATERIAL AND METHODS
1. Isolation of human keratinocyte and
melaninocyte.
Adult human skin keratinocytes and
melaninocytes were isolated and cultured
following routine procedures which have

In order to isolate melininocyte, the
epidermis layer was cut into small pieces of
2 - 3 mm2 and then placed in flask T-25
(Nunc, Denmark) with 5 - 10 pieces per flask.
2 mL medium 254CF supplemented with
HMGS-2 (Invitrogen, USA) was then added

2


Journal of military pharmaco-medicine 7-2013
to the flask for culturing at 37 oC, 5% CO2
in an incubator. The medium was replaced
every four days. Sub-culture was done when
the cells reached 70 - 80% confluence using

trypsin/EDTA 0.25% (GeneWorld, Hochiminh
City, Vietnam).
Keratinocytes and melaninocytes of the
3 passage were analyzed for expression
of specific markers, CD24 for keratinocyte
and CD117 for melaninocyte with the following
protocol. Once the cells reached 70% confluence,
they were treated with trypsin/EDTA 0.25%
(GeneWorld, Hochiminh City, Vietnam) then
collect the single cell suspension. 106 cells
were stained with specific antibody for 30
minutes in dark at 40C. The antibody stained
cells were then washed two times with
FACS flow to remove unbound antibody then
resuspened in 500 µL FACSflow. The cells
were then analyzed with FacsCalibur system
(BD Bioscience). All data were analyzed by
CellQuest Pro with 10.000 cells.
rd

130 newborn umbilical cords samples that
voluntarily donated by biological mothers
were collected for research purpose. Mothers
and the cords were selected following a
strictly screening tests followed NetCord
standards and had negative results to all
HIV, HBV, HCV, CMV as described in our
previous paper [1]. After collection in hospitals
and transferred to the lab, each cord was
taken a portion with the length ranging from

18 to 22 cm. The cords were then stored at
-800C till further usage.
* Preparation of extracellular extract:
Frozen cords were defrosted in the 37oC
water bath then washed with physiological
saline; sliced, add buffer (saline supplemented
with protease inhibitor, Sigma-Aldrich, USA)
to protect protein from hydrolysis during the
preparation process. The mixture was milled
thoroughly then centrifuged at 3,000 rpm for
20 minutes at 4oC. The supernatant (namely as
cord tissue extract or extracellular extract) was

2. Umbilical cord stem cell sorting.

then serially diluted to different concentrations

Umbilical cord tissues were processed
mechanically to cell suspension. Stem cells
were sorted by fluorescence activated cell
sorting (FACS) method on FACSJazz system
(BD Bioscience, USA). Each stem cell type
was sorted as it stained with antibody to a
specific marker: CD90 for mesenchymal
stem cell; CD117 for epithelial stem cell;
CD113 for vascular endothelial stem cell.

and kept at -80oC till the next usage.

3. Preparation of cord cell and tissue

extracts.

extract or intracellular extract) was then serially

* Umbilical cord collection and storage:

* Preparation of cellular extract:
The pellet obtained from above was quickly
frozen with liquid nitrogen and defrosted
with warm buffer (saline supplemented with
protease inhibitor) then centrifuged at 3,000
rpm for 20 minutes at 4oC. The supernatant
obtained after this step (namely as cord cellular
diluted to different concentrations and kept
at -80oC till the next usage. The cord tissue
extract and cord cellular extract were mixed

59


Journal of military pharmaco-medicine 7-2013
together in different ratio to form various
formulas for further activity testing.
* Preparation of stem cell extract:
Stem cell cellular extract was also prepared
as described above.
* Cord extracts combinations:
5 main preparations were formulated: cord
tissue extract, total cord cellular extract, cord
stem cell extract, mixture of cord tissue and

cord cellular extracts, control (physiological
saline). Each preparation was diluted into
five different concentrations and form 25
testing formulas coded as followed: Cord
tissue extract: solution 1-1; 1-2; 1-3; 1-4;
1-5; Cord cellular extract: solution 2-1; 2-2;
2-3; 2-4; 2-5; Mixture of cord tissue and
cord cellular extracts: solution 3-1; 3-2; 3-3;
3-4; 3-5; Cord stem cell extract: solution
4-1; 4-2; 4-3; 4-4; 4-5; Control: solution 5-1;
5-2; 5-3; 5-4; 5-5.
4. Cell proliferation assessment by MTT
assay.
The cell proliferation was evaluated following
the instruction of manufacturer (Cell proliferating
kit, GeneWorld, Hochiminh City, Vietnam)
as followed: Take the 96 well cell culture
plates out of the incubator and move to the
clean cell culture area, add sterile MTT
equal to 10% of final volume, put the 96 well
cell culture plates back into the incubator for
another 3 - 4 hours; after the incubation,
take the plates out of the incubator and
dissolved the MTT formazan crystals by
adding equal volume of solvent. The absorbent
was measured within 1 hour after adding
the solvent with a spectrometer (Multimode

Reader DTX880, Beckman-Coulter, USA)
570 nm (measure wavelength) and 690 nm

(referent wavelength).
5. Realtime RT-PCR analysis.
Total RNA was extracted as described
in our previous study [6]. The Real-time
RT-PCR analysis was performed on Eppendorf
gradient S thermal Cycler system (EppendorfAG, Hamburg, Germany).
6. Data analysis.
All tests were repeated three times. Value
p ≤ 0.05 is considered as statistical significant.
Data was analyzed by Statgraphics software
7.0 (Statgraphics Graphics System, Warrenton,
VA).
RESULTS AND DISCUSSION
1. Effects of different extraction formulas
on the proliferation of keratinocyte.
Skin softening is one of the properties in
the modern beauty products. While wrinkle
is results of structural breakdown of skin
extracellular proteins, large hair bulb micro
scar leading to rough skin, skin softening
will stimulate keratinocytes of epidermis to
develop to fill up micro defects on skin. With
the aim to develop a product, which has
skin softening effetc, we test the effects
of main ingredient on the proliferation of
keratinocyte.
After testing the effects of 25 formulas
and identified that supplementation of 7.5%
of formula 3-5 having anti-wrinkle effect in
vitro. Based of that data, we continue to test

