TISSUE-SPECIFIC STEM CELLS

Immune Properties of Human Umbilical Cord Wharton’s

Jelly-Derived Cells
MARK L. WEISS,a CAMERON ANDERSON,a SATISH MEDICETTY,c KIRAN B. SESHAREDDY,a RITA J. WEISS,a
IRENE VANDERWERFF,a DERYL TROYER,a KEVIN R. MCINTOSHb
a
Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas, USA; bCognate BioServices,
Baltimore, Maryland, USA; cNeoStem, Inc., Boston, Massachusetts, USA

Key Words. Adherent cells • Immunogenicity • Immunosuppression • In vitro culture • Stromal cell

ABSTRACT
Cells isolated from Wharton’s jelly, referred to as umbilical
cord matrix stromal (UCMS) cells, adhere to a tissue-culture
plastic substrate, express mesenchymal stromal cell (MSC)
surface markers, self-renew, and are multipotent (differen-
tiate into bone, fat, cartilage, etc.) in vitro. These properties
support the notion that UCMS cells are a member of the
MSC family. Here, the immune properties of UCMS cells
are characterized in vitro. The overall hypothesis is that
UCMS cells possess immune properties that would be per-
missive to allogeneic transplantation. For example, UCMS
cells will suppress of the proliferation of “stimulated” lym-
phocytes (immune suppression) and have reduced immuno-
genicity (e.g., would be poor stimulators of allogeneic lym-
phocyte proliferation). Hypothesis testing was as follows:
first, the effect on proliferation of coculture of mitotically
inactivated human UCMS cells with concanavalin-A-stimu-
lated rat splenocytes was assessed in three different assays.
Second, the effect of human UCMS cells on one-way and
two-way mixed lymphocyte reaction (MLR) assays was de-
termined. Third, the expression of human leukocyte antigen
(HLA)-G was examined in human UCMS cells using reverse
transcription-polymerase chain reaction, since HLA-G ex-
pression conveys immune regulatory properties at the ma-
Disclosure of potential conflicts of interest is found at the end of this article.
ternal-fetal interface. Fourth, the expression of CD40, CD80,
and CD86 was determined by flow cytometry. Fifth, the
cytokine expression of UCMS cells was evaluated by focused
gene array. The results indicate that human UCMS cells
inhibit splenocyte proliferation response to concanavalin A
stimulation, that they do not stimulate T-cell proliferation
in a one-way MLR, and that they inhibit the proliferation
of stimulated T cells in a two-way MLR. Human UCMS
cells do not inhibit nonstimulated splenocyte prolifera-
tion, suggesting specificity of the response. UCMS cells
express mRNA for pan-HLA-G. UCMS cells do not ex-
press the costimulatory surface antigens CD40, CD80,
and CD86. UCMS cells express vascular endothelial
growth factor and interleukin-6, molecules previously im-
plicated in the immune modulation observed in MSCs. In
addition, the array data indicate that UCMS cells make a
cytokine and other factors that may support hematopoi-
esis. Together, these results support previous observa-
tions made following xenotransplantation; for example,
there was no evidence of frank immune rejection of un-
differentiated UCMS cells. The results suggest that hu-
man UCMS will be tolerated in allogeneic transplanta-
tion. STEM CELLS 2008;26:2865–2874

stimuli [4 – 6]. MSCs suppress lymphocyte proliferation in vitro

INTRODUCTION
The Mesenchymal and Tissue Stem Cell Committee of the Inter-
national Society for Cell Therapy has suggested three defining
criteria for mesenchymal stromal cells (MSCs). MSCs (a) are a
plastic-adherent cell population, (b) have specific surface markers
(e.g., CD14-, CD34-, CD45-, and human leukocyte antigen [HLA]
class II-negative and CD73-, CD29-, CD105-positive), and (c)
have the capacity to self-renew and differentiate into various lin-
eages, including bone, cartilage, and adipose, in vitro [1].
MSCs possess the immune properties of immune suppres-
sion and immune avoidance (reviewed in [2, 3]). MSCs exert
immunosuppression by inhibiting T-cell responses to polyclonal
and prolong skin graft survival [4]. Two different general mech-
anisms, one cell contact-dependent and one -independent, have
been advanced to explain this immunosuppression capability
[7]. The effect is most likely due to soluble factors secreted by
the MSCs [8], such as transforming growth factor ␤-1, hepato-
cyte growth factor, interleukin-6, prostaglandin E2, indoleamine
2,3-dioxygenase-mediated tryptophan depletion, or nitric oxide
(reviewed in [9]). MSCs downregulate the interleukin (IL)-2
receptor (CD25) and CD38 on phytohemagglutinin-activated
lymphocytes [10]. Dendritic cells (DCs) cocultured with MSCs
develop into tolerogenic DCs [11].
In addition to MSCs derived from bone marrow, MSCs from
other tissues are reported to be immunosuppressive (e.g., MSCs

Author contributions: M.L.W.: conception and design, assembly of data, data analysis and interpretation, manuscript writing and final
approval of manuscript, financial and administrative support; C.A. and K.R.M.: design, assembly of data, data analysis and interpretation,
manuscript writing; S.M., K.B.S., R.J.W., and I.V.: collection and assembly of data; D.T.: administrative support, data interpretation.
Correspondence: Mark L. Weiss, Ph.D., Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas 66506, USA.
