Placenta 35 (2014) 994e1000

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Placenta
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Expression of epithelial markers by human umbilical cord stem cells.

A topographical analysis
 n a, b, C.A. Alfonso-Rodríguez a, b, c, C. Martínez-Go
I. Garzo  mez d, V. Carriel a, b,
M.A. Martin-Piedra a, b, R. Fernandez-Valade s b, e, M.C. Sa
nchez-Quevedo a, b,
a, b, *
M. Alaminos
a
Department of Histology (Tissue Engineering Group), University of Granada, Spain
b n Biosanitaria ibs. Granada, Spain
Instituto de Investigacio
c
PhD programme in Biomedicine, University of Granada, Spain
d
PhD programme in Clinical Medicine and Public Health, University of Granada, Spain
e
Division of Pediatric Surgery, University Hospital Virgen de las Nieves, Granada, Spain

a r t i c l e i n f o a b s t r a c t

Article history: Introduction: Human umbilical cord stem cells have inherent differentiation capabilities and potential
Accepted 13 September 2014 usefulness in regenerative medicine. However, the epithelial differentiation capability and the hetero-
geneity of these cells have not been fully explored to the date.
Keywords: Methods: We analyzed the expression of several undifferentiation and epithelial markers in cells located
Human umbilical cord in situ in different zones of the umbilical cord ein situ analysise and in primary ex vivo cell cultures of
Human Wharton's jelly stem cells
Wharton's jelly stem cells by microarray and immunofluorescence.
Epithelial markers
Results: Our results demonstrated that umbilical cord cells were heterogeneous and had intrinsic
capability to express in situ stem cell markers, CD90 and CD105 and the epithelial markers cytokeratins 3,
4, 7, 8, 12, 13, 19, desmoplakin and zonula occludens 1 as determined by microarray and immunofluo-
rescence, and most of these markers remained expressed after transferring the cells from the in situ to
the ex vivo cell culture conditions. However, important differences were detected among some cell types
in the umbilical cord, with subvascular zone cells showing less expression of stem cell markers and cells
in Wharton's jelly and the amnioblastic zones showing the highest expression of stem cells and epithelial
markers.
Conclusions: These results suggest that umbilical cord mesenchymal cells have intrinsic potential to
express relevant epithelial markers, and support the idea that they could be used as alternative cell
sources for epithelial tissue engineering.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction Histologically, the UC is covered by an amnion-derived simple

In recent years, the umbilical cord stem cells (UC) have become a
topic of great interest as cell source for Tissue Engineering and
Regenerative Medicine. The UC is connecting fetus and mother
during pregnancy, the UC is formed around the 26th week of
gestation and gradually grows to form a coiled cord of 60e65 cm
length and 40 g weight at birth [1].
* Corresponding author. Department of Histology, Faculty of Medicine, University
of Granada, Avenida de Madrid 11, E18012 Granada, Spain. Tel.: þ34 958243514;
fax: þ34 958244034.
E-mail address: malaminos@ugr.es (M. Alaminos).
epithelium comprising amnioblast cells (AM) [2,3], two arteries
and a vein, spirally wound and immersed in a cellular mucous
connective tissue without capillaries or lymphatic vessels. First
described in 1656 by Thomas Wharton, Wharton's jelly is now an
important source of mesenchymal stem cells (MSC) [4,5]. Human
Wharton's jelly stem cells were first isolated and cultured ex vivo by
McElreavey [6]. According to their original location, there are three
distinct cell populations in the Wharton's jelly [7]: (1) the sub-
amnioblastic population (SAM cells), in the outer most UC mucous
tissue, directly underneath the AM cells; (2) subvascular (SV) cells
situated around the umbilical blood vessels, and (3) remaining
cells, located below the SAM region and between the SV regions
(this last population are the Wharton's jelly stem cells, which are

