Supplementary methods
Liver tissue and hMSC
Tissue was obtained with ethical approval and consent from the liver unit at
Queen Elizabeth Hospital Birmingham, UK. Hepatectomy specimens were
obtained from patients with primary biliary cirrhosis (PBC) or alcoholic liver
disease (ALD). Donor tissue surplus to requirements served as normal
controls (NL). hMSC from normal healthy donors were purchased from Lonza
Group Ltd, and cultured in MSCGMTM Mesenchymal Stem Cell Growth
Medium. Cells were designated P1 upon arrival and were used at P4. All
experiments were repeated with multiple hMSC/liver donors.
Human Hepatic Sinusoidal Endothelial Cell (HSEC) Isolation
HSEC were isolated from 30g human liver tissue as described previously.1
HSEC were cultured in rat-tail collagen coated flasks in human basal
endothelial media (Invitrogen) containing 10% heat inactivated human serum
(HD Supplies, UK), glutamine, penicillin and streptomycin (292µg/ml,
100U/ml, 100µg/ml respectively, all Sigma, UK), hepatocyte growth factor
(HGF) and vascular endothelial growth factor (VEGF) (both at 10ng/ml
Peprotech).
Differentiation Assays
hMSC multipotency was demonstrated by tri-lineage differentiation to
adipocytes, osteoblasts and chondrocytes using specific induction media from
Lonza under manufacturer-specified conditions. Calcium production during
osteoblastic differentiation was assayed using a Calcium Liquicolor Kit
(Stanbio Laboratory). Adipogenic differentiation efficiency was assessed by
Oil Red O staining of the lipid formed by the cells according to standard
protocols. Chondrogenic differentiation was measured by immunostaining
using a Collagen II antibody.
Flow cytometric analysis of hMSCs
To ensure the criterion for the definition of hMSCs, set by the Mesenchymal
and Tissue Stem Cell Committee of the International Society for Cellular
Therapy (ISCT)2 was met, hMSCs surface receptor expression was
determined cytometrically using a hMSC Phenotyping kit (Miltenyi Biotec). To
characterise hMSC integrin, chemokine receptor and CD44 expression, the
hMSCs were also analysed by flow cytometry. hMSC were removed from the
flask using TrypLE (Invitrogen) and resuspended in FACs buffer consisting of
PBS plus 1% Foetal Calf Serum (FCS). 1x105 cells were transferred to FACs
tubes and the recommended concentration of fluorescently-conjugated
antibody was added and incubated for 20 minutes. Isotype matched
antibodies determined background staining levels. See Supplementary
Table 1 for antibodies used. Data collected was analyzed (Dako Cytomation
CyAn-ADP) using Summit software (Dako Cytomation, UK).
Modified Stamper-Woodruff/static adhesion assay on liver tissue
sections
10µm sections were cut from snap frozen tissue. 1x105 hMSC were used per
section. For blocking experiments, hMSC or sections (or combinations) were
incubated with pre-determined optimal concentrations of blocking antibodies
or isotype controls (Supplementary Table 1). To block G-protein coupled
signalling, hMSC were incubated with a pre-determined optimal concentration
of pertussis toxin (PTX; 100ng/ml; Sigma), shown to be effective upon other
cell types in our laboratory,3, 4 for 30mins at 37°C. hMSC were incubated
statically on sections for 30mins at room temperature, washed with
phosphate-buffered saline (PBS) to remove non-adherent cells and acetone
fixed. Some sections were immunostained for CD31 (Supplementary Table 1).
Mayers Haematoxylin was used to identify the cells, which appear dark
compared to the rest of the section and are in a different focus-plane.
Adherent cells in 10 fields of view of both the parenchyma and portal tracts
were counted on each section (magnification x200; Axioskop 40 Zeiss, UK).
Sirius red staining of sections
Sections were hydrated with distilled water then placed into 0.5%
phosphomolybdic (PMA) for 5mins, before staining with 0.1% Sirius red
(Direct red 80; % w/v in saturated picric acid) for 2hours. The slides were
dipped in 0.01M HCL, before washing in distilled water. The sections were
counterstained with Meyers Haematoxylin and mounted.
Immunohistochemistry
Sections that have previously been through the static adhesion assay were
stained for CD31. Briefly, sections were incubated with CD31 antibody for
30minutes, washed to remove unbound antibody, detected with HRPconjugated secondary antibody and ImmPACT NovaRed substrate
(Vectorlabs). Mayers Haematoxylin was used to identify hMSCs as per static
adhesion assay method.
Immunofluorescence
5µm frozen sections from the in vivo experiments were used for
immunostaining. After equilibrating to room temperature, sections were
blocked for 5 mins with 10% serum. Next the sections were incubated for 30
mins with primary antibody (Supplementary Table 1) at appropriate
concentration. After washing, sections were incubated with fluorescentlyconjugated secondary antibody followed by DAPI as a nuclear counterstain.
Sections were mounted in fluorescent mountant (DAKO), before viewing on a
Zeiss LSM 510 UV confocal microscope.
Chemotaxis assay
Migration of hMSCs was assessed using modified Boyden chamber as
previously described.15 The optimal determined concentration of soluble
recombinant chemokines (CXCL12/SDF-1α; 10ng/ml, CCL22/MDC; 500ng/ml,
CCL17/TARC; 100ng/ml, CCL8/MCP2; 100ng/ml, CCL4/MIP1β; 500ng/nl,
CCL5/RANTES; 5-500ng/ml, CXCL11/ITAC; 500ng/ml) or control buffer
(media alone) were placed into 6 replicate lower wells, separated by a
polycarbonate membrane (8µm pores; Whatman International) from the
hMSCs (1.5x106 cells/ml) in 50µl aliquots in the upper wells. The chamber
was incubated overnight at 37°C. Non-migrated cells were scraped from the
upper side of the membrane and the migrated cells on the lower surface were
fixed in methanol and counterstained. Migrated cells were counted in 2 fields
per well (200X magnification) and the average for the six replicates
calculated. The chemotactic index (CI) or ratio of cells migrated in the
presence versus absence of chemoattractant was determined for each
optimal concentration of chemokine.
Fluorescence intravital microscopy (IVM)
IR injury was induced for 90minutes as previously described.5 The left lobe of
the liver was exteriorised and one region of interest containing both a postsinusoidal venule and surrounding vessels was identified before systemic
introduction of 1x106 CFSE-labelled hMSCs. This area was monitored
throughout the experiment in order to determine adhesion dynamics.
Recordings were made of the pre-selected area every 5 minutes from
introduction of the cells at 30minutes post-reperfusion for 90minutes. At the
end of the experiment, an additional six fields of view were selected to ensure
that cell kinetics observed in the initial field were representative of events
occurring in the entire liver. Images were analysed off-line (Slidebook;
Intelligent Imaging Innovations, Denver, Colorado, USA).
Determination of hMSC size
Images of hMSCs in culture immediately after trypsinisation were taken and
the size determined using a graticule (Pyser-SGI LTD). Size of hepaticresident hMSCs was determined using a scale bar and measuring diameter in
a minimum of 10 fields of view.
Statistical analysis
Data were deemed normally distributed, then analysed by student T-test to
determine statistical significance. Data are expressed as mean with standard
errors. Bonferroni correction was used where multiple comparisons were
undertaken. p=0.05 was considered significant and denoted by *.
1.
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