Telephone: 785-532-4520; Fax: 785-532-4557; e-mail: weiss@vet.ksu.edu Received December 10, 2007; accepted for publication July 28,
2008; first published online in STEM CELLS EXPRESS August 14, 2008. ©AlphaMed Press 1066-5099/2008/$30.00/0 doi: 10.1634/
stemcells.2007-1028

STEM CELLS 2008;26:2865–2874 www.StemCells.com

2866
from dental pulp [12], adipose tissue [13, 14], and amniotic
tissue [15]). The immune-suppressive property of MSCs has
been exploited for the successful treatment of severe human
graft-versus-host disease [16].
MSCs have immune avoidance mechanisms that reduce
immunogenicity. Reduced immunogenicity is suggested by the
lack of acute rejection response by the host following xenoge-
neic transplantation into immune-competent animals [17, 18].
Mechanisms that may allow MSCs to escape from the immune
system in allogeneic hosts include modulation of host dendritic
and T-cell function and promotion of regulatory T-cell induc-
tion, as well as limited expression of alloantigen [19]. Low
immunogenicity is also suggested to be due to the induction of
divisional anergy in T cells [6], as well as secretion of the
soluble factors mentioned above by MSCs [19]. Coculture of
bone marrow MSCs with T lymphocytes downregulated T-cell
alloresponsiveness via the TH2 path and resulted in the T cells
assuming a T-regulatory phenotype [20]. Adipose-derived
MSCs demonstrate reduced immunogenicity [13, 14]. Thus, the
immune properties described above are characteristics of MSCs
derived from several tissue sources.
We identified a population of CD45-, CD34-, and HLA-DR-
negative and CD73-, CD105-, CD90-, and CD29-positive cells
derived from human umbilical cord Wharton’s jelly, termed
umbilical cord matrix stromal (UCMS) cells [21–24]. Human
UCMS cells can be isolated rapidly in large numbers from
⬎90% of human cords. Thus, UCMS cells may be a robust
source of MSC-like cells for therapeutic use since they can be
frozen/thawed, clonally expanded, engineered to express exog-
enous proteins, and extensively expanded in culture. We spec-
ulate that UCMS cells have therapeutic potential since they
resemble MSCs in many respects, including surface phenotype,
plastic adherence, and multipotency; in the latter case, they can
be induced in vitro to become bone, cartilage, adipose cells [22,
25–30], neural cells [22, 30, 31], skeletal muscle cells [25], and,
possibly, types of muscle cells [30, 32]. Following xenotrans-
plantation of pig or human UCMS cells into immune-competent
rats, little to no host immune cell infiltration was observed,
suggesting low immunogenicity of UCMS cells [21, 23, 24].
Our hypothesis is that UCMS cells have immune properties
that are permissive to allogeneic transplantation. MSCs derived
from bone marrow [2, 4, 5, 16, 33, 34] and adipose tissue [13,
14] are immune-suppressive and have low immunogenicity in
vitro. Our hypothesis would be supported if UCMS cells share
these properties.
Furthermore, in some tissues, such as the chorionic plate of
the placenta and perhaps parts of the umbilical cord, additional
immune avoidance mechanisms are found, because the early
embryo must avoid immune-based destruction, especially in
hemochorial species. One well-characterized mechanism is the
expression by the trophoblasts of a nonclassic HLA class I gene,
HLA-G. Six or seven splice variants of HLA-G have been
identified: HLA-G1 through HLA-G4 are membrane-bound iso-
forms; HLA-G5 and HLA-G6 are soluble forms. The soluble
forms of HLA-G have been shown to have immunoregulatory
functions, including inhibition of T-cell activation [35]. Al-
though HLA-G expression is downregulated and not found in
the fetus, its expression in the umbilical cord is unknown. The
hypothesis would be supported if UMCS cells express HLA-G.
Here, the immune properties of human UCMS cells were
assessed in vitro using standard assays, including splenocyte
proliferation assays and one-way and two-way mixed lympho-
cyte assays, and their expression of HLA-G isoforms were
assessed using reverse transcription (RT)-polymerase chain re-
action (PCR). The expression of costimulatory molecules CD40,
CD80, and CD86 by UCMS cells was characterized by flow
cytometry, and their expression of cytokines was examined by
Immune Properties of UCMS Cells
focused gene array. The results indicate that UCMS cells are
immunosuppressive, have reduced immunogenicity, express
HLA-G, do not express costimulatory molecules, and express
cytokines that may modulate immune function. Together, these
results suggest that human UCMS will be tolerated in allogeneic
transplantation.
MATERIALS AND METHODS
Approvals
The Kansas State University Institutional Review Board reviewed
and approved this protocol. The Kansas State University Institu-
tional Animal Care and Use Committee reviewed and approved
animal use.
Human Umbilical Cord Matrix Stromal Cells
The protocol for isolation and characterization of umbilical cord
matrix stromal cells has been previously described [22, 24]. Using
previously described protocols, a subset of the isolates was charac-
terized to confirm that they met the ISCT minimal marker set for
MSCs (e.g., they were shown to be plastic-adherent and to express
surface markers CD105, CD90, and CD44) and that they did not
express surface markers CD34 and CD45, and they were verified to
differentiate along two or three mesenchymal lineages (e.g., chon-
drogenic, osteogenic, and adipogenic lineages) [22, 28, 29, 36].
Splenocyte Proliferation Assay
Rat splenocytes were obtained from euthanatized rats using stan-
dard protocols. Briefly, the spleen was removed aseptically, washed
thoroughly, and diced, and spleen cells were released by trituration.
The splenocyte preparation was cleaned up by centrifugation and
resuspending the pellet in RPMI 1640 (Invitrogen, Carlsbad, CA,
http://www.invitrogen.com), and the erythrocytes were lysed. The
splenocyte preparation was again centrifuged, and splenocytes
were washed two more times with RPMI. Cell count and viability
were assessed by trypan blue exclusion prior to plating. Cells were
resuspended in splenocyte medium (i.e., RPMI containing 10% fetal
bovine serum [FBS; HyClone, Logan, UT, http://www.hyclone.
com], 2 mM glutamine, 0.1 mM nonessential amino acids, 1 mM
pyruvate, 20 mM HEPES, and 5 ⫻ 10⫺5 M 2-mercaptoethanol).
Human umbilical cord matrix stromal (hUCMS) cells were plated in
a 12-well plate in low-serum medium (as described previously). At
70% confluence, the medium was removed, and fresh medium
containing mitomycin-C (20 ␮g/ml) was added for 2 hours at 37°C
to mitotically inactivate the hUCMS cells, followed by two washes
and the addition of low-serum medium. In some cases, the hUCMS
cells were inactivated by irradiation (7–9 Gy; Kansas State Univer-
sity CVM linear accelerator). Several hours later or the next day, the
medium was removed from the inactivated hUCMS cells, and 1 ml
of splenocyte medium containing 5 ⫻ 104 concanavalin A (Con-
A)-treated (10 ␮g/ml) splenocytes was added to three wells with
hUCMS cells and three wells without hUCMS cells (each trial was
done in triplicate, and each trial was performed with a different
splenocyte isolate).
Assessing Splenocyte Proliferation
Splenocyte proliferation was assessed three different ways. First,
live splenocytes were counted using a hemocytometer 3 days after
plating using trypan blue exclusion. Live cell splenocyte counts
were done in triplicate and averaged for each well. Here, the effect
of hUCMS cell coculture on splenocyte proliferation was evaluated
by comparing the number of viable splenocytes in wells cocultured
with inactivated hUCMS cells with the number of viable spleno-
cytes in control wells (wells without an hUCMS cell layer; data
were averaged from five independent trials).
Second, in completely independent experiments, a colorimetric
assay using tetrazole reduction was used to assess splenocyte pro-
liferation following Con-A stimulation using the manufacturer’s pro-
tocols (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium [MTT]
Weiss, Anderson, Medicetty et al.
Figure 1. Immune suppression by coculture of human umbilical cord matrix stromal (hUCMS) cells with ConA-activated splenocytes. (A): Using
the trypan blue method to determine the number of live cells, the average suppression in splenocyte growth when cocultured with hUCMS cells was
40%. (B): Using the MTT assay, the average suppression after 4 days was 25%. Thus, both methods indicated immune suppression by coculture with
mitotically inactivated hUCMS cells. Data are presented as means ⫾ SE. Statistically significant differences are indicated. Abbreviations: ConA,
concanavalin A; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; UCMS, umbilical cord matrix stromal.
assay; Roche Diagnostics, Basel, Switzerland, http://www.roche-
applied-science.com). The plate was evaluated on an enzyme-linked
immunosorbent assay reader at 550 – 690 nm. The effect of hUCMS
cells on splenocyte proliferation was evaluated by comparing the
optical density (OD) in wells cocultured on inactivated hUCMS
cells with the OD of splenocytes grown without an inactivated
hUCMS cell layer (performed in triplicate and averaged from four
independent trials).
Third, in independent trials, the effect of coculture with inacti-
vated hUCMS cells on Con-A-stimulated splenocytes was assessed
followed dye-loading of splenocytes with carboxyfluorescein suc-
cinimidyl ester (CFSE). Briefly, splenocytes were incubated with 5
␮M CFSE for 10 minutes followed by two washes with phosphate-
buffered saline (PBS). The CFSE-labeled splenocytes were equally
divided into four experimental groups: splenocytes alone (no Con-A
stimulation), splenocytes cultured with mitotically inactivated
hUCMS cells in splenocyte medium (no Con-A), splenocytes stim-
ulated with Con-A (10 ␮g/ml; no hUCMS cells), and splenocytes
stimulated with Con-A cultured with inactivated hUCMS cells.
Twenty-four or 48 hours later the splenocytes were lifted, washed
with PBS, fixed with 4% paraformaldehyde, rinsed twice with PBS,
and stored at 4°C until flow cytometry (FACSCalibur; BD Bio-
sciences, San Diego, http://www.bdbiosciences.com). The flow cy-
tometry data were analyzed using ModFit (Verity Software House,
Topsham, ME, http://www.vsh.com) to estimate the proliferation of
the splenocytes. To compare the effects of the experimental vari-
ables, the proliferation index, a statistic generated by ModFit that
relates to the number of population doublings the splenocytes had
undergone following CFSE loading, was used.
In each case, a paired t test was used to compare means of
experimental groups to evaluate whether Con-A exposure increased
splenocyte proliferation (CFSE experiment; one-tailed test) and
whether culture with UCMS cells inhibited Con-A induced spleno-
cyte proliferation (all three experiments; one-tailed test). A two-
tailed test was used to evaluate whether culture of UCMS cells with
splenocytes affected splenocyte proliferation (compared with
splenocytes only; CFSE experiment).
Mixed Lymphocyte Proliferation Assays
The mixed lymphyocyte proliferation assays were carried out using
standard methods. The methodological details are found in the
supplemental online data.
RT-PCR
RT-PCR was carried out using standard methods. The methodolog-
ical details are found in the supplemental online data.
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2867
Flow Cytometry
Three different hUCMS cell isolates (isolates 19, 12, and 21) were
assessed via flow cytometry using standard methods described
previously [24]. Briefly, approximately 1–2 ⫻ 106 cells were sus-
pended in 2 ml of PBS containing 0.5% bovine serum albumin and
2 mM EDTA. Aliquots of cells (100 ␮l) were incubated with
labeled antibody (or isotype control) for 30 minutes at 4°C then
washed twice by centrifugation. The cells were protected from light
and held at 4°C until analysis using a FACSCalibur. The antibodies
used here can be found in supplemental online Table 3.
