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Open Science Journal of Clinical Medicine 2015; 3(5): 182-187
Published online September 28, 2015 (http://www.openscienceonline.com/journal/osjcm)
Isolation, Culture and Identification of Undifferentiated Wharton’s Jelly Mesenchymal Stem Cells (WJ-MSCs) Derived from the Human Umbilical Cord
Moustafa A. H. El-Nawasany1, Elsayed G. E. Khedr
1, Moustafa E. E. Motawee
1, Zeinab A-B Ali
2,
Hosam E. H. Kamel3, Tamer M. M. Abu-Amara
1, *
1Histology & Cytology Department, College of Medicine, Al-Azhar University, Cairo, Egypt 2Histology & Cytology Department, College of Medicine, Suiz Canal University, Ismailia, Egypt 3Obstetric & Gynecology Department, College of Medicine, Al-Azhar University, Cairo, Egypt
Email address
[email protected] (T. M. M. Abu-Amara)
To cite this article Moustafa A. H. El-Nawasany, Elsayed G. E. Khedr, Moustafa E. E. Motawee, Zeinab A-B Ali, Hosam E. H. Kamel, Tamer M. M. Abu-
Amara. Isolation, Culture and Identification of Undifferentiated Wharton’s Jelly Mesenchymal Stem Cells (WJ-MSCs) Derived from the
Human Umbilical Cord. Open Science Journal of Clinical Medicine. Vol. 3, No. 5, 2015, pp. 182-187.
Abstract
Wharton’s Jelly-derived Mesenchymal stem cells (WJ-MSCs) have been found to be an alternative source of stem cells for the
regenerative medicine. This is as WJ-MSCs have potential for differentiation, self-renewal and unique immunomodulatory
properties. Aim of the work: This work was conducted to assess our lab and staff capabilities in our Tissue Culture and Medical
Genetics Unit to isolate, culture, identify and characterize WJ-MSCs derived from the adult human umbilical cord to be used
for clinical applications in the future. Material and Methods: WJ-MSCs were isolated from the human umbilical cord collected
from late-gestation pregnancy after cesarean section. Isolated WJ-MSCs cultured as P0 and recultured as P1. After confluency,
WJ-MSCs were collected and characterized for surface markers expression using flow cytometry technique. Results: WJ-
MSCs isolated in this study were positive for CD44, CD90, CD105 and negative for CD34. These findings suggest that WJ-
MSCs appeared to be readily obtainable and highly proliferative cell lines that come from noninvasive source. Conclusion:
isolated WJ-MSCs may be a good noninvasive source for stem cell therapy and regenerative medicine in the future. However,
further studies should be done to evaluate its use as an allogenic cell source and expression of other markers in relation to cell
immunogenicity.
Keywords
Stem Cells, Mesenchymal Cells, Wharton’s Jelly, Tissue Culture, Umbilical Cord, Flowcytometry
1. Introduction
Adult stem cells are undifferentiated cells that are found in
diverse tissues and are frequently multipotent cells [1].
Postnatal sources of stem cells can be reached from different
kinds of tissues including bone marrow, brain, adipose tissue,
umbilical cord blood and amniotic fluid [2-6]. The umbilical
cord is an extraembryonic structure that supplies feeding for
the fetus during the intrauterine life. The umbilical cord is
formed during early gestation and surrounds the yolk sac.
Yolk sac is an embryonic source for two different populations
of mesenchymal stem cells. It contains both mesenchymal
stem cells (unrestricted somatic stem) and WJ-MSCs [7, 8].
Mesenchymal stem cells can be isolated only from fresh
umbilical cord blood at the time of birth [7]. However, WJ-
MSCs can be isolated from umbilical cord matrix that can be
collected and stored [8]. Both types can be used for
therapeutic uses or biotechnology purposes [7, 8]. Wharton’s
Jelly is the gelatinous connective tissue that is present inside
umbilical cord. It contains my fibroblast-like stromal cells,
collagen fibers and proteoglycans matrix [9]. Earlier studies
have shown that WJ-MSCs derived from human umbilical
183 Moustafa A. H. El-Nawasany et al.: Isolation, Culture and Identification of Undifferentiated Wharton’s Jelly
Mesenchymal Stem Cells (WJ-MSCs) Derived from the Human Umbilical Cord
cord matrix are capable of expressing a diversity of stem cell
characteristics [8, 10]. WJ-MSCs of the umbilical cord have
many interesting properties such as; they are simple to
harvest via non-invasive methods, they can provide large
numbers of cells without any risk for the donor, their stem
cell population can be expanded, cryogenically stored,
thawed, manipulated genetically and differentiated in vitro
[8, 10-12]. This work was conducted to assess our lab and
staff capabilities in our Tissue Culture and Medical Genetics
Unit to isolate, culture, identify and characterize WJ-MSCs
derived from the adult human umbilical cord to be used for
clinical applications in the future.