60


Journal of military pharmaco-medicine 7-2013
if formula 3-5 also can increase skin softness.

culture medium 7.5% of each of all 25

Targeting for a skin care product that has

formulas. Data on keratinocyte proliferation

both anti-wrinkle and skin softening effects,

are shown in figure 1.

OD value

OD value

OD value

OD value

we then selected to supplement to the

61

Journal of military pharmaco-medicine 7-2013

Figure 1: Keratinocyte proliferation when adding 25 formulas at 7.5% after
(0, 2, 4, 6, 8 and 10 days from left to right).
Data from figure 1 show that supplementation
of 25 formulas have different effects on
keratinocyte proliferation. In general, it is
different from the effects on fibroblast, some
formulas has no clear effect on keratinocyte
in comparison to control group, esspecially
some formulas including 1-3, 1-4, 1-5, 2-1,
2-3, 2-5, 4-1, 4-3, 4-4 inhibite the proliferation
of keratinocyte. Among 25 formulas tested,
formula 3-5 also shows most positive effects
in stimulation the proliferation of human
keratinocyte.

enzyme tyrosine kinase. Since then in
evaluation of skin brightness we indirectly
test the ability to inhibit enzyme tyrosine
kinase by RT-PCR. The data show that
supplementation of formula 3-5 at 7.5% to
melaninocyte culture medium; the expression
of tyrosine kinase gene did not decrease as
compared to control (repeated three times,
p > 0,5) (Figure 2.). It is clear that formula
3-5 did not inhibit the expression of tyrosine
kinase gene; in the other words it did not
reduce melanin production in melaninocyte.

Similar to the effects of umbilical cord
extracts on fibroblast, keratinocytes also
requires both extracellular and intracellular
proteins in order to get optimum proliferation
rate in comparison to just adding either
extracellular or intracellular extract.
2. Evaluation of concentration of formula
3-5 melanin production in melaninocyte.
Melanin is natural pigment of skin, produced
by melaninocyte. Skin pigments make skin
darker; increasement of melanin production
in some places make black spots on skin.
Consequently, black spots are condition
caused by over melanin production.
All melanin are made from polyacetylene.
Most of them are products of tyrosine amino
acid. Melanin synthesis from tyrosin requires

Figure 2: Comparison of tyrosine kinase gene
expression between control and formula
3-5 supplemented group.
In short, formula 3-5 from umbilical crod
cells/tissue lack of factor that reduce melanin
production. In order to produce a brightening
skin care product, one may think of adding

62


Journal of military pharmaco-medicine 7-2013

a strong antioxidant or a factor to inhibit
melanin production or stimulation of melanin
breakdown.
CONCLUSION
This in vitro study demonstrates that
umbilical cord extracts have some effetcs
on human skin keratinocyte. Especially, formula
3-5 which is mixture of cord tissue and
cellular extracts, keratinocyte got strongest
stimulation effect at 7.5%. However, at this
concentration, no inhibition on melanin
production was observed. These data suggest
that umbilical cord extracts can be used for
development of skin softening but not
brightening product.
ACKNOWLEDGMENT
This research was partially financially
supported by research project “Development
and evaluation the effects of beauty product
from umbilical cord stem cell” Code: 1882010 sponsored by Department of Science
and Technology of Hochiminh City.

2. Agar N and Young AR. Melanogenesis: a
photoprotective response to DNA damage?.
Mutation Research. 2005, 571 (1-2), pp.121-132.
3. Claudinot. S, Nicolas. M, Oshima. H,
Rochat. A, Barrandon. Y. Long-term renewal of
hair follicles from clonogenic multipotent stem
cells. Proceedings of the National Academy of
Sciences of the United States of America. 2005,

102 (41), pp.14677-1482.
4. Ito. M, Liu. Y, Yang. Z, Nguyen. J, Liang.
F, Morris. RJ, Cotsarelis. G. Stem cells in the
hair follicle bulge contribute to wound repair but
not to homeostasis of the epidermis. Nature
Medicine. 2005, 11 (12), pp.1351-1354.
5. McGrath JA, Eady RAJ, Pope FM.
Anatomy and Organization of Human Skin. In
Burns T, Breathnach S, Cox N, Griffiths C. Rook's
Textbook of Dermatology (7th ed). Blackwell
Publishing. 2004, p.4190.
6. Phuc P.V. Nhung T.H. Loan D.T, Chung
D.C, Ngoc P.K. Differentiating of banked human
umbilical cord blood-derived mesenchymal stem
cells into insulin-secreting cells. In Vitro Cell Dev
Biol Anim. 2011, 47 (1), pp.54-63.

REFERENCES
1. Phạm Thúy Trinh, Lê Văn Đông và CS.
Nghiên cứu phân lập tế bào gốc trung mô từ
màng dây rốn trẻ sơ sinh. Tạp chí Thông tin Y
dược. Số Chuyên đề Miễn dịch học. 2010, tr1-6.

63


Journal of military pharmaco-medicine 7-2013

64