Focused Array
Nonradioactive GeArray cDNA expression array filters (OHS-022;
SuperArray Bioscience Corporation, Frederick, MD, http://www.
superarray.com) were used to evaluate cytokine gene expression,
and procedures were as described by the manufacturer. The meth-
odological details are provided in the supplemental online data.
Statistical Analysis
Data collection was conducted in an experimenter-blind fashion,
when possible. Following data collection, the group status was
decoded prior to statistical analysis. In general, the Student’s t test
was used to evaluate the differences in the means between groups,
and significance was set at p ⬍ .05. For the CFSE experiment,
significance was set at p ⬍ .1. Data are presented as mean ⫾ 1 SEM
or mean ⫾ 1 SD, as noted.
RESULTS
Characterization of hUCMS Cells
Human UCMS cells were rapidly extracted from the umbilical
cord and grown in culture. As shown in supplemental online
Table 1, the umbilical cord isolates yielded an average of
approximately 10,500 cells per centimeter of umbilical cord
length in the initial isolate; this fits within the 10,000 –15,000
cells per centimeter of length that has historically been obtained
in our laboratory [37, 38]. Here, a randomly selected subset of
the isolates was fully characterized by plastic adherence, flow
cytometry, and differentiation into mesenchymal lineages: os-
teogenic, chondrogenic, and adipogenic lineages (supplemental
online Table 1). Thus, hUCMS cells used here the minimal
definition of MSCs given by ISCT [1], as previously described
by our laboratory and others [22, 28, 29, 36].
2868 Immune Properties of UCMS Cells

Figure 2. Immune suppression by coculture of human umbilical cord matrix stromal cells (hUCMS cells) with ConA-activated splenocytes assessed
by the CFSE method. Splenocyte proliferation was assessed after 4 days of coculture with hUCMS cells and analyzed by flow cytometry. The
proliferation index (PI; a statistic generated by ModFit) correlated to the number of cell divisions the splenocytes had undergone. (A): Data from one
of the three independent trials. Top left: Splenocytes stimulated with ConA proliferated extensively (PI, 3.63; no coculture). Top right: Coculture of
stimulated splenocytes with inactivated hUCMS cells decreased splenocyte proliferation (PI, 2.63). Bottom left: Coculture of unstimulated splenocytes
with hUCMS cells tended to increase, and did not decrease, unstimulated splenocyte proliferation (compare PI of this panel, 1.57, with the PI of
splenocytes alone [bottom right], 1.46). Bottom right: Unstimulated splenocytes alone (PI, 1.46). (B): The data were normalized to the PI of
splenocytes only (100%). Shown are averaged results from three independent trials. Splenocyte proliferation was significantly enhanced by ConA (p ⬍
.001). In contrast, culture of inactivated hUCMS cells with ConA-stimulated splenocytes significantly suppressed splenocyte proliferation (p ⬍ .08).
The immune suppression effect of inactivated hUCMS cells was specific to stimulated splenocytes, and coculture with inactivated hUCMS cells was
not in itself harmful to splenocytes because coculture of hUCMS cells did not affect proliferation of splenocytes (p ⬎ .1). Data are presented as
means ⫾ SE. Statistically significant differences are indicated with an asterisk (ⴱ). Abbreviations: CFSE, carboxyfluorescein succinimidyl ester;
ConA, concanavalin A; NS, not significant; UCMS, umbilical cord matrix stromal.

Splenocyte Proliferation Assay Results
Both the trypan blue (shown in Fig. 1A) and MTT assay (Fig.
1B) methods indicated significant suppression of Con-A-in-
duced splenocyte proliferation by coculture with mitotically
inactivated human UCMS cells. The average suppression in
splenocyte growth when cocultured with UCMS cells was 40%
(Fig. 1A). Using the MTT assay, immune suppression ranged
from 8% to 69% after 3 days and from 15% to 55% after 4 days.
The average suppression was 30% after 3 days and 25% after 4
days (Fig. 1B). Thus, both methods indicated significant sup-
pression of splenocyte proliferation by coculture with inacti-
vated hUCMS cells. The MTT assay results indicated that
suppression was 25% after 4 days versus 40% suppression of
proliferation obtained by manual counting after 4 days.
The CFSE experiment was conducted to evaluate splenocyte
proliferation and to evaluate the specificity of the effect of
hUCMS on activated splenocytes. The results are shown in
Figure 2 and are similar to those obtained using the other
methods; for example, they indicate a suppression of Con-A-
induced splenocyte proliferation by coculture with mitotically
inactivated hUCMS cells (Fig. 1). Specifically, splenocytes
grown in culture without the mitogen Con-A yielded a prolif-
eration index of 2.17 ⫾ 0.77. Stimulation with Con-A signifi-
cantly increased the proliferation of splenocytes (proliferation
index, 5.38 ⫾ 2.17). This indicates that the splenocytes prolif-
erate in response to Con-A stimulation (positive control, p ⬍
.01). The coculture of Con-A-stimulated splenocytes with mi-
totically inactivated hUCMS cells produced a proliferation in-
dex of 3.80 ⫾ 1.08. This represents a significant suppression of
activated splenocyte proliferation (p ⬍ .08). To evaluate
whether coculture of splenocytes with inactivated UCMS cells
produced a nonspecific inhibition of splenocyte proliferation,
the proliferation index was assessed following coculture of
unstimulated (no Con-A stimulation) splenocytes with inacti-
vated hUCMS cells. In this case, the proliferation index was
2.39 ⫾ 1.38. Thus, splenocyte proliferation is mildly stimulated
(and not significantly stimulated) and was not inhibited by
coculture with human UCMS cells. This observation suggests
that the suppressive effect of hUCMS cells on splenocytes is
specific to mitogen-induced splenocyte proliferation. These re-
sults provide independent confirmation of the data obtained
from by manual counting and by MTT assays and extend those
findings to indicate that (a) hUCMS cells lack toxicity on
splenocytes when cocultured and (b) the immune-suppressive
effects of UCMS cells are specific to splenocytes stimulated by
Con-A.
One-Way Mixed Lymphocyte Reaction
(Immunogenicity Assay) Results
The data shown in Figure 3A and 3B are derived from indepen-
dent experiments performed on different cell isolates. In both
cases, the experiments were performed the same way. As indi-
Weiss, Anderson, Medicetty et al.
cated in Figure 3A and 3B, the purified T-cell response to
hUCMS cell isolates derived from four donors was not signif-
icantly higher than the response to autologous peripheral blood
mononuclear cells (PBMCs), indicating that hUCMS cells did
not elicit a proliferation response in vitro. Importantly, the
response of all four isolates did not change with passage: neither
early passage (P5 or P6) nor later passage (P9) cells elicited an
appreciable T-cell proliferation response. In contrast, vigorous
T-cell responses were detected to allogeneic PBMCs (the pos-
itive control).
Two-Way Mixed Lymphocyte Reaction (Immune
Suppression) Results
The data shown in Figure 4A and 4B were derived from inde-
pendent experiments performed on different cell isolates. In
both cases, the experiments were performed the same way. As
shown in Figure 4A and 4B, all four hUCMS cell isolates
suppressed the mixed lymphocyte reaction (MLR) response to
various degrees, generally in a dose-dependent manner. In the
first set of experiments (Fig. 4A), control splenic fibroblasts
were not suppressive at the highest dose of 20,000 cells per well.
At the same dose, hUCMS cells suppressed the MLR by an
average of 35% ⫾ 14% (range, 18%–53%). This dose of cells
corresponds to a 1:10 ratio of hUCMS to responder cells,
assuming that half of the PBMCs are T cells. In the second set
of experiments (Fig. 4B), splenic fibroblasts were suppressive at
10,000 cells per well (34% suppression) but not at 5,000 cells
per well (8% suppression). At 5,000 cells per well, hUCMS cells
suppressed the MLR by an average of 36% ⫾ 15% (range,
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2869
Figure 3. Immunogenicity of human um-
bilical cord matrix stromal (hUCMS) cells
by one-way mixed lymphocyte reaction as-
say. Four different hUCMS cell isolates at
early (P5–P6) or later (P9) passage were
tested for their ability to stimulate prolifera-
tion of allogeneic T cells derived from two
different donors, responder 008 and re-
sponder 009. The proliferation of T cells was
determined in the presence of autologous
irradiated PBMCs (negative control, back-
ground), in the presence of allogeneic irra-
diated PBMCs (positive control), or in the
presence of UCMS cells. Results are shown
for hUCMS donors 4-3 and 4-5 in (A) and
for hUCMS donors 4-6 and 4-7 in (B). The
stimulator cells were tested at densities of
5,000, 10,000, or 20,000 cells per well. Only
the highest cell dose is shown here since the
lower doses of positive control allogeneic
PBMCs induced poor T-cell proliferation.
Data are presented as mean counts per
minute ⫾ SD. An asterisk (ⴱ) indicates sig-
nificant T-cell stimulation above back-
ground responses by criteria described in
Materials and Methods. Abbreviations: P,
passage; PBMC, peripheral blood mononu-
clear cell; UCMS, umbilical cord matrix
stromal.
19%– 49%). This dose of cells corresponds to a 1:40 ratio of
hUCMS to responder cells.
The immune suppression data from both sets of experiments
is summarized in Figure 5, comparing passage number of
UCMS cells. In two of three isolates (4-3, 4-5) suppression
decreased with increased passage by approximately 19%. Im-
mune suppression by the third hUCMS cell isolate (4-7) was
unchanged by passage. Overall suppression by early passage
(P5–P6) hUCMS cells was 37% ⫾ 14%, and suppression by late
passage (P9) cells was 33% ⫾ 15%. The immune suppression
observed by allogeneic MLR (approximately 35%) was similar
to that observed by the xenogeneic splenocyte proliferation
assays (Figs. 1 and 2, above).
RT-PCR Results
Pan-HLA-G primers indicated that HLA-G mRNA was ex-
pressed by hUCMS cells at both passage 4 and passage 8 (data
not shown). As shown in Figure 6, hUCMS cells differed from
the term placenta in terms of the expression of mRNA for
HLA-G6 isoforms. Human UCMS cells weakly expressed
HLA-G6 and did not express HLA-G5 (Fig. 6). In contrast, term
placenta expressed both HLA-G5 and HLA-G6. Data from the
same PCR experiment depicting results from the JAR p745
negative control cells, and the jeg-3 HLA-G positive controls
are shown as well.
Flow Cytometry Results
As shown in Table 1, three different isolates of hUCMS cells
tested at passage 4 did not express costimulatory molecules
(e.g., CD40, CD80, or CD86).
2870
Figure 5. Suppression summary. The data from Figure 4 are sum-
marized and expressed as percent age of suppression of the control
mixed lymphocyte reaction response. The data used for this figure
were derived from doses of cells added in which fibroblasts were not
suppressive: 20,000 (isolates 4-3 and 4-5) and 5,000 (isolates 4-6 and
4-7) cells per well. Abbreviations: ND, not done; P, passage; UCMS,
umbilical cord matrix stromal.