2. Material & Methods
Materials: All the chemicals used and antibodies in this
study, except those otherwise indicated, were purchased from
Sigma-Aldrich company.
Umbilical cord acquisition and WJ-MSCs isolation:
Umbilical cords were obtained from the Obstetrics and
gynaecology Department, Faculty of Medicine Hospital, Al-
Azhar University. Then, umbilical cords were transported
within 2 h to the Tissue Culture and Medical Genetics Unit,
Histology and Cell Biology Department, Faculty of Medicine
in Cairo, Al-Azhar University. Umbilical cords were
obtained from the late gestation fetuses and placed in sterile
phosphate buffer saline (PBS, composition in mM: 140
NaCl; 2 KCl; 1.5 KH2PO4; 15 Na2HPO4) supplemented
with 200 IU/ml penicillin, 200 µg/ml streptomycin and 2
µg/ml amphotericin B (Bristol-Myers Squibb). Within the
next 12 hours, the umbilical cord segments (15-20 cm in
length) were longitudinally cut. After that, the umbilical cord
artery and veins were removed and the remaining umbilical
cord tissue, including Wharton's jelly, was cut into small
segments (2 × 2 mm2) by using small blades (Fig. 1).
Furthermore, each umbilical cord segment was transferred to
35 mm culture Falcon dish (Becton Dickinson & Company
Franklin Lakes) containing 1 ml of cell culture medium
[DMEM; Dulbecco's modified Eagle's medium supplemented
with 2 µg/ml amphotericin B, 20% fetal bovine serum (FBS,
Gibco), 200 IU/ml penicillin and 200 µg/ml streptomycin]
and maintained at 37°C in a humidified atmosphere of 5%
CO2. Cell culture media were filled to 3 ml and adherent WJ-
MSCs were observed 24 h after plating. The plates were left
to grow as passage 0. After the cells of primary culture
became nearly confluent (80-90%), some of these plates were
subcultured to P1. To subculture P0 WJ-MSCs to P1, adhered
WJ-MSCs were dissociated with 0.1% trypsin + 1.0 mM
EDTA in PBS for 2 min and then Fetal bovine serum (FBS)
was added to stop trypsinization. Then, the detached WJ-
MSCs subcultured in a new 250 ml Falcon flask (Becton
Dickinson & Company Franklin Lakes). For long term
storage, WJ-MSCs were cryopreserved in a freezing medium
consisting of 20% FBS and 10% dimethyl sulfoxide (DMSO)
in DMEM. The plates and/or flasks were incubated at 37C,
saturating humidity and 5% CO2. The plates and/or flasks
were checked for confluency every day and the cells were fed
every other day by removing half the medium and replacing
it with fresh medium.
Cell viability analysis and counting: Cell viability was
done after culture using trypan blue dye exclusion test
[13].This method is based on the principle that viable cells do
not take up certain dyes, whereas dead cells do. The cells are
counted by trypan blue exclusion assay using a
hemocytometer.
Fig. 1. A) Photograph showing a piece of human umbilical cord after
collection. B) Photograph showing removal of blood vessels from a piece of
umbilical cord tissue. C) Photograph showing human umbilical cord cut into
0.5 to 1 cm long pieces. D) Photograph showing crushing of umbilical cord
pieces and washing with PBS.
Flowcytometry and immunophenotyping: WJ-MSCs do
not express a unique surface marker but they express several
markers that determine their identity. These markers were
described in the guidelines recommendations of the
International Society for Cellular Therapy (ISCT) for WJ-
MSCs characterization [14]. For instance, WJ-MSCs are
positive for the homing receptor (hyaluronan receptor,
CD44), (thy-, CD90), and (endoglin, CD105) [15, 16, 17].
On the other hand, WJ-MSCs are negative for the expression
of the hematopoietic surface antigens CD14, CD45, and
CD34 [18, 19]. Immunophenotyping of positive; CD44,
CD90, CD105andnegativeCD34WJ-MSCs markers was done
within the samples by flow cytometry. To prepare MSC for
flow cytometry analysis, viable MSC was transferred into
three sterile tubes: the first tube was the control for the
procedure (with no mAbs and on which the machine was
adjusted in order to obtain the basic histogram showing the
main cell population and to adjust the auto-fluorescence
region), the second tube contains (10 µl of anti CD44, CD90
mAbs mixed with WJ-MSCs) and the third tube contains (10
µl of anti CD105, CD34 mAbs mixed with WJ-MSCs).The
second and third tubes were incubated for 15 minutes at room
temperature in the dark. At the end of the incubation the
tubes were washed once (by adding 2 ml of 5% PBS and
centrifuged for 5 minutes)and the packed cells were
resuspended in 5% PBS.
