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144 Current Stem Cell Research & Therapy, 2013, 8, 144-155

Mesenchymal Stem Cells Derived from Wharton's Jelly of the Umbilical

Cord: Biological Properties and Emerging Clinical Applications

Aristea K. Batsali1,2, Maria-Christina Kastrinaki1, Helen A. Papadaki1 and

Charalampos Pontikoglou*,1

1
Department of Hematology, University of Crete School of Medicine, Heraklion, Greece; 2Graduate Program, Univer-
sity of Crete School of Medicine, Heraklion, Greece

Abstract: In recent years there seems to be an unbounded interest concerning mesenchymal stem cells (MSCs). This is
mainly attributed to their exciting characteristics including long-term ex vivo proliferation, multilineage potential and im-
munomodulatory properties. In this regard MSCs emerge as attractive candidates for various therapeutic applications.
MSCs were originally isolated from the bone marrow (BM) and this population is still considered as the gold standard for
MSC applications. Nevertheless the BM has several limitations as source of MSCs, including MSC low frequency in this
compartment, the painful isolation procedure and the decline in MSC characteristics with donor's age. Thus, there is ac-
cumulating interest in identifying alternative sources for MSCs. To this end MSCs obtained from the Wharton's Jelly (WJ)
of umbilical cords (UC) have gained much attention over the last years since they can be easily isolated, without any ethi-
cal concerns, from a tissue which is discarded after birth. Furthermore WJ-derived MSCs represent a more primitive
population than their adult counterparts, opening new perspectives for cell-based therapies. In this review we will at first
give an overview of the biology of WJ-derived UC-MSCs. Then their potential application for the treatment of cancer and
immune mediated disorders, such graft versus host disease (GVHD) and systemic lupus erythematosus (SLE) will be dis-
cussed, and finally their putative role as feeder layer for ex vivo hematopoietic stem cell (HSC) expansion will be pointed
out.

Keywords: Graft versus host disease, hematopoietic stem cells, mesenchymal stem cells, umbilical cord, Wharton's jelly.

INTRODUCTION Friedenstein et al. demonstrated that a population of non-

Stem cells are defined by their unique capacity to self-
renew, differentiate into one or more cell lineages and tissue
types, and functionally repopulate a tissue in vivo [1]. On the
basis of their ability to give rise to one or more cell types,
known as potency, stem cells can be hierarchically classified
as unipotent, multipotent, pluripotent and totipotent. Unipo-
tent stem cells can produce only one cell type (e.g. neurons),
whereas multipotent ones can differentiate into several cell
types (e.g. hematopoietic stem cells, HSCs). An embryonic
stem cell (ESC), derived from the inner cell mass (ICM) of
the embryo is pluripotent as can differentiate into cells of the
three germ layers, but not into trophoblastic cells, whereas
the zygote is totipotent as it produces cells of embryonic as
well as extraembryonic tissue.
HSCs isolated from post-natal bone marrow (BM) and
umbilical cord blood (UCB) are generally acknowledged as
the prototype of multipotent adult tissue stem cells, being
able to differentiate into all mature blood lineages as well as
serially reconstitute hematopoiesis in lethally irradiated mice
at the single cell level [2]. In the late 60s and early 70s,
*Address correspondence to this author at the Department of Hematology,
University Hospital of Heraklion, P.O. Box 1352, Heraklion, Crete, Greece;
Tel: +30 2810 394629 ; Fax: +30 2810 394632 ;
E-mail: pontikoglou@yahoo.com
hematopoietic, plastic-adherent, fibroblast-like cells can also
be isolated from BM [3]. These cells, which were later
shown to be able to give rise to adipocytes, chondrocytes,
osteocytes, smooth muscle cells, fibroblasts and hema-
topoietic supportive stroma [3-6] and be self-renewing [7],
are currently referred to as mesenchymal stem/stromal cells
(MSCs) [4, 5, 8, 9]. So far MSC-like cells have been isolated
from several tissues, such as skeletal muscle, adipose tissue,
umbilical cord, synovium, dental pulp, amniotic fluid, as
well as fetal blood, liver, BM, lung and heart [10-12]. Never-
theless, these populations are not equivalent in terms of mul-
tipotency, when in vivo assays are used [13].
The field of MSCs has witnessed considerable progress
over the last decade. Due to the unraveling of many of their
exceptional characteristics and the encouraging preclinical
and clinical studies, it is now widely accepted that MSCs
will provide a revolutionary advantage in the therapeutic
intervention of various diseases or tissue damage in the near
future. More than forty years since their initial description,
BM-MSCs represent the most extensively studied population
of adult MSCs and are still considered as the gold-standard
for MSC clinical applications. Nevertheless it is now becom-
ing increasingly clear that BM may not represent the most
suitable source for MSC collection. Besides the invasive and
painful isolation procedure, MSCs are present at very low
frequency (approximately 0.001–0.01% of BM mononuclear

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Mesenchymal Stem Cells Derived from Wharton's Jelly Current Stem Cell Research & Therapy, 2013, Vol. 8, No. 2 145

cells [14]) in BM-aspirates and in addition their qualitative Human Umbilical Cord is a Rich Source of MSCs
and quantitative properties decline with donor's age [15].
The human UC (Fig. 1) contains two arteries and a vein
The low frequency implies that an extensive in vitro expan-
buried within a mucous or gelatinous connective tissue also
sion of cells is usually required to obtain sufficient cell num-
known as the Wharton’s jelly (WJ), enclosed by a simple
bers for therapeutic interventions, thereby enhancing the risk
of epigenetic damages as well as viral and bacterial contami- amniotic epithelium. WJ is largely made from proteoglycans
and various types of collagen, forming a sponge-like tissue,
nations. To circumvent these obstacles, much effort has been
within which stromal cells are embedded [19]. These cells,
invested over the last decade in identifying alternative, more
also referred to as umbilical cord matrix (UCM) cells are
abundant and easily attainable sources of MSCs for thera-
thought to be trapped into the WJ early in embryogenesis
peutic use. In this regard the umbilical cord (UC), which is
during their migration from the aortic-gonadotropin-
considered medical waste and is obtained with a painless,
simple and safe procedure during delivery, has aroused much mesonephric region to the fetal liver through the umbilical
cord [20]. UCM cells have exceptional properties in that
attention during the last years as a promising source for MSC
although they are bona fide MSCs [18, 21] as discussed be-
isolation. MSCs have actually been found in both UC blood
low in detail, possessing similar properties with their adult
(UCB) as well as in UC tissues. Nevertheless, the scarcity of
BM counterparts, they also retain characteristics of primitive
MSC in UCB (their frequency being much lower than in
stem cells, like the expression of embryonic stem cell mark-
BM) and the lack of a robust and reproducible technique for
harvesting and expanding MSCs from UCB units [16, 17] ers [22, 23]. In line with these observations MSCs derived
from WJ, hereafter referred to as "UC-MSCs", maintain their
have hampered their use in clinical applications. On the other
self-renewal capacity in vitro for a longer period of time, and
hand existing isolation protocols have constantly induced the
exhibit a shorter doubling time and a broader pluripotency as
harvest of large numbers of MSCs from UC-tissues with a
compared to BM-MSCs [18]. These unique developmental
hundred per cent success.
and operational characteristics along with the fact that UC-
UC-MSCs share many properties with adult BM-MSCs, MSCs can be easily isolated, extracorporeally without risks
but are generally considered as a more primitive population for the donor, from a tissue which is routinely discarded,
than the latter. In fact, while they possess several characteris- highlight the advantages of UC-MSCs over ESCs and adult
tics of ESCs, they do not form teratomas upon transplanta- MSCs and explain the rapidly growing interest in this field
tion [18] and their research does not raise ethical or legal of stem cell biology.
issues. All these features open attractive perspectives for the
The first report providing robust evidence that WJ stro-
propagation and engineering of UC-derived MSCs for cell-
based therapies. This review will first attempt to give a mal cells can be classified as MSCs was published in 2004
[24]. In that study, cells extracted from WJ, displayed a fi-
summary of the biology of MSCs derived from Wharton's
broblast-like phenotype when expanded in vitro and were
Jelly (WJ-MSCs), the most abundant region of UC and then
able to differentiate into osteoblasts, adipocytes and chon-
discuss their potential application for the treatment of cancer
drocytes upon appropriate stimulation [24]. Furthermore,
and immune mediated disorders such as graft versus host
akin to BM-MSCs they were shown to express high levels of
disease (GVHD) and systemic lupus erythematosus (SLE).
Finally, their putative role as feeder layers for ex vivo hema- CD29, CD44, CD51, CD73 and CD105, but lacked expres-
sion of the hematopoietic markers CD34 and CD45 (Table 1)
topoietic stem cell (HSC) expansion will be pointed out.
[24]. Subsequent reports have provided additional informa-

Umbilical arteries
Perivascular

Intervascular
Wharton’s Jelly

A
Amniotic epithelium Umbilical vein/ Subamnion
Umbilical cord blood
Fig. (1). Umbilical cord contains two arteries and a vein, within a mucous or gelatinous connective tissue, the Wharton’s jelly, enclosed by a
simple amniotic epithelium. Mesenchymal stem cells have been isolated from umbilical cord blood and also from the intervascular, perivas-
cular and subamniotic regions of Wharton's jelly.
146 Current Stem Cell Research & Therapy, 2013, Vol. 8, No. 2
tion on WJ stromal cell phenotype, demonstrating the ex-
pression of various membrane molecules known to be asso-
ciated with a MSC-like identity. These molecules include
CD90, CD146, CD166 and ganglioside 2 (GD2) [8, 23, 25,
26]. Collectively, the morphology, immunophenotype and
differentiation potential of WJ stromal cells fulfill the criteria
for MSCs proposed by the International Society for Cellular
Therapy (ISCT) [8]. Indeed, given that at present there is no
specific marker to define UC-MSCs, the expression of the
aforementioned set of membrane antigens combined with the
demonstration of in vitro multi-lineage differentiation poten-
tial is necessary to identify MSCs in UC-derived tissues
UC-MSCs have been isolated from three relatively indis-
tinct regions of the WJ: the perivascular, the intervascular
and the subamnion zones (Fig. 1) [27]. Cells from these
zones share a fibroblast-like morphology, a high proliferative
rate and multipotent differentiation capacity. However a
comprehensive head-to-head comparison between MSCs
obtained from different regions of the UC has not been per-
formed thus far, due to the great variation in cell isola-
tion/culture protocols. Hence the question whether cells har-
vested from different components of the UC represent differ-
ent populations is still open. Nevertheless one study combin-
ing both in situ and in vitro experiments [27] has demon-
strated that MSCs derived from the perivascular region com-
prise a distinct subpopulation than those derived from the
intervascular and subamniotic parts of the UC. It has thus
been reported that perivascular MSCs stain strongly for pan-
cytokeratin as compared to intervascular and subamniotic
counterparts and give rise in vitro to flat, wide cytoplasmic
cells, named type 1 cells, which tend to diminish in number
during protracted culture [27]. On the other hand intervascu-
lar and subamniotic MSCs generate fibroblast-like cells per-
sisting throughout culture (type 2 cells). In view of these
findings it appears that UC-MSCs are actually type 2 cells,
displaying adipogenic, chondrogenic, osteogenic and neu-
ronal capacity, upon appropriate differentiation stimuli [27].
Isolation of MSCs from UC perivascular zone has been re-
ported by other groups as well [28, 29]. These cells, termed
as Human Umbilical Cord Perivascular Cells (HUCPVCs),
have a colony forming unit (CFU) frequency of 1:333 [29]
and differ in their growth characteristics from UC-MSCs
(population doubling times ranging from 59.4±42.4h in pas-
sage 0 to nearly 20h in passage 6 for HUCPVCs versus
85±7.2h in passage 0 to 11±1.2h in passage 7 for a mixed
population of UC-MSCs [27, 29]. Furthermore, HUCPVCs
exhibit higher proliferative capacity and osteogenic potential
as compared to BM-MSCs [28]. The phenotype of
HUCPVCs shares many similarities with that of WJ-derived
MSCs (Table 1). One significant difference though, is the
high expression of CD146 by HUCPVCs. Interestingly this
marker is either absent [28] or weakly expressed by MSCs
derived form other regions of WJ [30].
Isolation of MSCs from Wharton's Jelly
In recent years, several methods (Fig. 2) have been de-
veloped for the isolation of MSCs from WJ. However, ex-
perimental approaches vary considerably and in some cases
harvested cells from UC tissue most likely consist of a het-
erogeneous population, thereby biasing comparisons. With
regards to protocols used to isolate UC-MSCs, some authors
Batsali et al.
remove umbilical cord vessels, scrape off WJ with a scalpel
and then treat the tissue enzymatically [24]. Others cut down
UC to smaller segments, strip the vessels and then directly
immerse the remaining UC to an enzymatic solution [25, 27,
31]. Interestingly, neither the enzymes used nor the incuba-
tion period have been standardized thus far. Alternatively,
instead of enzymatic digestion, several groups dissect the
cord segment into very small pieces, with or without discard-
ing the cord vessels and the segments are used as explants
from which cultures are subsequently established [32-34].
The major advantage of this method is that it minimizes cell
damage as it does not involve enzymatic treatment, however
it should be noted that primary cultures generated in this way
are quite heterogeneous [33]. UC-MSCs have also been iso-
lated from WJ by simply cutting UC segments longitudinally
and plating them with WJ side-down onto a plastic surface,
once again without enzyme digestion or dissection [35]. Fi-
nally, to collect HUCPVCs some groups remove blood ves-
sels together with surrounding WJ, tie up the ends of each
vessel into a loop and then immerse the vessel in enzyme,
thereby isolating cells from the perivascular tissue [28, 29].
The question as to which is the optimal method for effi-
cient isolation of a homogeneous and well defined popula-
tion of UC-MSCs has not been answered thus far. However,
unless an agreement is reached between laboratories regard-
ing the isolation and ex vivo expansion of UC-MSCs, there
will be no consensus on the characteristics and biological
properties of the cells, which is a prerequisite for the proper
design of preclinical studies, so as to pave the way for the
potential use of UC-MSCs in clinical applications.
Do differences in isolation procedures and ex vivo expan-
sion protocols have an impact on the antigens expressed by
UC-MSCs? This issue has not been properly investigated so
far, yet a recent study [36] demonstrated that MSC markers
CD73, CD90 and CD105 varied their expression patterns
according to whether enzyme cocktails or explants were used
for UC-MSC harvesting. A similar variation with respect to
the isolation method was also noted in the expression of the
ESC markers c-Kit and Oct-4 by UC-MSCs [36].
WJ-derived UC-MSCs may be Regarded as a Primitive
Stromal Cell Population
Porcine UC-MSCs have been reported to express the
ESC markers Oct-4, Sox-2 and Nanog [37] and equine UC-
MSCs express, apart from Oct-4, the embryonic markers
SSEA4 and c-Kit [38]. In line with these findings, human
UC-MSCs express Oct-4, Nanog, Sox-2, c-Kit, as well as
Dnmt3b and Tert, albeit at lower levels as compared to ESCs
[22, 34, 39]. Such a low expression of the aforementioned
pluripotency markers may explain why UC-MSCs do not
produce teratomas in vivo in immunodeficient mice and rats
[22, 25]. In addition human UC-MSCs express the ESC
markers Tra-1-60, Tra-1-81, SSEA-1 and SSEA-4 [22].
Many of these pluripotent stem cell markers retain their ex-
pression for at least nine passages during ex vivo expansion.
Comparative gene expression profiling between UC-MSCs
and BM-MSCs demonstrated that the former express higher
levels of Nanog, Dnmt3b and Gabrb3, and also
Mesenchymal Stem Cells Derived from Wharton's Jelly Current Stem Cell Research & Therapy, 2013, Vol. 8, No. 2 147

Table 1. Markers expressed by BM-MSCs and UC-MSCs derived from WJ and the perivascular zone

Markers WJ-derived MSCs Perivascular MSCs BM-MSCs

CD10 neutral endopeptidase + [25] + [103] + [101]

CD11b Integrin alpha-M - [23] - [102]
CD13 aminopeptidase N + [25] + [102]
CD14 LPS receptor - [25] - [49] - [102]
CD26 dipeptidylpeptidase 4 + [102]
CD29 intergrin 1 + [25] + [49] + [5]
CD31 PECAM-1 (platelet endothelial adhesion molecule-1) - [25] - [103] - [5]
CD33 sialic acid binding Ig-like lectin 3 - [81] - [101]
CD34 sialoprotein - [24] - [29] - [5]
CD38 ADP-ribosyl cyclase 1 - [47] - [101]
CD40 tumor-necrosis factor receptor superfamily 5 - [61] + [101]
CD44 hyaluronan receptor + [25] + [29] + [5]
CD45 pan-leucocyte antigen - [25] - [29] - [5]
CD49a integrin
1 - [104] + [5]
CD49b intergrin
2 + [25] + [5]
CD49c intergrin
4 + [25] + [5]
CD49d intergrin
3 + [25] - [5]
CD49e integrin
5 + [25] + [5]
CD50 ICAM-3 (Intercellular adhesion molecule 3) - [105] - [5]
CD51 integrin
V + [25] + [5]
CD53 Tetraspanin-25 - [104] - [102]
- [47]
CD54 ICAM-1 (intercellular adhesion molecule-1) + [29] + [5]
+ [105]
CD56 NCAM-1 (neural cell adhesion molecule) - [61] - [5]
CD58 LFA-3 (lymphocyte function-associated antigen 3) + [81] + [101]
CD71 transferin receptor - [106] + [100]
CD73 Ecto-5 nucleotidase + [104] + [29] + [5]
CD80 CD28 ligand - [81] - [102]
CD86 Activation B7-2 antigen - [81] - [102]
CD90 Thy-1 + [25] + [29] + [5]
CD105 TGF-RIII (transforming growth factor- receptor III) + [25] + [29] + [5]
- [49] - [29]
CD106 VCAM-1 (Vascular cell adhesion molecule-1) + [5]
+ [61] + [107]
- [104] - [49]
CD117 C-kit receptor - [102]
+ [108] + [29]
CD133 AC133 (prominin) - [25] - [109]
CD140
PDGF-R
(platelet-derived growth factor receptor
) - [54] + [102]
CD140 PDGF-R (platelet-derived growth factor receptor ) ND + [102]
CD144 VE-cadherin Low [30] - [5]
CD146 Mel-CAM (melanoma-cell adhesion molecule) Weak + [30] + [49] + [7]
148 Current Stem Cell Research & Therapy, 2013, Vol. 8, No. 2 Batsali et al.

Table 1. Contd….

Markers WJ-derived MSCs Perivascular MSCs BM-MSCs

CD166 ALCAM (activated lymphocyte cell adhesion molecule) + [104] + [5]

CD235
Glyocophorin - [29] - [102]
CD325 N-cadherin + [25] + [102]
HLA-I + [54] + [29] + [110]
HLA- II - [54] - [29] - [110]
HLA G + [34] + [111]
- [81]
STRO-1 + [113]
+ [112]
SSEA4 Stage-specific embryonic antigen 4 + [105] + [114]
a-SM actin a-smooth muscle actin + [54] - [103] + [10]
NANOG + [25] + [115]
OCT4 + [34] - [29] + [115]
SOX2 + [25] - [6]
GD2 Ganglioside + [26] + [10]

