Supplementary Data for “Factors Determining Successful Engraftment of Hepatocytes
and Susceptibility to Hepatitis B and C Virus Infection in uPA-SCID Mice.”
MATERIALS&METHODS
Liver tissue specimens, histochemistry and immunohistochemistry
Snap frozen or formalin-fixed liver samples from transplanted animals were processed using
standard techniques. Quantification of the fraction of liver parenchyma occupied by donor
human hepatocytes was performed on H&E-stained slides as described previously (2).
Four µm-thick cryostat sections were cut from the frozen liver tissue, fixed in acetone for 10
minutes and incubated with a monoclonal antibody against mouse H2-Kb (BD Pharmingen,
dilution 1/50) for 30 min. