Open Journal of Cell Biology, 2012, 2, 1-9
doi:10.4236/ojcb.2012.21001 Published Online March 2012 ( 1
Immunoresponse to Allogeneic Synovial or Xenogenic
Mesenchymal Stromal Cells in a Co-Culture Model
Seth S. Jump1,2, David S. Smith1, David C. Flanigan3, Alicia L. Bertone1,2
1Comparative Orthopaedic Research Laboratories, The Ohio State University, Columbus, USA
2Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, USA
3Department of Orthopaedics, College of Medicine, The Ohio State University, Columbus, USA
Received February 2, 2012; revised March 7, 2012; accepted March 16, 2012
The purpose of our investigation s was to measure, in a co-cu lture cond ition, th e immunoresponse to allog eneic or xeno-
genic cells, selected as potential sources for cell therapy of arthritis. We challenged human spleen-derived cells (hSpl)
by three different mechanisms: 1) exposure to donor allogeneic or xenogeneic cellular antigens; 2) exposure to donor
cells transduced with adenoviral antigens (Ad) and 3) lipopolysaccharide (LPS), a known inflammatory immunostimu-
lant. The immunoresponse to allogeneic human synovial-derived mesenchymal stromal cells alone or transduced with
adenoviral green fluorescent protein (hSD-MSC or hSD-MSC/GFP) or the immunoresponse to xenogeneic equine
mesenchymal stromal cells (eqMSC) or equine dermal fibroblasts (eqDFb), characterized by the proportion of CD3+,
CD4+, and CD8+ human splenocytes (hSpl), was measured on Day 0 and Day 6 of co-culture by flow cytometry. In
culture with hSD-MSC, hSD-MSC/GFP, eqDFb, or eqMSC, the proportion of CD3+ and CD8+ hSpl increased with time
in culture but not with expo sure to cell allo- or xeno-antigens. Both hSD-MSC and hSD-MSC/GFP increased in number
during culture and were not affected in viab ility or proliferation b y co-culture with allogeneic hSpl. In this in vitro, pri-
mary exposure stud y, hSpl demonstrated a natural selectio n and adap tation to a short-term cell culture env ironment, and
that neither allogeneic nor xenogeneic cell antigens incited a greater cellular immunoactivation than co-cultured hSpl
Keywords: Arthritis; Stem Cells; Allogeneic; Xenogeneic; Immunoresponse
1. Introduction
Cutting-edge advancements in regenerative medicine may
harness the potential of an engineered cell source as a
therapeutic vecto r for the repair and restoration of multi-
ple human tissues including articular cartilage damaged
by injury or degenerated through either osteoarthritis or
rheumatoid arthritis. Allogeneic cells, from a different
organism of the same species or xenogenic cells, from a
different species, could provide a nearly limitless supply
of therapeutic cells for use in tissue repair. Allogeneic or
xenogeneic cell sources, engineered to serve as a clinical
tool, could dramatically and irrevocably enhance current
medical practices that promote healing.
Allogeneic mesenchymal stem cells in Phase III clini-
cal trials, have been used as a treatment for inflammatory
conditions including acute Bone Versus Graft Disease
and Crohn’s disease [1]. In orthopaedic medicine, au tolo-
gus cell-based strategies for tissue repair and restoration,
including autologous chondroctye implantation and os-
teochondral grafts, are currently used in clinical practice
with some promising results [2-6]. A potentially promis-
ing alternative or ad junct strategy to current regenerative
techniques could use allogeneic cells or xenogenic cells.
To this end, a cell-based allogeneic therapy has been
provided for 1st generation, commercially-available car-
tilage neograft (DeNovo® ET Live Cho ndral Engineered
Tissue Graft, Zimmer Holdings, Inc). This product, ap-
proved last year, relies on allogeneic, juvenile chondro-
cytes and a proprietary cell-scaffold system to promote
healing. Potentially, allogeneic or xenogeneic cells could
serve as effective therapeutic vectors in vivo to integrate
into the native biological environment of tissue. The im-
munoresponse of allogeneic and xenogeneic cells needs
to be further investigated, and further observations of the
cellular mechanisms and interactions will help to eluci-
date and describe the immunoresponse for future clinical
New cell sources could improve the repair and restora-
tion of articular cartilage, such as in injured cartilage or
deteriorating cartilage as in osteoarthritis (OA), the most
common form of arthritis. It is believed that there are
immune processes that could be responsible for the de-
Copyright © 2012 SciRes. OJCB
generation of cartilage. In particular, activated immune
cell infiltration, including T-cells, has been associated
with the advancement of arthritis [7-9]. Finding a poten-
tial cell- or tissue-based treatment for damaged cartilage
is dependent upon further understanding the immunore-
sponse of cells from an articular joint, in cluding synovial
lining cells or chondrocytes. Organ or cell transp lantation
is characterized by an activation of host defenses includ-
ing the activation and proliferation of immune cell types
including cluster determination 3 (CD3+) mature lym-
phocytes, cluster determination 4 (CD4+) T-helper (TH1)
cells, and cluster determination (CD8+) natural killer/
cytotoxic T lymphocytes (CTL) [7-9].