http://dx.doi.org/10.1016/j.placenta.2014.09.007
0143-4004/© 2014 Elsevier Ltd. All rights reserved.
 n et al. / Placenta 35 (2014) 994e1000
I. Garzo
commonly isolated and cultured for research purposes as WH cells.
Several studies suggest that cells corresponding to different UC
zones could show different phenotypes and play different roles in
the intact UC [8].
In common with other mesenchymal stem cells, UC cells have
distinct characteristics, such as the expression of CD90, CD105,
CD73 surface markers, potential to differentiate into adipogenic,
chondrogenic and osteogenic mesodermic lineages, high telome-
rase activity, rapid proliferation and low expression of major his-
tocompatibility complex class I and II [5,9]. However, very few
studies have examined the differential behavior of the UC cell types.
Recent studies developed by our group have demonstrated the
unique potential of cultured Wharton's jelly stem cells, corre-
sponding to the WH zone, to differentiate into non-mesodermic
tissues such as skin and oral mucosa epithelia [10].
Our hypothesis is that each population of UC cells may express
key epithelial cell markers. Therefore, the objective of the present
work was to analyze the expression of several undifferentiation and
epithelial markers in cells allocated in situ in different zones of the
umbilical cord -in situ analysis- and in primary ex vivo cell cultures
of Wharton's jelly stem cells to shed light on the intrinsic capability
of UC cells to differentiate into epithelial-like cells and to determine
what specific cell population could be the most adequate for ex vivo
epithelial induction.
2. Materials and methods
2.1. Human umbilical cord samples and cell cultures
Six human umbilical cords (UCs) were obtained from full-term newborns
delivered by previously programmed caesarian section due to obstetric reasons,
with the consent of the parents. For histological analysis, specimens of the 6 UCs
were obtained, fixed in formaldehyde and embedded in paraffin for the study of cells
in situ. For the generation of ex vivo Wharton's jelly stem cell cultures (C-WH), UC
cells -cells corresponding to the WH zone-were isolated and cultured from the same
6 UCs. Briefly, arteries and veins were removed from the UC, and the exposed WH
zone Wharton's Jelly tissue was surgically dissected, sectioned into small pieces and
digested with a type I collagenase (Gibco BRL Life Technologies, Karlsruhe, Germany)
and trypsin 0.5 g/L-EDTA 0.2 g/L solution (Gibco BRL). Once isolated, these cells were
seeded an expanded in 75 cm2 culture flasks using Amniomax™ culture medium
(Gibco BRL) as previously described [10]. C-WH were kept in culture under saturated
humidity conditions at 37  C, 5% (v/v) CO2.
Ethical approval for this experimental protocol was granted by the Institutional
Ethical and Research Review Board.
Fig. 1. Samples analyzed in this study. A: UC section stained by using H&E. Identification of four different zones of the UC around the two arteries and the vein. SAM: UC cells
allocated at the subamnioblastic zone of the umbilical cord sections. SV: UC cells localized at the subvascular zone of the UCs sections. WH UC cells allocated at the Wharton's jelly
zone of the UCs sections. AM: Amnioblast cells in the surface of UC sections. B. ex vivo cultured cells obtained from WH zone.
995
2.2. Histological analysis and immunofluorescence
UC sections and C-WH were analyzed to determine the histological pattern of UC
cells both in situ and ex vivo. The following cell types were analyzed (Fig. 1): 1-
amnioblast cells in the surface of UC sections (amnioblasts zone or AM); 2- UC cells
allocated at the subamnioblastic zone of the umbilical cord sections (SAM); 3- UC
cells localized at the subvascular zone of the UCs sections (SV); 4- UC cells located at
the Wharton's jelly zone of the UCs sections (WH); 5- Ex vivo C-WH.
For each cell type, immunofluorescence assays was used to identify relevant
mesenchymal stem cell markers (CD90 and CD105) and epithelial markers,
including cytokeratins (CK3/12, CK4, CK7, CK8, CK19, pan-cytokeratin, keratins
AE5/AE3) and cellecell junctions (desmoplakin dDSPd, zonula occludens
dZO1d). As summarized in Supplementary table S1, UC tissue sections were
treated with specific antigen retrieval solutions for antigenic unmasking for
20 min, and blocked in 2.5% horse serum (S-200 Vector laboratories, Inc., Burlin-
game, CA) in a humidity chamber during 30 min. Subsequently, primary antibodies
were diluted and applied to tissue sections. After washing in PBS, samples were
incubated in FITC anti-mouse or CY3 anti-rabbit secondary antibodies (both at
1:500). Finally all samples were washed and contrasted with 25 ml of a 1.5 mg/ml
DAPI fluorescent mounting solution (Vectashield®, Vector laboratories, Burlingame,
USA) on coverslips to allow the mounting medium to disperse over the entire
section. In the case of the ex vivo samples immunofluorescence (C-WH cells
cultured on chamber slides), 0.1% triton X-100 was used as a permeabilizing so-
lution and blocked using 2.5% horse serum. All samples were incubated with pri-
mary and secondary antibodies (Supplementary table S1), washed and contrasted
with DAPI fluorescent mounting solution. All results were analyzed using a Nikon
eclipse 90i microscope (Nikon, Tokyo, Japan). Positive and negative controls are
shown in Supplementary table S1.
Protein expression as determined by immunofluorescence was semi-
quantitatively analyzed by four independent histologists. The expression was
scored as (þþþ): Strong expression; (þþ): Mild expression; (þ): Slight expression;
(þ/): Very slight expression; (): Negative expression.
2.3. Microarray analysis
Total RNA was isolated from six different samples corresponding to C-WH of six
different individuals by using Qiagen RNeasy System™ (Qiagen, Mississauga,
Ontario, Canada). The total RNA was converted into cDNA using a reverse tran-
scriptase (Superscript II, Life Technologies, Inc., Carlsbad, California, EEUU) and a T7-
oligo(dT) primer. Subsequently, biotinilated cRNA was generated by using a T7 RNA
polymerase and biotin-11-uridine-5'-triphosphate (Enzo Diagnostics, Farmingdale,
Nueva York, EEUU). Labeled cRNA was chemically fragmented to facilitate the pro-
cess of hybridization and hybridized to Affymetrix Human Genome U133 plus 2.0
oligonucleotide arrays for 6 h at 45  C. Genes corresponding to the mesenchymal
stem cells markers and the epithelial markers analyzed in this work were selected
and the average expression was calculated for each gene as Affymetrix Fluorescent
Units (AFU). Each transcript was classified as present (P) or absent (A) for each
sample by using the Affymetrix Suite software.
996 n et al. / Placenta 35 (2014) 994e1000
I. Garzo