Focused Array Results
As shown in supplemental online Fig. 1, cytokines implicated in
hematopoietic stem cell proliferation, cell proliferation factors,
or rescue functions or with specific cytokine activity were
expressed by hUCMS cells. Specifically, mRNA for interleu-
kins 1A, 1B, 6, 8, 11, and 14 was expressed by hUCMS cells. In
addition, mRNA was present for BMP1, CSF3, FAM3C,
GDF15, PDGFB, and tumor necrosis factors 4, 11b, 12, and
vascular endothelial growth factor (VEGF) was expressed by
hUCMS cells (supplemental online Fig. 1).
Immune Properties of UCMS Cells
Figure 4. Suppression of two-way MLR
by human umbilical cord matrix stromal
(hUCMS) cells. Four different hUCMS cell
isolates were tested at early (P5 or P6) or
later (P9) passage for their ability to sup-
press T-cell proliferation in a two-way MLR.
hUCMS cells or splenic fibroblasts were
added to MLR cultures at the numbers indi-
cated, ranging from 5,000 to 20,000 cells per
well. T-cell proliferation, expressed as cpm,
was determined 7 days later by pulsing the
cells with [3H]thymidine during the final 16
hours of culture. Data are represented as
mean counts per minute ⫾ SD. Significant
suppression (p ⬍ .05) of the MLR by
hUCMS cells is denoted by an asterisk for
the blocking of responses at the highest dose
of splenic fibroblasts that did not mediate
significant suppression. This dose was
20,000 cells per well in (A) and 5,000 cells
per well in (B). Abbreviations: MLR, mixed
lymphocyte reaction; P, passage; UCMS,
umbilical cord matrix stromal.
DISCUSSION
Here, the first description of the immune properties of Whar-
ton’s jelly-derived cells (here called hUCMS cells) was pro-
vided. Five observations were reported. First, human UCMS
cells suppressed the proliferation of stimulated immune cells.
Con-A-stimulated rat splenocytes (xenograft model) or acti-
vated human peripheral blood mononuclear cells (allogeneic
transplant model) were cocultured with human UCMS cells.
Second, human UCMS cells did not stimulate proliferation of
allogeneic or xenogeneic immune cells. Human UCMS cells
were cocultured with two different isolates of purified T cells,
and no proliferation of the T cells was found. When human
UCMS cells were cocultured with rat splenocytes, no prolifer-
ation was observed. Third, RT-PCR indicated that hUCMS cells
produced an immunosuppressive isoform of human leukocyte
antigen (HLA), HLA-G6. Fourth, flow cytometry revealed that
the expression of immune response-related surface antigens
CD40, CD80, and CD86 was absent on hUCMS cells. Fifth, the
presence of mRNA for cytokine genes by hUCMS cells was
evaluated, and it indicated that hUCMS cells make several
cytokines, such as IL-6 and VEGF. Together, these results
indicate that human UCMS cells have immunosuppressive prop-
erties in vitro and that human UCMS cells have low immuno-
genicity. These findings indicate that UCMS cells that have
been expanded for four to nine passages in vitro appear to have
the same immune properties as MSCs derived from other tissue
sources. Furthermore, these results suggest that UCMS cells
would be tolerated in an allogeneic transplant.
Weiss, Anderson, Medicetty et al.
Figure 6. Reverse transcription polymerase chain reaction (RT-PCR)
for HLA-G isoforms. RT-PCR for HLA-G5 and HLA-G6 isoforms
revealed that human umbilical cord matrix stromal cells express mRNA
for HLA-G6. Positive controls were human term placenta and jeg-
HLA-G cells. Negative controls were JARp745 cells. Abbreviations:
GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HLA, human
lymphocyte antigen; P, passage; UCM, umbilical cord matrix.
Table 1. Flow cytometry results
FACS hUCMS 21, hUCMS 12, hUCMS 9,
antibody passage 4 passage 4 passage 4
CD40 2% ND ND
CD80 ND ND ND
CD86 ND ND ND
Abbreviations: FACS, fluorescence-activated cell sorting;
hUCMS, human umbilical cord matrix stromal; ND, not detected.
Properties of UCMS Cells
The umbilical cord matrix, also known as Wharton’s jelly, is the
loose connective tissue that surrounds and cushions the umbil-
ical vessels. The Wharton’s jelly contains hyaluronic acid, col-
lagen, and primitive multipotent cells [21, 24, 26, 28, 29, 36, 39,
40]. Human UCMS cells used here met the definition estab-
lished for MSCs by the ISCT based upon their plastic adherence,
morphology, surface phenotype, and multipotency (supplemen-
tal online Table 1). The differences between adult-derived
MSCs and UCMS cells are that (a) UCMS cells have the
potential for faster expansion ex vivo, and (b) UCMS cells have
telomerase activity [22, 24, 28, 40], and the umbilical cord is
relatively enriched for CFU-F compared with bone marrow [36].
Several laboratories have reported that 10,000 –15,000 nucle-
ated cells can be isolated per centimeter of human umbilical
cord length [24, 29, 34]; another laboratory has reported 106
nucleated cells per centimeter [36]. Thus, Wharton’s jelly is
enriched for MSC-like cells. Furthermore, UCMS cells are
www.StemCells.com
2871
multipotent and can be differentiated into bone, cartilage, fat,
and neural cells [28 –30, 40].