Allogeneic synovial-derived cells (SDSCs) have been
successful in the repair of full-thickness defects of the
femoral condyle in rats; however, contaminating macro-
phages provided evidence of a delayed immune reaction
to the transplantated allogeneic cells [10]. Furthermore,
xenogeneic SDSCs failed to repair cartilage defects in
vivo, and an enhanced immune response, characterized
by detection of major histocompatability complex anti-
gen II (MHCII) in foreign bodies found in the repair tis-
sue [11]. In cell culture, a limited number of studies have
used a mixed immune cell design to evaluate host versus
donor reactions [12-16]. Allogeneic human MSCs de-
rived from bone marrow successfully reduced CD8+ ex-
pansion in cell culture providing support for the benefi-
cial immunomodulation of MSCs [17], and allogeneic
fibroblast-like synoviocytes have also reduced prolifera-
tion in T-cells found in bone marrow [12]. On the other
hand, allogeneic peripheral blood mononuclear cells elic-
ited an activation of both CD4+ and CD8+ cells [14].
Further characterization of the potential cellular activa-
tion of allogeneic or xenogeneic cells with the host im-
mune system is needed to further develop clinical tools to
control or monitor this reaction for the future of cell
therapy as a treatment for joint disease. Our study will
extend the findings in the literature by using another
source of human immune cells, splenic tissue, to investi-
gate the potential immune activation of CD3+, CD4+, and
CD8+ cell types to allogeneic or xenogeneic mesenchy-
mal stromal cells.
The purpose of our investigatio ns was to measure, in a
co-culture condition, the immunoresponse to allogeneic
or xenogenic cells, selected as potential sources for cell
therapy of arthritis. We challeng ed hu man spleen-d erived
cells (hSpl) by three different mechanisms: 1) exposure
to donor allogeneic or xenogeneic cellular antigens; 2)
exposure to donor cells transduced with adenoviral anti-
gens (Ad); and 3) lipopolysaccharide (LPS), a known
inflammatory immunostimulant. For the allogeneic ex-
periment, human synovial-derived mesenchymal stromal
cells (hSC-MSC) or hSC-MSC transduced with adeno-
virus expressing green fluorescent protein (hSC-MSC-
GFP) were co-cultured with allogen eic hSpl. In the xeno-
geneic experiment, equine (eq) bone-marrow derived
MSCs (eqMSC) or equine dermal fibroblasts (eqDFb)
were co-cultured with hSpl. Our hypothesis was that a
cell-mediated challenge of either allogeneic or xenoge-
neic cells would stimulate the formation and develop-
ment of CD3+ and CD8+ hSpl in co-culture compared to
unchallenged hSpl and that adenoviral challenge may
further enhance this effect. Additionally, the viability of
the allogeneic or xenogeneic donor cells was expected to
be reduced when co-cultured with human host cells.
2. Methods
2.1. Human and Equine Donor Tissue Harvest
and Digestion
Synovial biopsies were obtained from the knee joint of
orthopedic pa tients undergoing anterio r cruciate ligament
reconstruction by an author surgeon (DCF). Tissue har-
vest was conducted in accordance with the guidelines set
by the Institutional Review Board (IRB Protocol 2009H0256)
at The Ohio State University and only by consent of the
Synovial biopsies were digested in sterile-filtered (0.2
um) media containing collagenase type I (0.2% m/v) for
90 min (Gibco, Carlsbad, CA). Following digestion, cells
were filtered through a cell strainer (70 µM) and washed
twice in Gey’s Balanced Salt (GBSS) (Sigma-Aldrich, St.
Louis). Before initial seeding, cell samples from syno-
vium were exposed to trypan blue exclusion stain and
cell count and cell viability was determined using a he-
Bone marrow aspirates were obtained from the ster-
num of adult horses immediately after euthanasia for rea-
sons unrelated to the immune system. A bone marrow
aspiration needle (MD Tech Inc, Gainesville, FL) was
inserted into a sternebral body from the ventral aspect of
the sternum, and marrow was aspirated into a sterile,
heparin-flushed (American Pharmaceutical Partners Inc.,
Schaumburg, IL), 12-mL syringe. The procedure was
repeated until a minimum of 1 0 mL of bone marrow was
collected. Primary BMD-MSCs were isolated via cen-
trifugation of marrow specimens and cultured in a mono-
layer, as has been described [18]. Derived eqBMD-MSCs
(eqMSC) were confirmed as pluripotent by culturing in
controlled osteogenic, chondrogenic, and adipogenic me-
dia containing dexamethasone with ascorbate, recombi-
nant human transforming growth factor-I, and dex me-
thasone with insulin and indomethacin (Gibco, Grand
Island, NY) [19-21].
Dermal fibroblasts (DFb) were obtained via skin bi-
opsy as part of another equine study [20,22]. Full-thick-
ness skin tissue was harvested using a 5 mm diameter
biopsy punch from the pectoral region (10 - 12 punches
Copyright © 2012 SciRes. OJCB
per horse) from each of six adult horses. The dermal
layer was dissected from the epidermis under a micro-
scope, and DFbs were isolated by type-1 collagenase
digestion (GIBCO, Grand Island, NY) and cultured in
DMEM supplemented with L-glutamine (300 mg/mL),
penicillin (30 mg/mL), streptomycin (30 mg/mL), and
10% fetal bovine serum at 37˚C in a 5% CO2 atmosphere.
Synovial-derived mesenchymal stromal cells were pas-
saged a minimum of 4 times, but no more than 7 times.
Previous studies in the literature have demonstrated that
hSD-MSCs cells maintain a consistent phenotype be-
tween passages 3 and 8 [23,24]. Equine BMD- MSC and
Dfb were low passage ( <3 passages ).
2.2. Tissue Harvest of Host Mixed Immune Cells
Human spleens were selected as the host mixed cell
population for co-culture experiments. Approval for re-
ceiving portions of human spleens was granted by Life-
line of Ohio, an organ donor center in Columbus, Ohio.
Splenocytes were h arvested and p repared with h igh yield
and successfully grown in cell culture using methods
adapted from murine splenocyte culture [25,26]. Briefly,
tissue was trimmed into small pieces and thoroughly
minced using a syringe plunger inside of a nylon cell
strainer (70 µm) in a 35 mm cell culture plate. Cells were
digested using Ack lysing buffer (Gibco, Carlsbad, CA),
and the samples were subjected to consecutive washes in
ice cold PBS. Isolated splenocytes were immediately
allocated to cultures as described below in experimental
2.3. Adenoviral Transduction of Donor
Allogeneic Synovial-Derived Mesenchymal
Stromal Cells
Recombinant, E1-deficient, serotype-5 adenovirus vec-
tors containing the open reading frame segment of hu-
man GFP (AdGFP) under the control of the cytomega-
lovirus promoter were generated. Successful transduction
of AdGFP was verified in cell culture. Viral titer [infec-
tion units per mL, (IFU/mL)] was determined (Adeno-X
Rapid TiterKit; Clonetech, Mountain View, CA, USA)
and SD-MSCs were transduced at 100 multiplicities of
infection (MOI) or 1 × 102 infectious units per cell [19-
2.4. Cell Co-Culture Conditions and
Experimental Design
For the overall experimental design, three sources of do-
nor cells, hSD-MSC (allogeneic; Experiment 1) and eqMSC
and eqDFb (xenogeneic; Experiment 2), were seeded at
10,000 cells/cm2 onto 24-well cell culture plates at Day
–2 in 10% FBS containing 1% penicillin streptomycin in
DMEM (Invitrogen). Human SD-MSC cultures were
transduced with Ad-GFP on Day –1. For Experiments 1
and 2, isolated hSpl were obtained from 3 different hu-
man spleens for each experiment (6 spleens) and cultured
in triplicate alone or added to donor cultures at a 50:1
ratio or 500,000 hSpl/cm2 on Day 0. For Experiment 1,