Umbilical cord

Removal of umbilical
Dissection in vessels
small segments

Scrapping off
mesenchymal tissue
Cut tissue into
very small pieces

Enzymatic treatment

Explant culture Filter

Stromal cell culture

Fig. (2). A scheme depicting the different approaches followed for the establishment of WJ-derived UC-MSC cultures (modified from [100]).
Mesenchymal Stem Cells Derived from Wharton's Jelly
show an increased expression of the pluripotent/stem cell
markers Brix, CD9, Gal, Kit and Rex1 [39]. The implication
of these markers in the biology of UC-MSCs is currently
unknown, yet their expression is thought to reflect the more
primitive nature of UC-MSCs as compared to their adult
counterparts within the BM. However, not all UC-derived
MSCs express ESC markers. In fact, it has been demon-
strated that primitive markers are expressed only by a subset
of UC-MSCs [26, 40], thereby suggesting that these cells are
rather hetereogeneous in terms of stemness. It would there-
fore be very challenging to identify that specific marker
which would permit the isolation of the primi-
tive/multipotent MSCs from UC.
Human UC-MSCs exhibit also higher expression of
genes implicated in the phosphoinositide 3-kinase (PI3K)-
AKT survival/proliferation pathway, including GAB2,
NFKBIA, FOXO1A, YWHAB, FOXO3A, CDKN1A,
MAP3K8, NFKB1, NOS3, DDR1 and TEK [41]. Further-
more, the NF
-B pathway has been reported to be more ac-
tive in UC-MSCs than in BM-MSCs [41]. These findings
together with the fact that human WJ-MSCs express the
primitive stem cell marker TERT [25] explain, at least in
part the observation that UC-MSCs have a shorter doubling
time [27] and a more extensive ex vivo expansion capacity
[18].
Multilineage in vitro Differentiation Potential of WJ-
derived UC-MSCs
Accumulating evidence suggests that UC-MSCs can be
successfully induced to differentiate into mesodermal and
non-mesodermal lineages. As mentioned above, several
groups have established that UC-MSCs can generate os-
teoblasts, adipocytes and chondrocytes in vitro [24, 27, 42,
43], thereby fulfilling one of the minimal criteria proposed
by ISCT for MSCs [8]. However, lipid accumulation takes
much longer in UC-MSCs and the adipocytes are less mature
than those derived from BM-MSCs [27, 41]. In addition,
UC-MSCs have been shown to possess weaker osteogenic
[41] and chondrogenic [44, 45] potential as compared to
BM-MSCs.
UC-MSCs have also been reported to successfully differ-
entiate into endothelial cells [43, 46] and actually demon-
strate higher endothelial differentiation potential when com-
pared to BM-MSCs [43]. In accordance with this observation
non-differentiated UC-MSCs exhibit higher expression of
endothelial associated genes including FLT1, GATA4,
GATA6, ISL1, LAMA1, SOX17 and SERPINA1 [39].
One group has suggested, that UC-MSCs can give rise to
skeletal muscle cells when cultured in the appropriate me-
dium and that non-differentiated UC-MSCs may display
myogenic potential in vivo [47]. Differentiation of UC-MSCs
to cardiomyocytes has also been reported but is still contro-
versial [24, 31, 48-50]. For instance, one group [24] demon-
strated that UC-MSCs could be appropriately induced to
form cells demonstrating cardiomyocyte morphology and
expressing cardiac markers, including N-cadherin and car-
diac troponin. Another team [49], however, was unable to
detect cardiomyocyte marker expression after in vitro induc-
tion of MSCs isolated from WJ and perivascular tissues. De-
Current Stem Cell Research & Therapy, 2013, Vol. 8, No. 2 149
finitive evidence that UC-MSC-derived cardiomyocytes rep-
resent functional and terminally differentiated cells is still
scarce. To our knowledge, only one group has thus far re-
ported that UC-MSC-derived cardiomyocytes exhibit slight
spontaneous beating after 21 days of induction [31].
Finally, several groups have shown that UC-MSCs can
acquire morphological and biochemical properties of neural
cells when exposed to appropriate differentiation media [27,
32, 51, 52]. Mitchell et al. [32] were the first who observed
UC-MSC neuronal differentiation, following a complex and
multi-step neuronal induction process. Differentiation was
assessed by changes in morphology and expression of neu-
ron specific enolase, bIII-tubulin, neurofilament and tyrosin
hydroxylase. Subsequent studies have further corroborated
these findings. It has also been suggested that UC-MSCs
may be more primed for neuronal differentiation as com-
pared to BM-MSCs, since they express, higher levels of neu-
ronal-associated genes including NESTIN, GFAP and
SEM3A [39] in a non–differentiated state. However, it
should be kept in mind that definitive evidence for electro-
physiological activity of UC-MSC-derived neurons is still
missing. Therefore the issue whether the neuronal-like phe-
notype of UC-MSCs reflects a genuine trans-differentiation
is still pending.
Recent studies suggest that UC-MSCs can also differen-
tiate into endodermal lineages (reviewed in [53]). In this
context, Campard et al. [54] reported that non-differentiated
UCM cells constitutively expressed markers of hepatic line-
age, such as albumin, alpha-fetoprotein, cytokeratin-19, con-
nexin-32, and dipeptidyl peptidase IV. After in vitro hepatic
induction, cells exhibited hepatocyte-like morphology and
upregulated the expression of certain hepatocyte-associated
markers, but lacked important functional properties of ma-
ture liver cells, thereby questioning whether UCM cells have
actually undergone hepatocyte maturation [54]. Other groups
have also claimed that UC-MSCs can be induced to form
isle-like cells [55, 56] (reviewed in [57]), able to express
pancreatic associated genes and release insulin in response to
physiological glucose levels [56]. Interestingly, UC-MSCs
have been reported to have a higher pancreatic differentia-
tion potential than their BM counterparts [55].
The Immunomodulatory Properties of UC-MSCs
Apart from the multilineage potential, UC-MSCs possess
various immunoregulatory properties, rendering them as
appealing candidates for cell based therapies [58]. More pre-
cisely UC-MSCs have been found to exhibit very low ex-
pression of HLA class I, and lack the expression of HLA-DR
[59, 60]. Notably, in comparison to BM-MSCs derived from
aged donors, UC-MSCs express significantly lower levels of
HLA-I, and exhibit significantly higher intracellular concen-
trations of the immunosuppressive molecule HLA-G [59].
These findings together with the observations that UC-MSCs
do not express the co-stimulatory molecules CD40, CD80
and CD86 [59, s61], and do not elicit proliferative responses
from allogeneic T cells in vitro [61], suggest that UC-MSCs
may be characterized by inherently low immunogenicity.
