Page 95 - Power of Stem Cells- arthritis and regeneration

Liu et al. Arthritis Research & Therapy 2010, 12:R210 Page 4 of 13
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For evaluation of delayed-type hypersensitivity (DTH) cells were stained for anti-IFN-g-PE, anti-IL-4-PE or
reactivity, CIA mice treated with UC-MSCs or not were anti-IL-17-PE in FACS buffer containing 0.1% saponin.
intradermally injected with 10 μgCII/10 μlPBS in the An appropriate isotype-matched control antibody was
right ear and with 10 μl PBS in the left ear. Ear swelling used in all FACS analyses. All antibodies were from BD
was measured 48 hours later with a spring-loaded Pharmingen except anti-Foxp3 (eBioscience). Cells were
micrometer. analyzed on a FACS Calibur flow cytometer using Cell
Quest software (Becton, Dickinson and Company).
Histologic analysis
Formalin-fixed limbs were decalcified and paraffin- Statistical analysis
embedded using standard histologic techniques. Serial Data were presented as mean ± S.D. The difference
4 μm sections were cut and stained with hematoxylin between treatment and control groups was analyzed by
and eosin to examine morphologic features and assess Mann-Whitney U test. P < 0.05 was considered significant.
the histologic arthritis score. Histopathologic changes
are scored using the following parameters. Sections were Results
analyzed microscopically for the degree of inflammation Expansion of UC-MSCs in vitro
and for cartilage and bone destruction according to the The UC-MSCs were successfully isolated and expanded
method reported previously [26], using the following from all the umbilical cords. They had a fibroblast-like
scale: 0 = normal synovium, 1 = synovial membrane morphology, uniformly negative for CD14, CD45, CD34
hypertrophy and cell infiltrates, 2 = pannus and cartilage and HLA-DR, but positive for CD44, CD73, CD90 and
erosion, 3 = major erosion of cartilage and subchondral CD29 (Figure 1a, b). Functionally, they were capable of dif-
bone, and 4 = loss of joint integrity and ankylosis. Each ferentiating into adipocytes and osteocytes (Figure 1c, d).
joint was scored separately by two individuals unaware
of the treatment protocol. UC-MSCs inhibited proliferation of FLSs from RA patients
To trace the migration of transplanted cells in vivo, The FLSs are resident cells of synovial joints, recog-
analysis with mAb against human nuclei (Chemicon nized to play an important role in inflammation and
International, Temecula, CA, USA) was performed joint destruction of RA. Therefore, we attempted to
following the manufacturer’s instructions to detect determine the effects of UC-MSCs on the FLSs derived
UC-MSCs in heart, kidney, spleen and joints of mice from RA patients. The FLSs isolated from RA patients
treated with UC-MSCs. responded positively to TNF-a (20 ng/ml) when com-
pared with control (11,440 ± 2,452 vs. 1,985 ± 516,
Cytokine quantification P = 0.000). The UC-MSCs significantly inhibited the
After 72 hours of co-culture with or without TNF-a or proliferation of TNF-a-stimulated-FLSs in the cell-to-
PHA stimulation, fresh supernatant was collected. cell contact and the transwell system, and the effect
Quantitative analyses of IL-6 production were per- was dose dependent (Figure 2a). Moreover, such inhi-
formed by enzyme-linked immunosorbent assay (ELISA) bitory effects were profound even when UC-MSCs
using commercially available kits (R&D). TNF-a and were added on the fourth day in an experiment of five-
Matrix metalloproteinase 9 (MMP9) quantification were day coculture (11,110 ± 2,142 vs. 5,379 ± 1,435, P =
performed on the Luminex-100 system, and the R&D 0.000, Figure 2b).
Fluorokine MAP Human Base Kit A or Human MMP
MultiAnalyte Profiling Base Kit (R&D) was used. Super- Soluble factors involved in the suppressive effect of
natants of UC-MSCs, FLSs and T cells that were cul- UC-MSCs on the proliferation of FLSs from RA patients
tured alone served as controls. Cytokine and chemokine Since IDO, NO, PGE2, IL-10 and TGF-b1 are key factors
levels in the serum of mice with CIA were determined in MSCs-mediated inhibition [27-30], co-culture experi-
by sandwich ELISA using capture/biotinylated detection ments were performed using the corresponding inhibi-
antibodies obtained from BD PharMingen. tors. They included 1-MT (1 mM), an inhibitor of IDO
enzymatic activity, INDO (5 μM), an inhibitor of PGE2
Flow cytometric analysis synthesis, L-NAME (1 mM), a specific inhibitor of NO
Tregs were stained with anti-CD4-FITC. Then, cells synthase, anti-TGF-b1 monoclonal antibody (10 μg/ml)
were fixedand permeabilizedbyFix/Permbuffer and anti-IL-10 monoclonal antibody (10 μg/ml). As
(eBioscience, San Diego, CA, USA) and stained for anti- showninFigure2c, TNF-a-mediated FLSs proliferation
forkhead box P3 (Foxp3)-PE. Mice Th1, Th2 or Th17 could be sufficiently restored by anti-IL-10, 1-MT and
cells in spleen were stained for anti-CD4-APC, then anti-TGF-b1, respectively, suggesting that those soluble
washed with FACS buffer (PBS plus 1% BSA) and fixed factors were the key mediators in UC-MSCs-mediated
in PBS containing 2% paraformaldehyde. Subsequently, inhibition. However, IDO and PGE2 were not found