In vitro culture of keratinocytes from human umbilical cord blood mesenchymal stem cells the saigonese culture

Cell Tissue Bank (2011) 12:125–133
DOI 10.1007/s10561-010-9174-8

In vitro culture of Keratinocytes from human umbilical cord
blood mesenchymal stem cells: the Saigonese culture
Tran Cong Toai • Huynh Duy Thao • Ciro Gargiulo
Nguyen Phuong Thao • Tran Thi Thanh Thuy •
Huynh Minh Tuan • Nguyen Thanh Tung •
Luis Filgueira • D. Micheal Strong

•

Received: 6 January 2010 / Accepted: 3 March 2010 / Published online: 27 March 2010
Ó Springer Science+Business Media B.V. 2010

Abstract There have been many attempts to acquire
and culture human keratinocytes for clinical purposes
including from keratotome slices in media with fetal
calf serum (FCS) or pituitary extract (PE), from skin
specimens in media with feeder layers, from suction
blister epidermal roofs’ in serum-free culture and from
human umbilical cord blood (hUCB) mesenchymal
stem cells (MSCs) in media with skin feeder layers.
Conversely this study was designed to investigate
whether keratinocytes could be obtained directly from
hUCB MSCs in vitro. It is widely established that
mesenchymal stem cells from human umbilical cord
blood have multipotent capacity and the ability to
differentiate into disparate cell lineages hUCB MSCs

were directly induced to differentiate into keratinocytes by using a specific medium composed of primary

culture medium (PCM) and serum free medium (SFM)
in a ratio 1:9 for a period of 7 days and tested by
immunostain p63 and K1-K10. Cells thus cultured
were positive in both tests, confirming the possibility to
directly obtain keratinocytes from MSCs hUCB in
vitro.
Keywords Mesenchymal stem cell Á
UCB Á Keratinocyte culture Á Cell culture

Introduction
T. C. Toai (&) Á H. D. Thao Á N. P. Thao Á
T. T. T. Thuy Á H. M. Tuan
Department of Histo-pathology, Embryology, Genetics
and Biotechnology for Tissue Transplants, Pham Ngoc
Thach Medical University, Ho Chi Minh City, Vietnam
e-mail:
C. Gargiulo Á L. Filgueira
University of Western Australia School of Anatomy
and Human Biology, Crawley, WA, Australia
D. M. Strong
Department of Orthopaedics and Sport Medicine,
University of Washington School of Medicine, Seattle,
WA, USA
N. T. Tung
Department of Pathology, Children No 1 Hospital
in HCMC, Ho Chi Minh City, Vietnam

MSCs from human-UCB
Human UCB is a significant source of hematopoietic
stem cells and has been considered as a valid

alternative for hematopoietic stem cell transplantation (Toai et al. 2009; Lee et al. 2004; Park et al.
2006; Van de Ven et al. 2007; Maurice et al. 2007;
Musina et al. 2007; Sasaki et al. 2008). MSCs from
hUCB have been used in a wide range of diseases
such as liver disorders, myocardial infarction, central
nervous system condition or in degenerative pathologies such as diabetes, Crohn’s disease, osteogenesis
imperfect (OI), rheumatoid arthritis (RA) and osteoarthritis (OA) (Toai et al. 2009; Lee et al. 2004;

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Riordan et al. 2007; Kogler and Wernet 2006; Kim
et al. 2004; Reddi 2007; Koblas et al. 2005; De Bari
and Dell’Accio 2007; Tuan and Chen 2006; Waese
and Kandel 2007; Park et al. 2006). The most
valuable potential of MSCs is their ability to switch
into different cell phenotypes such as osteocytes,
chondrocytes, adipocytes, hepatocytes, neurons,
myocytes and keratinocytes with a great immunemodulatory and anti-inflammatory capacity that make
them a tool for clinical applications (Toai et al. 2009;
Lee et al. 2004; Goodwin et al. 2001; Chamberlain
et al. 2007; Kim et al. 2004; Bieback et al. 2004;
Musina et al. 2007; Jang et al. 2006; Rosada et al.
2003; Van de Ven et al. 2007; Maurice et al. 2007;
Tse and Laughlin 2005; Koc and Lazarus 2001; Chao
et al. 2004; Majhal et al. 2006; Sasaki et al. 2008;
Stocum 2006).
The skin is a barrier to the outside elements,