However, another study suggests that UC-MSCs may not be
as immune-privileged as thought, as they can trigger weak
allogeneic immune activation in vitro and will eventually
150 Current Stem Cell Research & Therapy, 2013, Vol. 8, No. 2
undergo rejection following xenogeneic transplantation [59].
Similar findings have been reported for BM-MSCs as well.
Interestingly the latter have been shown to be much more
immunocompetent compared to UC-MSCs [59].
Several studies have been published on the immunosup-
pressive potential of UC-MSCs on T lymphocytes [59, 60,
62-64]. One group reported that UC-MSCs target CD4+ and
CD8+ T subpopulations equally and are able to inhibit T-cell
proliferation regardless of the stimulus used to activate T-
cells [63]. Furthermore, UC-MSC-mediated inhibitory ef-
fects were higher than with BM-MSCs. In accordance with
previous studies suggesting that the immunomodulatory ef-
fect of MSCs on T cells may be mediated, at least in part, by
soluble factors (reviewed in [65]), such as hepatocyte growth
factor (HGF), prostaglandin E2 (PGE2), transforming growth
factor-
1 (TGF-
1) and indoleamine 2,3-dioxygenase (IDO),
the authors assessed whether any of these molecules may be
implicated in UC-MSC immunoregulation as well. In their
hands immune suppression exerted by both UC- and BM-
MSCs was mediated by cycloxygenase (COX) 1 and COX2
enzymes, regulating PGE2 synthesis, and by the production
of PGE2 per se, whereas HGF did not seem to be actively
implicated. These findings were further corroborated by an-
other study [64] which additionally showed that while UC-
MSCs intrinsically secrete very low levels of PGE2, its pro-
duction is upregulated when cells are co-cultured in the pres-
ence of activated human peripheral blood mononuclear cells
(hPBMCs). The authors attributed the PGE2 stimulation to
inflammatory cytokines (including IFN- and interleukin-1
)
produced by hPBMCs [64].
Interestingly, several studies [59, 62, 66] have reported
that UC-MSC immune characteristics may be modified fol-
lowing exposure to inflammatory cytokines and/or activated
PBMCs/T-cells so as to display immunosupressive proper-
ties, a process described by the term licensing or priming. In
this context, Deuse et al. [59] reported that IFN- at doses
below 50ng/ml upregulated HLA-DR and HLA-I expression,
thereby increasing UC-MSC immunogenicity. IFN- treat-
ment also induced tolerogenicity by increasing intracellular
HLA-G expression, TGF-
and IL-10 release, and stimulated
the production of IDO [59]. Higher doses of IFN- however
enhanced UC-MSC immunosuppressive phenotype by
downregulating HLA-DR expression and further increasing
IDO. These results suggest that appropriate priming may be
crucial for the fine adjustment of MSC characteristics, ren-
dering them suitable for a given therapeutic application.
The immunomodulatory effects of MSCs are not only
apparent in T lymphocytes but also concern B lymphocytes.
To our knowledge however, the impact of UC-MSCs on B
cells has been poorly investigated and undoubtedly awaits
further elucidation, as the information that will be gathered is
expected to provide crucial clues for the introduction of WJ-
MSCs into the clinic. In the single study reported until today
[67] UC-MSCs were found to inhibit B cell proliferation,
differentiation and antibody production in vitro. This sup-
pressive effect is mediated, at least in part by soluble factors,
which may downregulate Blimp-1 and upregualate PAX-5,
two master regulators of B cell differentiation [67]. Further-
more UC-MSCs have been demonstrated to inhibit Akt and
p38 MAPK phosphorylation. Interestingly, these signal
Batsali et al.
transduction pathways are implicated in the regulation of B
cell proliferation and differentiation [67].
The immunoregulatory potential of HUCPVCs has also
been assessed in a recent study [68]. More precisely it has
been demonstrated that when HUCPVCs are co-cultured
with resting or stimulated allogeneic lymphocytes they fail to
induce their proliferation. Furthermore, HUCPVCs have
been found to reduce in vitro alloreactivity in mixed lym-
phocyte reactions. Notably, the immunosuppressive effect of
the cells is exerted, at least in part via soluble factors [68].
UC-MSCs as Feeder Layers for ex vivo Umbilical Cord
Blood Expansion
Despite the fact that BM has been the most extensively
studied source for HSC transplantation, the importance of
UCB as an alternative source of transplantable HSCs has
been widely acknowledged since the first successful UCB
transplant in 1988 [69]. From a practical point of view UCB
transplantation has several advantages over BM, including
the non-invasiveness of the procedure, the ease of procure-
ment, the reduced risk of transmissible infections and the
availability for immediate use [70]. In addition, UCB grafts
allow for a greater tolerable HLA disparity with a decreased
frequency of acute or chronic graft versus host disease
(GVHD) as compared to BM grafts [70]. Finally UCB con-
tains a higher proportion of immature and committed hema-
topoietic progenitor cells (HPCs) in comparison to adult BM
[71].
However UCB transplantation has also some significant
restrictions, limiting its broad applicability. The most sig-
nificant limiting factor of UCB use is probably the low total
cell dose harvested from a UCB unit, which in turn is associ-
ated with delayed neutrophil and platelet engraftment and
higher engraftment failure [72]. For this reason UCB trans-
plantation remains significantly more successful in children
[73], whereas poor outcomes have been reported in patients
>45 kgr who receive only one unit of UCB [74].
To circumvent these obstacles, so as to improve out-
comes and extend the application of UCB transplantation to
older and/or larger patients, several groups have explored the
possibility of in vivo expansion of HSCs from UCB units.
Various protocols are under investigation over the last 30
years, including those based on cultures on feeder layers.
Among the different cell types that have been used as stro-
mal support for UCB expansion, the most extensively stud-
ied thus far are BM- MSCs.
The rationale behind the use of MSCs as feeder cells for
UCB expansion lies on a large body of evidence, clearly
demonstrating the BM-MSC promoting role on hematopoie-
sis (reviewed in [65]). More precisely BM-MSCs have been
shown in vivo to enhance HSC engraftment and repopula-
tion, as evidenced by the observation that co-transplantation
of human HSCs and MSCs results in increased chimerism
and/or hematopoietic recovery, in both animal models and
humans [65, 75]. On the other hand, MSCs have been shown
in vitro to maintain long-term culture initiating cells (LT-
CICs) and expand lineage-specific colony forming units