Flowcytometric analysis: Fluorescence Activated Cell
Sorter (FACS) flowcytometer (Coulter Epics Elite, Miami,
Open Science Journal of Clinical Medicine 2015; 3(5): 182-187 184
FL, USA) was used for analysis. After warming up the
argon laser (488 nm) for 30 minutes, the full alignment
procedures were performed using standard immunocheck
alignment flurospheres for adjusting forward scatter, side
scatter and photomultiplier tube (PMT2) for greencolor.
The proper protocol for CD44, CD90, CD105 and CD34
monoclonal antibodies was already loaded and ready for its
interpretation. Control sample was introduced in the
machine and forced in the sheath by the sample pressure
(run button) where the laser scatter was received on both
forward scatter detectors and scale to show the cell
population in a basic histogram and to adjust auto
fluorescence region. Ten thousands events (cells) were
passed in front of the laser for each case from which the
mononuclear cells were selectively gated for
immunophenotypical analysis. The second sample tube was
then introduced and processed in the same way as the
control, where the MSCs antibodies tagged with FITC was
analyzed on PMT2. The fraction of cells coated with MSCs
antibodies was determined inside the gated population of
mononuclear cells and assessed in a single histogram. After
10,000 events were counted, the number of cells expressing
the receptors will emit fluorescence signals which will be
summated and multiplied in PMT2, and the computer will
analyze the data as a single colored frequency histogram.
The results were declared as percentage of (CD44, CD90,
and CD105) +ve and (CD34) –ve cells inside the gated
population of mononuclear cells. Typically for each tube,
10,000 events are collected and the data are analyzed using
Cell Quest software.
3. Results
Morphological identification of human UC-derived MSCs
using phase contrast microscope: Twenty four hours from the
primary culture (passage 0 = P0) of human umbilical cord
mesenchymal stem cells, the cultured cells appeared crowded
and suspended (Fig 2A). They were variable in size and
shape. Most of the cells appear rounded. Three days from the
primary culture, the native MSCs were seen attached to the
culture flasks sparsely and sporadically (Fig 2B). The
hematopoietic stem cells were not attached to the culture
flasks, remained suspended and appeared rounded and
refractile. In contrast, MSCs were arranged in the form of
small colonies. The hematopoietic stem cells appeared non-
adherent, rounded and refractile. Seven days from the
primary culture, the MSCs of cultured flasks were
proliferated and reached 70% confluency (Fig 2C).The cells
exhibited different shapes with well-developed cytoplasmic
processes, granular cytoplasm and vesicular nuclei (Fig 2D).
Twelve days from the primary culture, the adherent cells
reached 80-90% confluency and appear triangular, star
shaped and spindle shaped. Some of the cells exhibited two
vesicular nuclei(Fig 3A, 3B, 3C).As the cells of primary
culture became nearly confluent (80-90%), the cells were
subcultured and designated as first passaged cells (P1).The
first passaged WJ-MSCs (P1) appeared spindle and triangular
shaped (Fig 3D). The cells were obviously homogenous in
morphology and appeared small star shaped forming
colonies. The expression of the CD- surface markers: Flow
cytometry immune profiling, cell passage 0–1, revealed
positive expression for the putative mesenchymal stem
markers CD44, CD90, CD105(Fig 4, 5). On the other hand,
CD45 marker showed negative expression alone and when it
as combined with CD105 marker(Fig 5).
Fig. 2. A) Phase contrast micrographs of human umbilical cord WJ-MSCs
24 hours from the primary culture (passage 0 = P0) of human umbilical cord
mesenchymal stem cells, the cultured cells appeared crowded and
suspended. They were variable in size and shape. Most of the cells appear
rounded. (P0 ˟ 200). B) Phase contrast micrographs of human umbilical
cord WJ-MSCs on 3th day from the primary culture (passage 0 = P0) of
human umbilical cord mesenchymal stem cells, some cells appear rounded,
refractile and non-adherent (hematopoietic stem cells).(P0 ˟ 200). C) Phase
contrast micrographs of human umbilical cord WJ-MSCs on 7th day from the
primary culture, the MSCs of cultured flasks were proliferated and reached
70% confluency.(P0 ˟ 200). D) Phase contrast micrographs of human
umbilical cord WJ-MSCs on 7thday from the primary culture. The cells
exhibited different shapes with well-developed cytoplasmic processes,
granular cytoplasm and vesicular nuclei. (P0 ˟ 400).