temperature loss, pathogens and trauma (Markowicz
et al. 2005). The use of skin substitutes for skin
replace in cases of burns and ulcers is a developing
field, however nothing works better than patient’s
own skin (Markowicz et al. 2005). The inconvenience
of using bio-engineered materials for skin graft
replacement is connected to the allogeneic origin of
these cells hence these bio-products can only be used
for wound coverage and not as a graft for tissue
substitution (Markowicz et al. 2005). Nevertheless,
many studies have confirmed a conspicuous advantage of UCB engraftment related with a very low rate
of transplant mortality and no increase of rejection or
graft versus host disease (GVHD) due to a high rate
of tolerance across 1 or 2 HLA-A, B and DR
mismatches and a lower risk of infectious disease
transmission (Van de Ven et al. 2007; Toai et al.
2009; Lee et al. 2004; Riordan et al. 2007; Kogler and
Wernet 2006; Tse and Laughlin 2005). The idea to
obtain keratinocytes from hUCB MSCs is mainly due
to their particular ability to differentiate into different
cell phenotypes and their immune-modulatory and
anti-inflammatory nature that is crucial in the case of
allograft procedures for skin regeneration (Van de
Ven et al. 2007; Toai et al. 2009; Lee et al. 2004;
Riordan et al. 2007; Kogler and Wernet 2006; Tse
and Laughlin 2005; Kamolz et al. 2006). Human
UCB have been shown to have a very limited number
of graft lymphocytes and hUCB MSCs are able to
secrete inhibitory cytokines such as IL10 and TFG-b
whilst maintaining the ability of presenting antigens

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to T cells, a condition that eventually confirm a
tolerogenic antigen capacity of this group of stem
cells (Toai et al. 2009; Lee et al. 2004; Riordan et al.
2007). Moreover, UCB T cells are distinctively
CD45RA? with low intensity of activation markers,
both of which are related with naăve Th0 phenotype
that show a restricted response triggered by recipient
alloantigens (Tse and Laughlin 2005). When isolated
CD34? cells from allogeneic cord blood were
inserted in an autologous fibrin glue of patients with
non-healing wounds, it was noted that a significant
wound repair was achieved without any sign of
GVHD from 3 to 7 months subsequent to the
procedure (Riordan et al. 2007). Then again, Kamolz
et al. successfully used male hUCB stem cells
together with skin from female donors to obtain
keratinocytes in vitro. Using PCR they confirmed the
presence of keratinocytes among the hUCB stem cell
population and by FISH histochemistry they revealed
Y-positive cells within the keratinocytes layer. In
addition, they detected hUCB cells among all layers
of cultured epidermis (Kamolz et al. 2006). Overall,
these data eventually confirm the capacity of hUCB
stem cells as a budding resource for cultivating
human epithelium under vitro conditions. Therefore,

this study was undertaken to demonstrate the ability
of hUCB to be directly influenced to produce
keratinocytes in vitro.

Materials and methods
Cell collection
Umbilical cord blood cells were collected and
isolated from consenting patients from normal full
term and pre-term deliveries. The material was
serology tested for HIV, HBV, HCV and syphilis
by VDRL. The blood was collected with heparin
anticoagulant, 15000UI/1 ml.
Cell processing
Processing UCB primary cells
Mononuclear cells from hUCB were isolated at a
density of 1 9 106 at room temperature using FicollPaque (Amersham, Freiburg-Germany) in a ratio of
1 part of Ficoll-Paque and 3 parts of blood and