from CD34+ marrow cells in long-term BM cultures (re-
viewed in [65]). Even though the exact mechanisms that me-
Mesenchymal Stem Cells Derived from Wharton's Jelly
diate the hematopoiesis promoting effect of MSCs have not
been elucidated, it is now widely accepted that MSCs supply
HSCs and their progeny with signals for survival, prolifera-
tion and differentiation through direct cell-to-cell contact
and/or production of cytokines, adhesion molecules and ex-
tracellular matrix proteins (reviewed in [65]).
These observations have provided the theoretical back-
ground for the investigation of the potential use of MSCs as
scaffolds for the ex vivo expansion of HSCs. Based on the
hypothesis that MSCs could provide many of the molecular
cues of the hematopoietic microenvironment necessary to
favor HSC self-renewal and regulate their proliferation, CB
hematopoetic stem/progenitor cells have been co-cultured on
BM-MSC preformed layers in a medium containing (FBS)
and various combinations of growth factors including G-
CSF, stem cell factor (SCF), thrombopoietin (TPO), Flt3-
ligand or fibroblast growth factor-1 (FGF-1) [76]. Using this
ex vivo model several groups have demonstrated an increase
in the number of total nucleated cells (TNC), clonogenic
cells or CD34+ cells [77, 78]. These findings were further
corroborated in a recent study [79], where CB CD34+ cells
were co-cultured with BM-MSCs in a serum free medium
supplemented with SCF, TPO and FGF-1. BM-MSCs were
also shown to maintain the primitive phenotype of CB hema-
topoietic stem and progenitor cells even after massive expan-
sion [79]. However co-culture with BM-MSCs did not result
in a significant expansion of long-term repopulating cells. A
clinical trial is underway at M.D. Anderson Cancer Center
combining an unmanipulated UCB unit with an expanded
UCB unit for 14 days in the presence of a cytokine cocktail
on layer of family member donor-derived BM-MSCs or "off-
the-self" BM-MSCs (Angioblast Systems Inc.). Preliminary
results suggest a beneficial role for these stromal cell-based
HSC expansion [80].
Given the promising role of BM-MSCs in supporting ex
vivo UCB expansion and since UC-MSCs share many char-
acteristics with their BM counterparts, but in contrast to the
latter can be readily and painlessly harvested from UC, they
soon emerged as attractive candidates for HSC expansion. In
this regard, the first report demonstrating that akin to BM-
MSCs, UC-MSCs can also support hematopoiesis was pub-
lished in 2007 [81]. In that study the authors showed that
UC-MSCs secrete significantly higher amounts of several
important cytokines and hematopoietic growth factors in-
cluding G-CSF, GM-CSF, LIF, IL-1, L-6, IL-8 and IL-11,
compared to BM-MSCs. Furthemore co-culture of UC-
MSCs with UCB CD34+ cells significantly increased hema-
topoietic colony formation as compared to clonogenic pro-
genitor cell assays performed in the absence of MSCs [81].
Finally, in agreement with previous reports demonstrating
that co-transplantation of BM-MSCs with HSCs can acceler-
ate hematopoietic engraftment, co-injection of UC-MSCs
with UCB cells or CD34+ cells in a NOD/SCID mouse
model accelerated human HSC recovery. The authors as-
sumed that the improved engraftment might be attributed to
the presence of UC- MSCs within the BM of the recipient
animals and also to the production of hematopoietic cytoki-
nes by these cells. Subsequent reports have confirmed the
capacity of UC-MSCs and UC-MSC conditioned medium to
Current Stem Cell Research & Therapy, 2013, Vol. 8, No. 2 151
support the growth and maintenance of UCB hematopoietic
stem/progenitor cells in long term culture [82, 83], thereby
establishing an ex vivo model that could enable the study of
the interactions between HSCs and their supportive micro-
environment within the UC. However, whether UC MSC-
based HSC expansion will have possible future clinical util-
ity is currently unknown and awaits further clarification.
UC-MSCs for GVHD Treatment
Graft versus host disease (GVHD) is one of the most
severe complications of allogeneic BM transplantation. First-
line treatment with corticosteroids is the gold standard for
this T-cell mediated process, however second line treatment
for steroid-resistant GVHD has not been clearly established
thus far and the outcome is poor, with one year overall sur-
vival (OS) of only 30% [84, 85].
The in vitro and in vivo immunomodulatory properties of
BM-MSCs have provided the theoretical background for the
use of these cells for treatment of acute steroid-resistant
GVHD. Several reports have been published thus far (re-
viewed in [86]) suggesting that clinical responses can actu-
ally be attained in a significant number of patients, especially
in children. BM-MSCs have also been used for the treatment
of chronic GVHD showing likewise promising results [86].
To our knowledge, there has been only one recent study
clinically testing UC- MSCs for GVHD [87]. In that particu-
lar report third-party culture expanded UC- MSCs were
transplanted in 2 children with severe steroid-resistant acute
GVHD. The first patient received three infusions of UC-
MSCs. Each time acute GVHD improved significantly and
eventually disappeared. The second patient received only
one infusion of UC-MSCs that led to complete remission of
acute GVHD. No child experienced any adverse events what
so ever and both children were well and remained off immu-
nosuppressive therapy at the time the study was published.
The authors concluded that UC-MSCs may be considered as
a feasible, safe and effective treatment modality for acute
GVHD [87]. These encouraging results need obviously to be
confirmed by larger trials, which will include adult patients
as well. Furthermore, additional studies are needed in order
to optimize the use of UC-MSCs in treating GVHD. For ex-
ample, it is currently unknown which is the optimal UC-
MSC dose and schedule, or whether the use of platelet-
lysate, or other substitutes of the standard FBS-enriched me-
dium (so as to comply with regulatory guidelines for the
production of MSCs for clinical use, which recommend
avoiding the use of animal derivatives) might influence the
therapeutic effect of UC-MSCs.
UC-MSCs Support the Expansion of Natural Killer Cells
As previously mentioned a major toxicity associated with
Bone Marrow Transplantation (BMT) is GVHD, an attack
by the T cells in the graft against the recipient organs includ-
ing the skin, gastrointestinal tract, liver, and eyes [88]. Ag-
gressive depletion of T cells in the allogeneic graft can
markedly reduce the incidence of acute GVHD, however,
this is associated with significant drawbacks, including re-
duction of the graft versus tumor effect of the allogeneic-