Table 1
Expression of undifferentiation markers (stemness), cytokeratins and cellecell junctions expression in the five umbilical cord zones analyzed in this work as determined by
immunofluorescence. SAM: UC cells allocated at the subamnioblastic zone of the umbilical cord sections. SV: UC cells localized at the subvascular zone of the UCs sections. WH:
UC cells allocated at the Wharton's jelly zone of the UCs sections. AM: Amnioblasts cells in the surface of UC sections. C-WH: ex vivo cultured cells. (þþþ): Strong expression;
(þþ): Mild expression; (þ): Slight expression; (þ/): Very slight expression; (): Negative expression; (*): Most cells show negative expression, with part of the cells
showing positive signal.

Stemness Cytokeratins CelleCell junctions

CD90 CD105 CK7 CK8 CK19 CK4 CK13 CK3/12 PANCK AE5/3 DSP ZO1

SAM þþþ þþþ þ þþ þ þþ þ/ * þþþ þ/ e þþ

SV þþ þþ þþ þþþ þþ þþ þ þ þþþ þþ þ þþ
WH þþþ þþþ þþ þþþ þþ þþ þ þ/ þþþ þþ þ þþ
AM þþþ þþþ þþþ þþþ þþþ þþþ þþþ þþþ þþþ þþ þ þþþ
HWJSC þþþ þþþ * þþþ þþþ e þ/ * þþþ þþþ þþ þþþ