Cell Tissue Bank (2011) 12:125–133

centrifuged, 300g for 5 min. Cells were collected and
seeded in flasks (Nunc, Wiesbaden-Germany) containing IMDM (Gibco, Grand Island NY-USA) with
15% fetal bovine serum-FBS (Gibco USA). The total
number of nucleated and viable cells was counted
using trypan blue stain.
Culture procedure for hUCB mononuclear primary
cells
Mononuclear derived cells were incubated at 37°C
with 5% CO2. During the first week, medium was

changed every 2 days and cells washed twice by
buffer solution (PBS). Primary mononuclear cells
began to attach at day 2, cells were passed at day 15
at 70–80% confluence. After each passage, cells were
washed twice with PBS and immersed in a 2 ml
solution of Trypsin–EDTA (Gibco, Brl-USA) and
incubated for 5 min at 37°C with 5% CO2. After
5 min, 2 ml of IMDM plus 15% FBS was added,
cells were removed, aspired and transferred in a tube
for centrifugation for 5 min at 200g. Suspended cells
were removed and seeded in new flasks at a density
of 105/1 ml c.ca.
Culture procedure for direct keratinocytes
differentiation
MSCs were induced to differentiate into keratinocytes by seeding them into a medium composed of a
combination of PCM plus SFM for a period of 7, 14
and 17 days. Human UCB MSCs were subcultured 3
times, after which medium was changed and MSCs
were directly induced to differentiate by using a
keratinocytes medium composed of PCM plus SFM
in a ratio of 1:9. At the day 7 cells were trypsinized
(using Trypsin–EDTA) and collected for K1-10
immune-stain. At the 14th–17th days, other cell
samples were collected and stained with p63.
Skin collection for keratinocyte staining
with p63 antibody
A serum collected from previous consented patient’s
was serology tested for HIV, HBV, HCV and syphilis
by VDRL. The sample was collected with heparin
anticoagulant, 15000UI/1 ml.

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Keratinocyte medium composition
Keratinocyte medium is composed of 1 part of PCM
and 9 parts of SFM.
PCM medium
DMEM medium (Gibco Grand Island NY-USA),
HEPES 1 M (Sigma Ultra), FBS 15%, EGF 100 mg/
ml (Gibco Invitrogen Corporation), Cholera toxin
10-7M (List biological Laboratories Inc.), Hydrocortisone 0,1 mg/ml (Westcort USA), Penicillin/Streptomycin 2009.
Defined keratinocyte-SFM Medium (Gibco Invitrogen Corporation).
Immunohistochemical stain
P63 histochemical stain procedure
Immunohistochemical staining was performed
according to standard procedures. Sample slides and
positive control were stored in an incubator at 37°C
overnight. Slides and control were deparaffinerized
by xylene twice for 5 min and washed by alcohol
100, 90, 80%, respectively for 1 min each passage.
For antigen retrieval, slides were immersed in buffer
solution at pH 9 and steamed in microwave oven for
25 s (S2368-Dako). Endogenous peroxidase activity
was blocked by PBS and incubated in 3% hydrogen
peroxide (H2O2) for 10 min. The antibody used for
p63 was obtained commercially from Neomarkers
(Fremont, CA, USA) and was used at a 1:25 dilution.
The detection step was performed using an LSAB2
System—HPR- Dako ? kit (DAKO; Carpinteria,
CA) as chromogen at 1:20 dilution rate for 20 min.

Samples were counterstained with streptavidin HRP
and hematoxylin (Biomeda-M10).
K1-10 Keratinocyte immunostain fluorescence
staining procedures
The cells were fixed in 1% paraformaldehyde in
culture medium before they were mechanically
detached and spun onto slides using a Shandon
centrifuge (4 min, 600 rpm). Thereafter the cells
were treated for 1 min with 0.1% triton 1009 (ICN
Biomedicals; Aurora, OH) in PBS, before staining
with the mouse anti-human keratin 1/10 monoclonal