BMT [89]. Limiting therefore GVHD while maintaining the
152 Current Stem Cell Research & Therapy, 2013, Vol. 8, No. 2
antitumor effect, would be critical to improve transplant out-
come [90]. Given that natural killer (NK) cells have been
found to control leukemia relapse while protecting patients
from GVHD [91, 92], the possibility of expanding and po-
tentially using these cells for adoptive immunotherapy ap-
pears an attractive approach in the setting of allogeneic
BMT. Provided that UCB is rich in NK cell progenitors and
since UC-MSCs can produce significant amounts of cytoki-
nes, Boissel et al. [93] hypothesized that a feeder layer of
UC-MSCs might actually effectively support ex vivo expan-
sion of UCB-NK cells. Indeed when CD3-depleted UCB
mononuclear cells were cocultured with UC-MSCs and a
cytokine cocktail, the proportion of CD3-CD56+ NK cells
increased significantly as compared to cell cultured with
cytokines only. Interestingly direct contact between UCB
mononuclear cells and UC-MSCs resulted in a significantly
greater NK cell expansion. Notably, expanded NK cells
maintained an elevated cytotoxic profile [93]. The possibility
of further optimizing this experimental protocol should be
evaluated in additional studies and of course the cytotoxicity
of the expanded NK cells awaits assessment in animal mod-
els.
UC-MSC Transplantation in Systemic Lupus Erythema-
tosus
To our knowledge, apart from GVHD, in vivo therapeutic
efficacy of UC- MSC transplantation has also been clearly
illustrated in the setting of systemic lupus erythematosus
(SLE). The theoretical background for assessing the clinical
impact of UC-MSCs in patients with this autoimmune disor-
der has been provided by the immunomodulatory properties
of UC-MSCs and also by the beneficial effects of BM- MSC
administration in patients with treatment-refractory SLE
[94]. In this context, it has recently been reported that trans-
plantation of UC-MSCs in patients with severe and refrac-
tory SLE results in clinical and serologic improvement and is
not associated with mortality or other treatment-related ad-
verse events [95]. Even though the mechanisms mediating
the therapeutic effect of UC-MSCs in SLE patients remain
unclear, it has been suggested that disease remission may be
associated with an increase in the proportion of Tregs in pe-
ripheral blood along with a decrease in IL-4 serum levels,
probably resulting in inhibition of humoral activity [95].
Another recent study has demonstrated that UC-MSCs can
also provide clinical benefit in diffuse alveolar hemorrhage,
a rare complication of SLE with high clinical mortality [96].
These findings suggest that UC-MSC transplantation may
hold promise as a treatment modality in selected SLE cases.
However, the efficacy and safety of this strategy need to be
further validated in larger clinical trials with longer periods
of follow-up.
Therapeutic Potential of UC-MSCs in Cancer
An emerging clinical application of UC-MSCs that re-
cently gained much attention is their use as cellular therapy
to combat cancer. One group has thus administered UC-
MSCs engineered to secrete interferon-
, in combination
with low-dose intraperitoneal injections of 5-fluorouracil in
severe combined immunodeficiency (SCID) mice, bearing
human breast carcinoma tumors in their lungs [97]. Inter-
estingly, UC-MSCs were only found in the lungs and not in
Batsali et al.
other tissues. This combined approach resulted in significant
metastatic tumor reduction, much greater than compared to
each treatment alone. A more recent study has corroborated
the ability of UC-MSCs to reduce the growth of human
breast carcinoma cells both in vitro as well as in vivo, in a
mouse xenograft model [98]. Notably, the antitumor proper-
ties of UC-MSCs do not seem to be specific only to breast
cancer cells, but have also been demonstrated in osteosar-
coma and ovarian carcinoma cell lines in vitro [99]. The
mechanisms mediating UC-MSC anticancer effects are still
under investigation. However, it has been suggested that UC-
MSCs exert their antitumor activity both via cell-to-cell con-
tact and humoral factors, through induction of apoptosis, cell
cycle arrest and autophagy [97, 99]. Taken together, the
aforementioned preliminary data imply that UC-MSCs may
hold promise as a therapeutic modality in cancer and high-
light the need for the development of additional well-
designed pre-clinical studies so as to properly investigate the
potential of this cell-based approach.
CONCLUSION
Within recent years considerable interest has been given
to the exciting properties of UC-MSCs, derived from the WJ,
because of their primitive nature as compared to BM-MSCs
(the prototype of adult MSCs), their multilineage potential
across germ layers, the extensive in vitro proliferation and
their immunosuppressive capacity. These characteristics
along with the ease by which UC-MSCs are isolated from a
tissue with unlimited availability, have generated much en-
thusiasm regarding their potential applications in cell-based
therapies. This expectation is being supported by both pre-
and clinical data. In particular, in this review we reported on
the promising results obtained in recent studies regarding the
use of UC-MSCs for the treatment of cancer and immune
mediated disorders, such as graft versus host disease and
systemic lupus erythematosus and discussed UC-MSC role
as feeder layers for ex vivo HSC expansion. Nevertheless
much work has to be done so as to fully unravel the biologic
features of UC-MSCs and especially those distinguishing
them from adult MSCs. Furthermore, properly designed
comparative studies need to be undertaken in the near future
so as to clearly identify and characterize MSC subpopula-
tions originating from different regions of WJ, in order to
answer the long-standing question: whether different ana-
tomical locations within WJ are associated with distinct
qualitative and quantitative MSC properties. Notably, avail-
able global expression technologies, like gene expression
and comparative genomic hybridization (CGH) microarrays,
proteome and secretome profiles, methylation mapping and
ChIP-on-ChiP technologies are anticipated to contribute sig-
nificantly to the better understanding of UC-MSC biology. It
is anticipated that the data obtained by these studies will not
only provide the theoretical background for the eventual in-
troduction of UC-MSCs in the clinic, but would also be ma-
jor contribution to stem cell biology in general.
CONFLICT OF INTEREST
The authors confirm that this article content has no con-
flicts of interest.
Mesenchymal Stem Cells Derived from Wharton's Jelly
ACKNOWLEDGEMENTS
Declared none.
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Accepted: December 28, 2012