It is important to note that in vitro data must be confirmed
with in vivo data. Although in vitro data appear to be fairly
consistent among laboratories (showing suppression and low
immunogenicity of MSCs), the animal data have been contra-
dictory. Recently, it has been shown that porcine MSCs, which
appeared to have low immunogenicity in vitro, elicited an im-
mune response after intracardiac transplantation into allogeneic
recipients, although subcutaneous transplants required a second
injection to effect a similar reaction [41]. However, multiple
injections of large numbers of allogeneic baboon MSCs (1 ⫻
106 cells per kilogram of body weight) via multiple routes
resulted in significant engraftment and the induction of host
T-cell hyporesponsiveness, although all recipient baboons pro-
duced alloantibodies [42]. In contrast, it was reported that mul-
tiple injections of allogeneic swine UCMS cells could elicit an
immune response [43]. This change is likely due to changes in
major histocompatibility complex (MHC) class II expression,
since human or swine UCMS cells could be induced to express
MHC class II following a 48-hour exposure to interferon-␥ in
culture [43]. Similarly, allogeneic swine UCMS cells activated
by interferon-␥ exposure were immunogenic upon their first
injection into swine, and nonactivated UCMS cells were not.
These results do not discount the idea of using hUCMS cells in
an allogeneic setting and suggest that care may be needed when
multiple injections of UCMS cells are given.
Work from several laboratories indicates that UCMS cells
have therapeutic potential for neurodegenerative disease [24,
27], stroke [44], photoreceptor degeneration [40], and breast
cancer [45]. Finally, UCMS cells may be useful for tissue
engineering [25, 29, 46 – 49]. These factors together suggest that
hUCMS cells may be an important source of cells for cell
therapy in the future. The ability to use UCMS cells in alloge-
neic transplantation would enable their therapeutic use. For
example, this would simplify the “stock” of cryogenically stored
UCMS cells required for off-the-shelf therapeutic use.
Do MSCs Have Specific Immune Properties?
In the Introduction, the minimal marker set of MSCs was given
as plastic adherence, a particular surface marker set, and mul-
tipotency. MSCs may have additional properties. For example,
it is well documented that (a) MSCs act as support cells for
hematopoietic stem cells [50, 51], (b) they migrate to sites of
pathology and infiltrate tumors [52, 53], and (c) they may have
particular immune properties of low immunogenicity and im-
mune suppression [2, 4, 5, 34]. A similar set of immune prop-
erties is reported here with UCMS cells. Furthermore, the cur-
rent work suggests that the UCMS cells act via to suppress
proliferation of activated splenocytes, specifically in allogeneic
and xenogeneic formats. The suppression of the proliferation
was 25%– 40% (in Fig. 1) to 38% (Fig. 2), showing good
agreement among three methods used to evaluate xenogeneic
suppression. The suppression in the allogeneic human MLR
averaged 38% (range, 17%–54%; Fig. 5). UCMS cells did not
inhibit proliferation of cultured splenocytes, and there is no
evidence that UCMS cell coculture caused splenocyte death.
Interestingly, a recent publication reports that VEGF and IL-6
secreted by MSCs result in immunosuppression [54]. VEGF is
thought to cause this effect by blocking the emigration or
differentiation of bone marrow lymphoid precursors [55]. IL-6
has been postulated to inhibit the differentiation of monocytes to
dendritic cells [56]. The focused array data indicated that
hUCMS cells express IL-6 and VEGF, as was reported previ-
ously [24]. The role of these factors will require confirmation to
2872
determine whether they are playing a role in the immune mod-
ulation observed here.
MSCs have been reported in most publications to be MHC
class II-negative (e.g., [1, 57]), but in at least two publications
they have been reported as weakly positive [34, 58]. To date,
cultured UCMS cells have been reported to be MHC class
II-negative [28, 29, 36, 40]. As discussed above, exposure to
interferon-␥ stimulates increased MHC class I and induces
MHC class II expression by human and swine UCMS cells [43].
Importantly, UCMS cells express IL-6 and VEGF genes (sup-
plemental online Table 1; [24]), and since, as stated above, these
two proteins have recently been shown to be pivotal in the
immunosuppressive capability of MSCs [54], their expression
by the UCMS cells may be a mechanism for the immunosup-
pressive properties demonstrated here. In support of the cyto-
kine gene expression profile provided here, the previous gene
expression analysis of UCMS cells confirms the focus array data
provided here [24], and Friedman et al. have reported that
UCMS cells release hematopoietic and immune regulatory cy-
tokines into the medium [59]. In addition, MSCs do not express
costimulatory molecules (e.g., CD80, CD86, or CD40) that
contribute to immunogenicity [34, 60]. Human UCMS cells do
not express these molecules either (Table 1). Together, these
findings may explain the lack of an apparent host immune
response observed following transplantation of either porcine
[21, 23] or human [24] UCMS cells into the rat brain, and they
support the notion that UCMS cells share properties of MSCs.
HLA-G
The reduced immunogenicity of MSCs has been likened to
maternal acceptance of the fetal allograft [19]. The human MHC
class I molecule HLA-G has long been known as a molecule
selectively expressed by cytotrophoblastic cells. By inhibiting
the cytolytic function of decidual natural killer cells, HLA-G
protects the fetal tissue from the mother’s immune system.
HLA-G molecules have 15 alleles [61– 63]; we tested for
pan-HLA-G (data not shown) and for the soluble HLA isoforms
HLA-G5 and HLA-G6. Since UCMS cells are fetal cells derived
from postnatal tissue anatomically continuous with the placenta,
we examined HLA-G expression in UCMS cells. The expres-
sion of pan-HLA-G (data not shown) by both early (P4) and
later (P8) passage human UCMS cells and expression of the
HLA-G6 isoform suggest that UCMS cells share some of the
gene expression profile expressed by placental cells. Therefore,
the importance of the presence of HLA-G in the UCMS cells in
immune suppression and immunogenicity is currently unknown.