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antibody (1ug/ml, CBL266, Chemicon/Millipore,
North Ryde, NSW, Australia) for 4 h, followed by
AlexaFluor488 donkey-anti mouse (1:100, Molecular
Probes/Invitrogen, Mulgrave, Victoria, Australia) for
1 h. The nuclei were counterstained with DAPI (1ug/
ml, Roche Diagnostics, Mannheim, Germany) and
the F-actin filaments with AlexaFluor546 labeled
phalloidine (0.3 Units/ml, Molecular Probes/Invitrogen). Control staining was done similarly without the
primary antibody. The cells were mounted with Dako
fluorescence mounting medium (DakoCytomation;
Carpinteria, CA). The specimens were analyzed and

documented with a Nikon Eclipse 90i microscope
with fluorescence and conventional setting, including
corresponding digital cameras and imaging software.
Fig. 1 Human UCB monoclonal primary stem cells after
2 days of culture in IMDM ?10% FBS, inverse microscope
9100

Results
In vitro culture of keratinocytes from MSCs from
hUCB and their morphology
To confirm the keratinocytic potential of hUCB
derived stem cells, low density mononuclear cells
were isolated from the original source and cultured
under proper condition with IMDM plus 15% FBS
(Figs. 1, 2, 3, 4). In line with other studies, after a few
days of culture, mononuclear cells started to form
clusters of adherent cells with typical fusiform and
elongated fibroblast shape (Figs. 3, 4). After 2 weeks
cells reached 70–80% of confluence forming a dense
monolayer of polyclonal cells, at this stage cells were
trypsinized and cultured for a total of 3 times. At the
3rd passage the old medium was removed and a new
keratinocyte medium was added composed of PCM
plus SFM. Cells started to change shape at day 2
assuming a more round-cuboidal conformation typical of keratinocyte like cells (Figs. 5, 6, 7, 8, 9, 10).
Cells were constantly monitored by inverse microscope and compared with cells from different studies
(data not shown). At day 7, part of samples were
selected and tested for immunohistochemical staining
to confirm the presence of K1-10 (Figs. 15, 16). At
day 14 and 17, the rest of samples were collected and

tested for p63 antibody reactivity (Figs. 11, 12, 13,
14). A sample of human skin was stained with p63
antibody by immunohistochemical staining as a
positive control (Fig. 17).

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Fig. 2 Primary cells from hUCB after 5 days of culture start
assuming fibroblast like shape, inverse microscope 9100

Discussion
By definition, MSCs show regular features includinga
fibroblast like morphology, a high rate of selfrenewal aptitude, an unusual capacity of differentiating into different cell phenotypes and the ability to
play a prominent role in tissue repair and the growth
process (Toai et al. 2009; Lee et al. 2004; Bieback
et al. 2004; Minguell et al. 2001; Reddi 2007; Sasaki
et al. 2008). Because MSCs, in our experience,
qualify by these criteria, we named these cells
mesenchymal stem cells in the current article.


Cell Tissue Bank (2011) 12:125–133

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Fig. 5 MSCs from UCB in keratinocyte medium culture after
3 days (2009)
Fig. 3 After 7 days of culture cells assumed a complete
mesenchymal morphology, inverse microscope 9100

Fig. 6 MSCs from UCB in keratinocyte medium culture after
3 days (1009)

Fig. 4 MSCs at day 10 start forming a compact and dens
monolayer the confluence is nearly 60–70%, inverse microscope 9100

A number of attempts have been performed to use
MSCs in clinical trials in order to regenerate tissues
in spinal cord injuries, myocardial infarction, bone
diseases, neurological diseases and skin (Mazzini
et al. 2006; Chernykh et al. 2006; Waese and Kandel
2007; Kamolz et al. 2006). Meanwhile, there have
been many efforts to attain and culture human
keratinocytes for clinical purposes including: for skin
replacement or wound repair for use as bio-material
or skin substitutes from keratotome slices in media
with fetal calf serum (FCS) or pituitary extract (PE)

from skin specimens in media with feeder layers and
from suction blister epidermal roofs in serum-free
culture (Sasaki et al. 2008; Kamolz et al. 2006).
However, only a few have directly obtained keratinocyte cultures from hUCB MSCs without the support
of any exogenous feed layers in vitro. The results from
those studies eventually validate the possibility of
using these cells as tools in skin regeneration therapy
in vivo (Kamolz et al. 2006; Sasaki et al. 2008).
The microenvironment is of great importance for
the recruitment of circulating MSCs at the affected
site. The inflammation mechanism plays a crucial
role in the wound healing process because of the

accrual of multiple inflammatory factors and cells
which promote tissue recovery and the regeneration