3. Results strongly positive in C-WH, with high expression as determined by

3.1. Stemness characterization of umbilical cord cells
Immunofluorescence analysis of two key markers of undiffer-
entiated mesenchymal stem cells revealed that all cells in the
umbilical cord were strongly positive for CD90 and CD105 in situ,
although SV cells showed lower expression of both markers. Once
cultured ex vivo, C-WH cells also expressed high levels of CD90 and
CD105 (Table 1, Fig. 2). In agreement with this, microarray analysis
of C-WH showed positive expression of CD90 (average
1490.57 ± 198.7 AFU, present expression) and CD105 (154.7 ± 3.2
AFU, present expression) in these cells (Table 2).
3.2. Cytokeratin expression in umbilical cord cells
As summarize in Tables 1 and 2, the expression of cytokeratins
varied between specific cells analyzed. First, the immunofluores-
cence analysis of simple epithelial cytokeratins in UC sections
(Fig. 3) demonstrated that CK7 was strongly positive in the AM
zone and slightly positive in the SAM zone with mild signal in SV
and WH zones. In addition, the analysis of C-WH revealed that most
cells showed negative expression of CK7 ex vivo, although some
cells were positive, what was confirmed by microarray analysis
(92.3 ± 143.9 AFU, present expression in 33.3% of the cell cultures
analyzed and absent in 77.7%). Regarding CK8, the AM, SV and WH
zones were strongly positive, similar to C-WH, which showed high
positive expression of CK8 also at the mRNA level (1342.9 ± 1147.6
AFU, present expression). Cells allocated at the SAM zone showed
mild expression of CK8. Whilst CK19 was mainly expressed in the
AM zone with the lowest expression corresponding to the SAM
region. CK19 expression was positive in SV and WH zone but
microarray (1039.3 ± 1743.8 AFU, present expression) (Fig. 3).
Analysis of CK4 expression showed that all UC cells were posi-
tive in situ, in contrast to C-WH that were negative at the protein
and the mRNA levels (3.0 ± 1.9 AFU absent expression). Addition-
ally, CK13 was strongly expressed in the AM zone, with very slight
expression in SAM and slight expression in SV and WH zones and
very slight in C-WH (which had 7.4 ± 3.0 AFU, absent for this
cytokeratin). CK3/12 was strongly expressed by AM, slightly
expressed by SV cells, and very slightly expressed in cells at the WH
zone. Interestingly, the expression of CK3/12 in C-WH was het-
erogeneous, with approximately half of the cells showing negative
protein expression and half of the cells had positive signal (Fig. 4).
The mRNA levels of CK3/12 as determined by microarray were very
low (7.4 ± 3.0 and 1.6 ± 2.7 AFU, absent expression).
Finally, the immunofluorescence analysis of cells in situ samples
using two broad spectrum anti-cytokeratin antibodies revealed
strong positive expression of pan-cytokeratin in all UC section zones
and a mild expression of AE5/AE3 cytokeratin in AM, SV and WH
zones, being slightly positive in SAM zones of the UC sections (Table 1
and Fig. 5). A strong signal for both markers was found in C-WH.
Analysis of the cellecell junction protein DSP revealed that the
AM, SV and WH zones of the UC were slightly positive for this
epithelial marker, whilst the SAM zone did not show any signal
(Fig. 6). In turn, ex vivo analysis of C-WH revealed mildly positive
expression of DSP at both the protein and the mRNA levels
(223.7 ± 299.0 AFU, present expression). In situ expression of ZO1
protein was strong in AM cells but limited in SAM, SV and WH zones.
In addition, the expression of ZO1 was highly positive ex vivo, with
high immunofluorescence signal and mRNA expression in C-WH
(578.9 ± 83.0 AFU, present expression) (Tables 1 and 2). Strikingly,
the expression of ZO-1 did not mimic the typical belt-like pericellular

Fig. 2. Analysis of expression of the undiferentiation markers CD90 (upper panel) and CD105 (lower panel) in the different zones of the umbilical cord sections and in cultured cells.
SAM: UC cells allocated at the subamnioblastic zone of the umbilical cord sections (red arrow). SV: UC cells localized at the subvascular zone of the UCs sections. WH: UC cells
allocated at the Wharton's jelly zone of the UCs sections. AM: Amnioblasts cells in the surface of UC sections (white arrow). C-WH: in vitro cultured Wharton's jelly stem. Scale bars:
10 mm.
 n et al. / Placenta 35 (2014) 994e1000
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Table 2
mRNA expression of several stem cell markers and epithelial markers as determined by microarray in 6 different C-WH samples (C-WH1 to 6). For each sample, the presence (P)
or absence (A) of each transcript and the absolute mRNA expression levels are shown. Means and standard deviations (SD) correspond to the 6 samples. Expression values are
shown as Affymetrix fluorescent units (AFU).