A more complete examination of the role of HLA-G6 in the
immune physiology of UCMS cells is slated for the future.
An important physiological variable for cells under consid-
eration for cellular therapy is how well those cells are tolerated
by the host’s immune system. For example, will complete
haplotype matching be required for engraftment, or will mis-
matched (e.g., allogeneic) transplantation be permitted? This is
important information for two independent reasons. First, since
graft rejection and graft-versus-host disease seen in bone mar-
row transplants are serious, life-threatening complications, it is
important for engraftment, reduction of risk, and therapeutic
efficacy to understand the immunogenicity and immune sup-
pression function of therapeutic cells. Second, the tissue match-
ing requirements will define commercial variables, such as how
many samples the stem cell bank must contain to serve a diverse
population. This mathematical exercise has been conducted by
embryonic stem cell scientists, since embryonic stem cells will
require tissue matching [64]. If allogeneic transplantation is safe
and effective, a smaller bank of human UCMS cells would be
required, compared with embryonic stem cells.
Immune Properties of UCMS Cells
Here, the one-way and two-way MLR assays were examined
as a function of passage (time in cell culture). Thus, additional
information was obtained. Specifically, the immunogenicity of
human UCMS cells was low and did not change from passage 5
through passage 9 in four isolates examined. In contrast, human
UCMS cells were found to be immunosuppressive at passages
5– 6 and passage 9, but the degree of immune suppression
observed in two of three isolates may have decreased slightly
during passage. Since chondrogenic differentiation alters the
immunosuppressive properties of MSCs [65], it is possible that
differentiation of UCMS cells may explain why two of the three
isolates lost immunosuppressive properties at passage 9. This is
not likely, though, because UCMS cells were not exposed to
medium with specific differentiation factors, and there were no
data to suggest that the UCMS cells were differentiating (e.g.,
no lipid in cells, no slowing down of proliferation, no evidence
of matrix deposition, and no changes in cellular morphology to
indicate differentiation of UCMS cells). Further work is needed
to determine whether prolonged culture or, specifically, differ-
entiation plays a role in UCMS cell immunophysiological prop-
erties. In this regard, recent work by Cho et al. indicates that
culture of umbilical cord-derived cells with inflammatory cyto-
kine interferon-␥ may increase immunogenicity by expression
of MHC molecules [43]. The mechanism for this change in
immune properties is unknown but may be due to changes in
cytokine profile or nitric oxide release (Dr. L. Wang, Auburn
University, personal communication).
Xenotransplantation Studies with hUCMS Cells
UCMS cells, unlike ESCs, do not induce tumors or death after
1 ⫻ 106 to 6 ⫻ 106 undifferentiated hUCMS cells were trans-
planted either i.v. or s.c. into Beige/SCID mice [24, 45]. In
addition, UCMS cells are not acutely rejected when transplanted
as xenografts in immune-competent rats. For example, pig
UCMS cells undergo a moderated expansion following trans-
plantation into rat brain without obvious untoward behavioral
effects or host immune response [21, 23]. Pig UCMS cells were
recovered from rat brain more than 6 weeks after transplantation
without immune suppression. Similarly, human UCMS trans-
plantation into rats did not trigger obvious host immune re-
sponse around the transplantation site, although cells were not
recovered more than 1 week after transplantation [24]. This
degree of tolerance may be due to either immune property tested
here: that is, either immunosuppressivity or low immunogenic-
ity. In either case, the lack of infiltration by host immune cells
bodes well for engraftment of UCMS cells. To date, there are no
reports of engraftment, expansion, or differentiation of UCMS
cells. Therefore, UCMS cells have not met the definition of true
stem cells. It is possible that syngeneic grafting, perhaps to-
gether with opening of the niche via irradiation, will be needed
to permit UCMS cells to engraft.
SUMMARY
Human UCMS cells from passages 4 –9 have low immunoge-
nicity and suppress the proliferation of activated immune cells.
The mRNA for HLA-G6, an immunosuppressive HLA-class I
marker synthesized by cytotrophoblasts at the maternal-fetal
interface, was found in UCMS cells. In contrast, HLA-G5
expression was not found. This differentiates umbilical cord matrix
stromal cells from cytotrophoblastic cells, which express both
HLA-G5 and HLA-G6, and from bone marrow-derived MSCs,
which do not express HLA-G. Human UCMS cells do not express
the costimulatory surface antigens CD40, CD80, and CD86. Fo-
cused gene array data revealed that UCMS cells express IL-6 and
Weiss, Anderson, Medicetty et al.
VEGF, molecules thought to play a role in MSC immune regula-
tion. These data, taken together with previous observations follow-
ing xenotransplantation of UCMS cells, suggest that hUCMS cells
would be tolerated in allogenic transplantation.
ACKNOWLEDGMENTS
We acknowledge the support of K-INBRE, Kansas State Uni-
versity (KSU) Developing Scholars program, KSU Department
of Anatomy and Physiology, KSU College of Veterinary Med-
icine Dean’s office, KSU Provost’s office, KSU Terry C. John-
son Center for Basic Cancer Research, NIH (NS34160), and
support from the State of Kansas to the Midwest Institute for
Comparative Stem Cell Biology. The Northeast Kansas Parkin-
son Association is thanked for its generous support. We thank
Robert Deans, Tony Ting, Kathy Mitchell, Alan Smith, Mahen-
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OF INTEREST
M.L.W. is a paid consultant for the Regenerative Medicine
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