hSpl were added to one of three co-culture immune chal-
lenges at Day 0; allogeneic hSD-MSC, allogeneic hSD-
MSC-GFP, or lipopolysaccharide stimulation (LPS 10
µg/mL, Sigma-Aldrich, St. Louis, MO). For Experiment
2, hSpl were assigned to one of three co-culture immune
challenges at Day 0; xenogeneic eqMSC, xenogeneic
eqDFb, or lipopolysacch aride stimulation (LPS 10µg/mL,
Sigma-Aldrich, St. Louis, MO). On Day –1, hSD-MSC
wells were assigned to receive adenoviral vector trans-
duction. Cells were transduced with AdGFP at 100 MOI
for 2 h in Gey’s Balanced Salt Solution. Effective trans-
duction methods with AdGFP have been validated in
previously published work [19]. On Day 0, prior to the
addition of the allogeneic hSpl cells, GFP expression was
confirmed by fluorescent microscopy and recorded. In
addition to co-cultures, hSpl were cultured alone. At day
0, hSpl were removed for cellular viability staining and
flow cytometry analysis approximately two hours after
co-cultures were established to serve as a baseline im-
mune status. At Days 0 and 6, hSpl were harvested for
cell surface marker content as measured by flow cytome-
2.5. Flow Cytometry, Cell Numbers, and Cell
Flow cytometry (C-Flow Plus, Accuri, Ann Arbor, MI)
was performed on hSpl subjected to extracellular staining
to determine the proportion of cells pos itively-stained for
binding of CD3, CD4, or CD8 antibodies. Human mono-
clonal antibodies for each immune cell subtype (FITC-
conjugated-anti-CD3, APC-conjugated anti-CD4, and PE-
conjugated anti-CD8, R&D Systems, Minneapolis, MN)
were used to assess the potential formation of immune
cell types. At Days 0 and 6, we investigated formation of
CD3+, CD4+, and CD8+ cells; an unstained control and an
antibody control for each respective antibody were used
on each of flow cytometry analysis.
Cell number and viability for both hSD-MSC and
hSD-MSC/GFP were determined on Day 6. Cells were
trypsinized, washed, and subjected to a trypan blue ex-
clusion stain. Cells were counted in a hemacyometer; the
number of live cells was recorded.
2.6. Statistical Analysis
For co-culture experiments, cells isolated from one hu-
man joint (hSD-MSC or hSD-MSC/GFP or one animal
(eqMSC or eqDFb) and an individual spleen (hSpl) were
Copyright © 2012 SciRes. OJCB
considered an n of 1. Data for quantitative outcomes of
hSpl CD3+, CD4+, CD8+ number and proportion, as well
as donor cell number and viability were presented as
means ± standard error of the mean (SEM). Two factor
analysis of variance (ANOVA) for Day (0 and 6) and
hSpl condition (alone, hSD-MSC, hSD-MSC-GFP; or
alone, eqMSC, eqDFb, LPS) was performed for hSpl
number, donor cell number, and hSpl or donor cell vi-
ability. All analyses were performed using a comer-
cially available statistical software package (Statistical
Analysis Software, SAS 9.1). Statistical significance was
accepted at p < 0.05
3. Results
Human spleens were obtained on ice, and splenoctyes
were harvested with initial high hSpl number and viabil-
ity (>95%) (Figure 1(A)) and co-cultured with donor
cells at Day 0 (Figure 1(B)). The proportion of gated
viable hSpl on the Day 0 of cultur e measurement by flow
cytometry was in the range of 65% - 74% (Figures 2(a)
and 2(b)). The proportion of CD+ hSpl (CD3+, CD8+,
plus CD4+) was <15% of total gated cells on Day 0 cul-
ture measurement by flow cytometry (Figures 3 and 4).
Culture of hSpl alone resu lted in a significan t decrease in
hSpl numbers and viability to an average of approxi-
mately 31% by Day 6 as measured by flow cytometry
(Figures 2(a) and 2( b) ). Co-culture of hSpl with alloge-
neic or xenogeneic donor cells did not further decrease
hSpl numbers (Figures 2(a) and 2(b)). Both allogeneic
and xenogeneic donor cells proliferated in cell co -culture
with hSpl (Figure 5) and were of normal morphology
with high efficiency of GFP expression demonstrated in
hSD-MSC-GFP cells for the entire 6 days. (Figure 1(B),
insert) Synovial-derived mesenchymal stromal cells were
successfully transduced (hSD-MSC-GFP) 24h after trans-
duction with AdGFP at 100 multiplicities of infection
(100 MOI). (Day 0).
In the allogeneic co-culture Experiment 1, the propor-
tion of CD3+ and CD8+ hSpl was significantly greater on
Day 6 (4-fo ld and 5-fold, respectively) r egardless of cul-
ture condition as compared to Day 0. (Figures 3(a) and 3
(b)) On Day 6, hSpl cultured in LPS had a greater
proportion o f CD3+ hSpl than hSpl cultured alone. There
was no change in the proportion of CD4+ hSpl on Day 6
versus 0 (Figure 3(c)).
In the xenogeneic co-culture Experiment 2, the propor-
tion of CD3+ and CD8+ hSpl was significantly greater on
Day 6 (3-fo ld and 6-fold, respectively) r egardless of cul-
ture condition as compared to Day 6 as in the allogeneic
co-culture experiment and compared to Day 0. (Figures
4(a) and 4(b)) On Day 6, hSpl co-cultured with eqDFb
had a lesser proportion of CD8+ hSpl than hSpl cultured
alone. There was no change in the proportion of CD4+
Figure 1. (A) Photomicrograph (100× magnification) of a
hemacytometer containing a cell dilution of human spleno-
cytes stained with trypan blue stain after being digested
from a human spleen. Viable hSpl are yellow and circular,
and non-viable human splenocytes (hSpl) are blue and py k-
notic. Each spleen sample that was digested had greater
than 95% living cells prior to the start of the co-culture
experiments (Day 0); (B) Photomicrograph (100× magnfi-
cation) of a co-culture containing hSpl (small circ ular cells)
and human synovial-derived mesenchymal stromal cells
(hSD-MSC) 100x magnification. Human SD-MSC trans-
duced with AdGFP at 100 infectious Ad particles per cell,
had >95% expression of GFP under fluore scent microscopy
at 24 hours (insert; 200× magnification).
cells between days (Figure 4(c)).
4. Discussion
This study provided evid ence that, in a short-term culture
environment, hSpl undergo a natural selection and adap-
tation to cell culture conditions. In the allogeneic or
xenogenic co-culture conditions, no particular evaluated
cell source (allogeneic synovial or xenogeneic bone-
marrow, or skin) or donor source (human allogeneic or
equine xenogeneic) promoted survival, inhibited death,
or promoted death of the lymphocyte cell fraction of hSpl.
In addition, these data show ed that neither allogene ic nor
Copyright © 2012 SciRes. OJCB
Figure 2. Number of gated (viable) hSpl on Day 0 and Day 6
in the allogeneic Experiment 1 (Panel (a)) and the xenoge-
neic Experiment 2 (Panel (b)). Abbreviations: hSpl = human
splenocytes, hSpl + hSD – MSC = human splenoctyes co-
cultured with human synovial-derived mesenchymal stromal
cells, hSpl + hSD – MSC/GFP = human splenocytes co-
cultured with human synovial-derived mesenchymal stromal
cells transduced with AdGFP, hSpl + eqMSC = human
splenoctyes co-cultured with equine mesenchymal stromal
cells, hSpl + eqDFb = human splenoc yte s co-culture d equine
dermal fibroblasts, hSpl + LPS = human splenocytes co-
cultured with lipopolysaccharide (LPS, 10 g/mL). Data are
mean SEM. The bracket (p < 0.05) denotes a statistical
difference at Day 6 compared to Day 0.
xenogeneic cells elicited a lymphocytic/dendritic cell
selection and no donor source promoted proliferation, at
least in vitro and for this short duration of primary expo-
sure. The short survival of immune cells in culture (Spl
or peripheral blood monocytes) limit in vitro studies.
However, within these limitations, the evidence sug-
gested that allogeneic or xenogeneic cells may be im-
munotolerated, at least initially, in a host environment.
Our study was a first to investigate the immunoresponse
of a mixed population of hSpl to different tissue sources
of both allogeneic and xenogeneic origin cells and as-
sessed three differing mechanisms of immune challenge,
including 1) cellular antig ens, 2) aden oviral antigens, and
3) lipopolysaccharide (LPS), a mediator of the inflam-
matory immune reaction. We were unable to document
Figure 3. Human Human splenocytes (hSpl) co-cultured with
allogeneic synovial-derived mesenchymal stromal cells (hSD-
MSC) and subjected to extracelluar staining using antibod-
ies specific for CD3, CD4, or CD8. The proportion of posi-
tive cells for each antibody was determined by flow cy-
tometry. (a) CD3+ hSpl on Day 0 and Day 6. (b) CD8+ hSpl
on Day 0 and Day 6. (c) CD4+ hSpl on Day 0 and Day 6.
