Isolation, Culture and Identification of Undifferentiated

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).



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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Isolation, Culture and Identification of Undifferentiated