Supplementary materials and methods
Flow cytometry
bEnd.3 cells were cultured on 60 mm tissue culture dishs in 10% FBS, high glucose
DMEM and treated with 20 ng/ml recombinant mouse TNFα (Peprotech, Inc., Rocky Hill, NJ)
for 4 and 16 h. The TNFα-treated bEnd.3 cells were detached from cell culture dish with cell
dissociation buffer (Versene, Invitrogen), incubated with 1% BSA in DMEM for 30 min at room
temperature, followed by 2 µg/ml goat anti-VCAM-1 or MAdCAM antibody (R&D systems,
Inc., Minneapolis, MN) on ice, and washed in DMEM twice. Cells were then incubated in 2
µg/ml of Alexa 488 donkey anti-goat IgG antibody. 10,000 events were analyzed by flow
cytometry (Coulter EPICS XL-MCL) with a 488 nm argon ion laser.
Cell coating and binding assay on mouse brain endothelial cells (bEnd.3)
Palmitated protein G (PPG) was derivitized as described previously by reacting
recombinant protein G (Pierce, Rockford, IL) and N-hydroxysuccinimide ester of palmitic acid
(Sigma, St. Louis, MO) followed by purification using Sephadex G-25 (Pharmacia, Piscataway,
NJ). Cell coating and cell binding assay was conducted as previously described [6]. Mouse brain
endothelia cells (bEnd.3; ATCC) were cultured at 37⁰C, 5% CO2 in high glucose DMEM
supplemented with 10% FBS, penicillin and streptomycin.
For cell attachment studies, 80 µl of 5 X 105 cells/ml of bEnd.3 were seeded onto
thermanox® plastic coverslips (13 mm round slips, Ted Pella, Inc, Rochester, NY), transferred
into a 24-well microplate, and cultured for 1 day prior to adherence assays. bEnd.3-seeded
coverslips were blocked with 1% BSA in DMEM in a 37⁰C, 5% CO2 incubator for 30 min, and
then incubated for 30 min with 80 µl of 5 X 105 cells/ml, followed by washing with DMEM
three times and counted with fluorescent microscope.
Incorporated Antibody release test from cell membrane
Cells were coated with PPG and human FITC-conjugated IgG according to standard
methods, and the fluorescent intensity measured using flow cytometry. To avoid loss of signal
due to cell spreading and the need to trypsinize, Immediately after coating, aliquots of 1.0 x 10^6
cells were placed in 1 ml aliquots of complete medium (10% FBS in DMEM) containing 2%
alginate. The cell/alginate mixture was then injected into complete medium containing 50 mM
calcium chloride and allowed to gel for 10 minutes at room temperature, the medium was then
exchanged to complete medium and the cells incubated at 37 degrees. For flow cytometric
analysis, the cells were incubated in complete medium containing 2 mM EGTA to release the
cells from the alginate.
Supplementary data
Figure S1. Preparation of Fluc-MSC. Murine MSCs (BMC9 cells) were transduced with a triplefusion reporter, fluc-mrfp-ttk (encoding firefly luciferase, monomeric red fluorescent protein
(RFP), and truncated herpes simplex virus type 1 sr39 thymidine kinase) by use of a lentiviral
vector showed, and showed 96.7% positive for RFP expression by flow cytometry at the
University of Pittsburgh) (Fig. S1-a).
Fluc-MSCs plated onto 96 well plates and assayed for
luciferase activity (Fig. S1-b) showed a linear correlation between cell numbers and luciferase
activity assay (Fig. S1-c).
Figure S2. MSC localization in lung and colon 2 hours post-injection. MSCs labeled with FarRed dye were injected into IBD treated mice and the mice harvested after 2 hr (not sure of this
time), tissues frozen, sectioned and immunostained. Arrows point to MSCs (red) and green
indicates immunostaining with for either laminin (A, B) to highlight basement membrane from
colon or von Willibrand factor (C, D) to label endothelial cells.
The MSC in A looks to be
inside of a small capillary, the MSC in D appear to be lining a large blood vessel or air space,
while the MSCs observed in B and C are localized to regions within the mesenchyme. C =
capillary; BV = blood vessel.
Figure S3. Quantification of antibody incorporation on MSCs. An FITC-labeled human IgG
standard curve was produced (a), and the fluorescent intensity of FITC-antibody coated-MSCs
was graphed vs cell number (b) and the amount of incorporated antibody on MSCs was
calculated by comparing fluorescent intensity from antibody-coated MSCs with that of standard
curve (c).
Figure S4. Schematic diagram of effective targeting Ab-MSCs to inflammatory sites. The
enhanced therapeutic effects are mainly mediated by increased MSC delivery to sites of
inflammation and also derived from blocking T cells adhesion to endothelium by locally released
antibody from MSC membrane.
Figure S5. MSC binding to bEnd.3. Cell binding assay of MSCs on bEnd.3 cells showed a 8fold increase in cell binding of AbVCAM-1-MSCs compared to MSCs only (* p<0.05) and PPGMSCs (** p<0.05) and a 3-fold compared to untreated and TNFα treated bEnd.3 cells († p<0.05).
Figure S6. Addressin expression on bEnd.3 cells. Flow cytometric results showing VCAM-1 (a)
and MAdCAM (b) expression in bEnd.3 cells, a mouse endothelial cell line, after 20 ng/ml of
TNFα treatment for 4 h or 16 h; VCAM-1 expression was upregulated while MAdCAM
expression was unchanged. (c) Quantification of addressin expression based on fluorescent
intensity from flow cytometric results. bEnd.3 cells showed 4-times higher expression than
untreated cells by TNFα treatment(* p<0.05): while MAdCAM expression was unchanged; (N=3,
Student’s t-test).
Figure S7. Antibody release test from cell membrane. Cells were coated with FITC-labeled
human IgG and after each incubation time at 37⁰C, FITC fluorescent intensity on cells was
analyzed by flow cytometry. The results show a FITC-IgG half-life of 3.4 hr; error bars show
standard deviation; N = 4.