Abbreviations: hSpl = human splenocytes, hSpl + hSD –
MSC = human splenoctyes co-cultured with human syno-
vial-derived mesenchy mal stromal cells, hSpl + hSD – M SC/
GFP = human splenocytes co-cultured with human syno-
vial-derived mesenchymal stromal cells transduced with
AdGFP, hSpl + LPS = human splenocytes co-cultured with
lipopolysaccharide (LPS, 10 g/mL). Data are mean SEM.
The bracket (p < 0.05) denotes a statistical difference at Day
6 compared to Day 0. # denotes an unexplained difference
compared to other groups at Day 0. * denotes that hSpl +
LPS at Day 6 is significantly greater than Day 0.
significant activation of CD+ immune cells or death of
immune cells or death of allogeneic or xenogeneic donor
Copyright © 2012 SciRes. OJCB
Figure 4. Human splenocytes (hSpl) co-cultured with xeno-
geneic equine bone-marrow derived mesenchymal stromal
cells (eqMSC) or dermal fibroblasts (eqDFb) and subjected
to extracelluar staining using antibodies specific for CD3,
CD4, or CD8. The proportion of positive cells for each an-
tibody was determined by flow cytometry. (a) CD3+ hSpl on
Day 0 and Day 6. (b) CD8+ hSpl on Day 0 and Day 6. (c)
CD4+ hSpl on Day 0 and Day 6. Abbreviations: hSpl = hu-
man splenocytes, hSpl + eqMSC = human splenoctyes co-
cultured with equine mesenchymal stromal cells, hSpl +
eqDFb = human splenocytes co-cultured equine dermal fi-
broblasts, hSpl + LPS = human splenocytes co-cultured
with lipopolysaccharide (LPS, 10 g/mL). Data are mean
SEM. The bracket (p < 0.05) denotes a statistical difference
at Day 6 compared to Day 0. *denotes that hSpl + EqDFb
was significantly lower than other groups at Day 6.
cells. Dermal fibroblasts have not been stated to be im-
munoprivileged as has been claimed for the synovial
fibroblast or bone-marrow derived mesenchymal pro-
Figure 5. Cell number of human synovial-derived mesen-
chymal stromal cells (hSD-MSC) and human synovial-de-
rived mesenchymal stromal cells transduced with adenovi-
rus containing green fluorescent protein (hSD – MSC/GFP)
on Day 0 and Day 6 as determined by hemacytometer live
cell count following a trypan blue exclusion staining Data
are mean SEM. The bracket (p < 0.05) denotes a statisti-
cal difference at Day 6 compared to Day 0.
genitor and stromal cells, but our data suggested that
xenogeneic dermal fibroblasts may be immunosuppres-
sive to CD8+ cells and would warrant further investiga-
Importantly, further experiments in vivo would be ne-
cessary to more critically assess immunotolerance, to
include longer exposure to antigens, and evaluate a sec-
ond exposure to elucidate the cellular contributions and
anamnestic response to isolated allogeneic or xenogeneic
cells. To our knowledge, our splenocyte co-culture sys-
tem is novel and contributed initial information on toler-
ance of these mixed immune cells to donor cells on short
term exposure and supports previous publications claim-
ing allogeneic juvenile chondroctyes exhibit immunotol-
erance [27]. Previous work has also provided evidence
that synovial cells have immune privilege an d will not be
rejected by a host organism [28,29]. Some studies sup-
ported the tolerance of allo- or xenotransplants for tissue
healing [30,31]. An additional study showed that xeno-
geneic chondrocytes can successfully repair full-thick-
ness cartilage defects [32]. However, in a recent 2010
study, xenotransplantation of porcine chondrocytes into
rabbits was unsuccessful for long term cartilage healing
Our study provided a unique contribution to the rela-
tively limited body of work in the literature using this
type of co-culture model. Our data is in agreement with
previous literature suggesting that mesenchymal stromal
cells maybe immunotolerated or immunoprivileged [15,
17,33]. Findings in our human allogeneic experiment
demonstrated that no detectable immune activation was
prompted; however, an immunosuppression was not ob-
served as in other studies. In cell culture, MSCs have
also been able to suppress T cell proliferation [13], sug-
Copyright © 2012 SciRes. OJCB
gesting MSC, including stem cells, are not only immu-
notolerated, but may be immunosuppressive [15]. Mac-
cario and colleagues observed a decrease in the forma-
tion of CD8+ T cells and natural killer lymphocytes in
favor of an expansion of CD4+ lymphocytes in response
to allogeneic MSC [17]. However, in our xenogeneic
experiments, the evidence suggested that equine dermal
fibroblasts may play a role in the inhibition of an im-
mune response. Our data showed less of an increase in
CD8+ cells after exposure to xenogeneic dermal fibro-
blasts. Additional evidence has shown that fibroblasts,
both autologous or allogeneic cells, resulted in no in-
crease in T-cell proliferation; however, this effect re-
quired a modification of CD80 expression on the surface
of the cell [12]. Data is accumulating that suggests an
immunotolerance to selected allogeneic and xenogeneic
isolated cells.