C-WH1 C-WH2 C-WH3 C-WH4 C-WH5 C-WH6 C-WH1 C-WH2 C-WH3 C-WH4 C-WH5 C-WH6 Mean SD Gene symbol Gene name

P P P P P P 177.4 165.4 189.2 113.8 172.8 109.6 154.7 34.2 CD105 CD-105
P P P P P P 1579.9 1629.0 1485.2 1183.4 1722.8 1342.7 1490.5 198.7 CD90 CD-90
P A A A P A 385.0 24.2 27.8 25.9 33.2 57.6 92.3 143.9 KRT7 keratin 7
P P P P P P 3659.7 1155.4 902.8 702.8 937.6 699.1 1342.9 1147.6 KRT8 keratin 8
P P P P P P 4595.4 328.2 316.1 459.2 223.4 313.2 1039.3 1743.8 KRT19 keratin 19
A A A A A A 1.1 3.8 2.6 1.2 6.2 3.2 3.0 1.9 KRT4 keratin 4
A A A A A A 3.3 1.4 0.7 5.0 0.9 2.4 2.3 1.7 KRT13 keratin 13
A A A A A A 2.4 5.7 7.9 8.0 11.1 9.2 7.4 3.0 KRT3 keratin 3
A A A A A A 0.3 0.5 0.3 7.2 0.9 0.5 1.6 2.7 KRT12 keratin 12
P P P P P P 823.9 138.2 51.7 67.7 194.0 66.9 223.7 299.0 DSP desmoplakin
P P P P P P 622.6 616.1 491.2 700.9 490.2 552.6 578.9 83.0 ZO1 Zonula occludens 1

expression pattern of epithelial cells. Instead, expression tended to
be scattered throughout the cell membrane (Fig. 6).
4. Discussion
Human UC stem cells can be easily isolated, with few ethical
concerns. HWJSC represent a primitive cell population as compared
to adult mesenchymal stem cells such as bone marrow or adipose
tissue stem cells, offering new perspectives for cell-based therapies
[4]. However, these cells are heterogeneous and several works
including the present study have found differences in the expres-
sion of cell markers among the different regions of the UC [11]. For
this reason, it is highly important to select an adequate cell popu-
lation for specific uses in cell therapy. In the present work, we have
demonstrated that UC cells are able to express key markers of
epithelial lineage both at the protein and the mRNA levels, with a
good correlation between most markers. Consequently, this study
helps identify specific cell populations with defined characteristics
for use in regenerative medicine.
First, this stemness analysis showed that cells corresponding to
AM, WH, and SAM zones of the UC expressed the typical markers of
undifferentiated cells in a better way than cells cells in the SV zone.
Moreover, there was evidence that C-WH obtained from UC did not
modify their stemness status upon transferring from in situ to
ex vivo conditions. As some authors suggest [12], all regeneration
processes are highly dependent on undifferentiated cells with the
potential to switch into other cell type by means of a differentiation
process. Therefore, the cell population with highest differentiation
potential appears to be AM, WH and SAM zones.
Several studies have confirmed that UC cells are multipotent or
even pluripotent stem cells which can differentiate into several
lineages, including adipose cells, chondrocytes, osteoblasts,
neuronal cells, cardiomyocytes, hepatocyte-like and pancreas beta
cells [13e16]. However, very few works have suggested the po-
tential of these cells to differentiate into the epithelial lineage [10].
The most surprising result of the present study was that expression
of epithelial markers not only occurs in the amnioblastic zone (AM)
which is epithelial in nature, but also in the Wharton's jelly zone
(WH), subamnioblastic zone (SAM) and sub-vascular zone (SV),
which are mesenchymal. These results are in agreement with
studies developed by Hayward et al. [17], that previously demon-
strated this very interesting pattern of cytokeratin expression
including cytokeratins of both simple and stratified epithelium,
although these studies were not carried out on UC sections. The
presence of cytokeratins, especially in the SV and WH zones, is
unusual and unexpected for these cell types and opens a door to the
use of UC cells for the generation of epithelial tissues. These results
could support the use of UC stem cells as alternative cell sources for

