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-Nawasany 1 , Elsayed G. E. Khedr 1 , Moustafa E. E. Motawee 1 , Zeinab A-B Ali 2 , Hosam E. H. Kamel 3 , Tamer M. M. Abu-Amara 1, * 1 Histology & Cytology Department, College of Medicine, Al-Azhar University, Cairo, Egypt 2 Histology & Cytology Department, College of Medicine, Suiz Canal University, Ismailia, Egypt 3 Obstetric & 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
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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
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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