Our data showed variability in human splenocyte ini-
tial cell proportions from human to human, demonstrated
as wide standard deviations in isolated hSpl numbers and
percentages of CD+ cells. The variability of splenocyte
populations under our conditions are likely explained by
differences in age, gender, and hematological parameters
of our human spleen donors. Human spleens were ac-
quired from an organ donor center, without exclusion for
age, gender, or cause of death. Previous health history of
the human spleen donor s may have altered the propor tion
of various cell types in the spleen including mature lym-
phocytes, T-helper cells, dendritic cells, or natural killer/
cytotoxic T cells. The heterogeneous human population
providing hSpl sources probably added variability to our
co-culture condition responses, but represent a typical
recipient population of humans. Despite the variability
inherent in using hSpl from multiple human donors, we
were able to demonstrate the natural selection and adap-
tation of hSpl to the cell culture environment in a short-
term cell culture study, minimal lymphocyte activation
by allogeneic and xenogeneic cell exposure and the abil-
ity of donor allogeneic and xenogeneic cells to thrive in
direct contact with a mixed population of immune cells.
The clinical applicability of our work extends to po-
tential cell-based strategies for the repair of human tissue.
Allogeneic or xenogeneic cell sources could serve as a
powerful clinical tool to enhance current medical prac-
tices that promote healing. Finding a potential cell- or
tissue-based treatment for cartilage is dependent upon
further understanding of the immunoresponse of cells,
including synovial lining cells or chondrocytes. Further
characterizing the immunoresponse may enable potential
molecular or other cellular modifications to enable spe-
cific cells to “tolerate” an immune response or modify
the native immune response. It is possible that specific
cells could be engineered to modify the acute immune
response in vivo, ahead of or in conjunction cellular in-
jection or transplantation
5. Conclusions
We uniquely investigated the immunoresponse of a mixed
population of hSpl to three different mechanisms of al-
logeneic or xenogeneic challenge: 1) cellular antigens 2)
cellular/adenoviral antigens and 3) lipopolysaccharide
(LPS). In our in vitro model, neither allogeneic nor xeno-
geneic cells elicited a lymphocyte/dendritic cell immu-
noresponse. Our study provided evidence that, in a short-
term culture environment, hSpl undergo a natural selec-
tion and adap tation to cell culture conditions. In hSpl co-
cultures with hSD-MSC, hSD-MSC-GFP, eqDFb, or
eqMSC, CD3+ and CD8+ hSpl increased in proportion
and decreased in number with time in culture, but not
with exposure to allogeneic or xenogeneic cell antigens.
The evidence suggested that allogeneic or xenogeneic
may be immunotolerated in the short term and upon first
exposure by human recipients. Further studies to charac-
terize the long-term response in vivo and the anemnestic
response is warranted
6. Acknowledgements
Dr. Jump was supported by a Fellowship at The Ohio
State University from the Sports Medicine Center. Dr.
Bertone was supported by NIH/NIAMS grant number
K08AR4920101. The work was supported by the True-
man Endowment. We would like to acknowledge and
thank Dr. Akikazu Ishihara for his assistance in the sta-
tistical and data analysis and for proofreading and editing
our mansucript. Additionally, we offer a special thanks to
Dr. Prosper Boyaka and his laboratory personnel, Dr.
Junbae Jee and Haley Steiner, for assistance with techni-
cal guidance and methodological expertise in performing
flow cytometry.
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