Fig. 3. Analysis of the expression of cytokeratins of simple epithelium CK7 (upper panel), CK8 (middle panel) and CK19 (lower panel) in the different zones of the umbilical cord
sections and in cultured cells. SAM: UC cells allocated at the subamnioblastic zone of the umbilical cord sections (red arrow). SV: UC cells localized at the subvascular zone of the UCs
sections. WH: UC cells allocated at the Wharton's jelly zone of the UCs sections. AM: Amnioblasts cells in the surface of UC sections (white arrow). C-WH: in vitro cultured Wharton's
jelly stem. Scale bars: 10 mm.
998 n et al. / Placenta 35 (2014) 994e1000
I. Garzo

Fig. 4. Analysis of expression of cytokeratins of stratified epithelium CK4 (upper panel), CK13 (middle panel) and CK3/12 (lower panel) in the different zones of the umbilical cord
sections and in cultured cells. SAM: UC cells allocated at the subamnioblastic zone of the umbilical cord sections (red arrow). SV: UC cells localized at the subvascular zone of the UCs
sections. WH: UC cells allocated at the Wharton's jelly zone of the UCs sections. AM: Amnioblasts cells in the surface of UC sections (white arrow). C-WH: in vitro cultured Wharton's
jelly stem. Scale bars: 10 mm.

Fig. 5. Analysis of expression of broad spectrum cytokeratins PANCK (upper panel), and AE5/3 (lower panel) in the different zones of the umbilical cord sections and in cultured
cells. SAM: UC cells allocated at the subamnioblastic zone of the umbilical cord sections (red arrow). SV: UC cells localized at the subvascular zone of the UCs sections. WH: UC cells
allocated at the Wharton's jelly zone of the UCs sections. AM: Amnioblasts cells in the surface of UC sections (white arrow). C-WH: in vitro cultured Wharton's jelly stem. Scale bars:
10 mm.

the generation of bioengineered tissues such as the human cornea,
skin and oral mucosa as recently reported [10,18]. In our work, the
pattern of cytokeratin expression in the UC cells is unforeseen,
since it combines cytokeratins of simple epithelia -CK7, CK8 and
CK19- with those of stratified epithelia such as CK4, CK13 and CK12.
Specifically, AM, SV and WH zone UC cells expressed high amounts
of CK7 and CK8. This could be explained by the fact that that CK8 is
considered as a primary keratin, because it is the first to be pro-
duced by simple epithelia in the embryo [19]. This expression can
also be associated with human fetal skin and could be considered as