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Fig. 7 Keratinocyte medium is composed of FSM and PCM,
ratio 1:9, the MSCs started changing shape at 3rd day of culture
gradually assuming a typical round cuboidal keratinocyte
shape. MSCs were induced without harvest procedure the old
medium was changed on situ with the new one

Cell Tissue Bank (2011) 12:125–133

Fig. 9 Keratinocytes monolayer after 17 days of culture 9200

Fig. 10 The MSCs completed their differentiation in 17 days
c.ca, 9200, once the cultures reached the 70–80% confluence
they were collected and tested for immunohistochemical stain
for p63 and K1-10
Fig. 8 MSCs, control group, 80% confluence, 15 days 9100.
MSCs in keratinocyte medium after 2 weeks culture c.ca. It’s
possible to visualize the presence of round shape keratinocyte
cells 9100

process by refilling of cells and extracellular components (Sasaki et al. 2008; Kamolz et al. 2006).
Keratinocytes at the wound site express Chemokine
(C–C motif) ligand 21 and secondary lymphoid-tissue

chemokine (SLC/CCL21) inducing a high presence
of MSCs that eventually promote the repair by
transdifferentiation into multiple skin cells (Sasaki
et al. 2008). This mechanism takes place because
MSCs express several chemokine receptors including
CCR7 which is a receptor of SLC/CCL21 that
enhances the recruitment of MSCs in loco (Sasaki
et al. 2008).

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Fig. 11 keratinocytes immunohistochemical stain with p63 at
day 14, red indicates presence of p63 9200


Cell Tissue Bank (2011) 12:125–133

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Fig. 12 Control group stained with Giemsa 9200

Fig. 15 Keratinocytes stained for keratin 1/10, green color,
nuclei blue color, by electronic microscope

Fig. 13 Keratinocyte immunochemical stain for p63 at day 14,
red indicates presence of p63 and blue indicates the nucleus,
inverse microscope 9400

Fig. 16 Control group stain, electronic microscope. Keratinocytes at 7 day culture were collected and stain by immunohistochemical stain for K1-10, it is clear the presence K1/10 in
green color and the presence of actin (red color)

Fig. 14 Keratinocyte immunochemical stain for p63 at day 17,
red indicates presence of p63, inverse microscope 9400

In the current study we have shown that is possible
to obtain keratinocytes from hUCB MSCs in vitro
culture, through direct induction. We isolated MSCs
from hUCB and we cultured up to the 3rd passage
and induced them into keratinocytes using a specific
medium composed of PCM-SFM. MSCs started to
change shape after few days of culture in a new
medium, assuming the typical round-cuboidal keratinocyte shape. Positive immunohistochemical stain

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Fig. 17 Immunohistochemistry result with p63 antibody,
positive control: keratinocytes from skin brown cells stain
positive for p63 as is indicated by black arrow

for K1-10 and p63 were found in confluent cultures,
the expression of these factors seems to rely on the
presence of keratinocytes in culture. In conclusion,
this method presents several advantages it is easy to
perform, there is no need for feeder layers and it can
be accomplished in typical closed culture flasks,
limiting the chances of bacterial contamination. In
addition, although this is still at the in vitro stage, the

results confirm two types of data. Firstly this
substantiates that MSCs are capable of keratinocyte
differentiation and secondly, it shows that MSCs
from hUCB retain a potential capacity in the skin
regeneration process that is of high value in clinical
applications.

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