Fig. 3. A, B, C) Phase contrast micrographs of human umbilical cord WJ-
MSCs on twelve days from the primary culture, the adherent cells reached
80-90% confluency and appear triangular, star shaped and spindle shaped.
Some of the cells exhibited two vesicular nuclei. (P0 ˟ 400). D) Phase
contrast micrograph of human umbilical cord WJ-MSCs. The first passaged
MSCs (P1) appeared spindle and triangular shaped. (P1 ˟ 400).
185 Moustafa A. H. El-Nawasany et al.: Isolation, Culture and Identification of Undifferentiated Wharton’s Jelly
Mesenchymal Stem Cells (WJ-MSCs) Derived from the Human Umbilical Cord
Fig. 4. Representative flow cytometry of WJ-MSCs (n = 3). Cells show positive expression for CD44 (represented by green color), CD90 (represented by
orange color) and combined CD44 andCD90 (represented by yellow color).
Fig. 5. Representative flow cytometry of WJ-MSCs (n = 3). Cells show negative expression for CD34 cells (represented by green color) and positive expression
for CD105 (represented by orange color).CombinedCD34 and CD105 show only positive expression for CD105 (represented by orange color).
Open Science Journal of Clinical Medicine 2015; 3(5): 182-187 186
4. Discussion
In this work we decided to choose extra-embryonic human
umbilical cord WJ-MSCs to deal with as it possess self-
renewal capacity, extended plasticity and long-term expansion
potential as well as immune-privileged characteristics in
comparison to other kinds of stem cells. The isolation of extra
embryonic WJ-MSCs is non-invasive and poses no potential
risk for the patient. In addition, extra-embryonic tissues is
normally discarded after birth represent so called “waste
material”, which. For that reason, their isolation is not
associated with any current ethical concerns [20]. Furthermore,
earlier studies have shown that umbilical cord-derived MSCs
can be differentiated into skin, bone, hepatocytes, neural
lineages and endothelium [21-25]. As the immunomodulatory
properties of the umbilical cord MSCs were shown to be
similar to the bone marrow-derived MSCs [26]. Therefore,
human umbilical cord WJ-MSCs can be a probable enormous
source for stem cells in the regenerative medicine applications.
In the current study, we chose human umbilical cord WJ-
MSCs as they can be harvested after birth with low cost, stored
cryogenically, thawed at any time, and efficiently expanded for
any therapeutic or research purposes. In many earlier studies,
MSCs seem to give promising results in regenerative
therapeutic applications [27, 28]. WJ-MSCs are increasingly
being considered as more robust than those from cord blood
itself and less invasive than those from the bone marrow [29].
In our present work, a piece of fresh umbilical cord (15-20 cm)
was collected from the theater in sterile phosphate buffer saline
and immediately transported into the lab. Within the next 12
hours, the umbilical arteries and veins were removed. After
that, Wharton’s jelly tissue was excised and manipulated as
mentioned before. As reported before, a single piece of 5–10
mm3Wharton’s jelly has the potential to yield as many as 1
billion MSCs in 30 days [30]. Therefore, the cultured cells
were examined triple/week by using the inverted phase
contrast microscope to assess the level of expansion of the
cultured MSCs and to detect the appearance of any bacterial or
fungal infection among the cultured cells. After successful
isolation and primary culture were performed we found freshly
isolated WJ-MSCs (fibroblast-like appearance) during the first
culture period (10-15 days) until first passage. Our results were
similar to other studies results that showed that WJ-MSCs have
self-renewal ability and can be expanded in in vitro culture
under the suitable conditions [31, 32]. Similar to Haasters et
al, 2009 [33] study, in our present study we chose to identify
WJ-MSCs by Flow cytometry using (CD44, CD90 and
CD105) as positive markers and (CD34) as negative marker
for cells due to all mentioned immunophenotypical properties.
Our Flow cytometry immune profiling was similar to several
pervious results [30, 33, 34]. In our work, we successfully
isolated fibroblastic-like cells from human full term umbilical
cord and demonstrated their MSC characteristics by analyzing
their morphology and immunophenotyping. However, further
studies should be done to evaluate its growth kinetics, multi-
lineage potentials, its use as an allogenic cell source and
expression of other markers in relation to cell immunogenicity.
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