Fig. 6. Analysis of the expression of cellecell junction proteins DSP (upper panel), and ZO1 (lower panel) in the different zones of the umbilical cord sections. SAM: UC cells
allocated at the subamnioblastic zone of the umbilical cord sections (red arrow). SV: UC cells localized at the subvascular zone of the UCs sections. WH: UC cells allocated at the
Wharton's jelly zone of the UCs sections. AM: Amnioblasts cells in the surface of UC sections (white arrow). C-WH: in vitro cultured Wharton's jelly stem. Scale bars: 10 mm.
 n et al. / Placenta 35 (2014) 994e1000
I. Garzo
an early promoter of skin differentiation. However, when cells are
isolated and transferred to cell culture conditions, some of the cells
may lose CK7 expression, which could indicate that cells may tend
to partially de-differentiate ex vivo. Along with the simple epithe-
lium markers, the positive expression of CK4, normally expressed
by oral mucosa and urinary system tissues and CK19 may confirm
the potential of UC cells to differentiate also into stratified epithelia,
especially in the case of AM cells followed by SV and WH. This
suggests that these cells have intrinsic potential to differentiate into
several tissue types, including well-differentiated stratified
epithelia such as the skin and oral mucosa, and simple epithelia
such as the vascular epithelium as we previously demonstrated
[10]. Regarding CK3/12 proteins expression, our results revealed
that although most cells were negative -also at the mRNA level-, a
percentage of UC cells are able to constitutively express these
cytokeratins. CK3/12 are usually found only in the cornea epithe-
lium [20] and they are important for cornea differentiation and
mechanical strength. This suggests that a small subpopulation of
UC cells could have intrinsic potential to differentiate into corneal
epithelial cells, although a high percentage of cells remained
negative. This finding supports the idea that UC cells are highly
heterogeneous and guarantee future studies in this field.
As we expected, immunofluorescent staining of cellecell junc-
tions' proteins and mRNA analysis revealed the higher expression
of these markers in AM cells. However, cells at the SV and WH
zones also reached high levels of DSP and ZO1 expression, which
have been particularly associated to stress-bearing epithelial tis-
sues. This finding could again support the idea that SV and WH
zones could have intrinsic potential to epithelial differentiation.
However, the fact that cells in the UC do not form real cellecell
junction complexes could explain the atypical diffuse expression
pattern found in these cells.
One of the limitations of the present study is that it is based on
non-quantitative immunolabeling studies. Although the results
were confirmed by microarray analysis, future works should
analyze protein expression of each UC cell type by flow
cytometry.
Although the expression of these epithelial markers was pre-
viously demonstrated in UC cells, our study revealed important
differences among the different cell types. In general, the highest
expression of the epithelial markers corresponded to AM zone
cells. This cell population is of epithelial origin and it has been
previously used for generation of artificial tissues [21]. However,
these cells are difficult to isolate and culture and their prolifera-
tion potential is limited as previously suggested [17]. For these
reasons, the UC cells that have been more widely used are the
mesenchymal cells. In this regard, our results suggest that WH
zone cells showed the highest expression of stem cells markers
and epithelial markers as determined by immunofluorescence and
microarray. Therefore, these cells, which are the most commonly
isolated and cultured UC cells worldwide, demonstrated to have
the highest epithelial differentiation potential among all mesen-
chymal UC cells.
In summary, our results confirm the heterogeneity of the UC
cells and the requirement to select the most appropriate cell pop-
ulation for differentiation studies according to the specific tissue or
organ to be regenerated. These findings may explain from a basic
point of view previous published works demonstrating the capa-
bility of UC cells to differentiate into skin and oral mucosa epithelia
[10,18]. As an easily accessible cell source with high proliferation
and differentiation potential, UC stem cells emerge as a potentially
source of cells for tissue engineering protocols in which epithelial
cells are difficult to obtain due to low proliferation capability or to
pathologies associated to the cell donor. In addition, in this study
we confirmed the potentiality of WH zone cells, as these could be
999
intrinsically committed to the epithelial lineage despite their
mesenchymal nature. Future works should confirm these results
using a greater number of stem cells markers and cell junctions in
order to fully characterize the capability of these cells to differen-
tiate in epithelial cells. Also, elucidation of the UC micro-
environmental conditions that are associated to the background
of plasticity of UC cells and its potential relationship with various
complications of pregnancy should be addressed.
Conflict of interest
None.
Acknowledgments
This study was supported by the Spanish Plan Nacional de
n Científica, Desarrollo e Innovacio
Investigacio  n Tecnolo gica
(I þ D þ I) from the Spanish Ministry of Economy and Competi-
tiveness (Instituto de Salud Carlos III), grant FIS PI11/1582 and PI11/
2668 (co-financed by FEDER funds, European Union) and by grant
P10-CTS-6060 from Consejería de Economía, Innovacio n, Ciencia y
Empleo, Junta de Andalucia (Proyectos de Excelencia). We thank Dr.
R. Brown for improving the scientific redaction of the manuscript.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.placenta.2014.09.007.
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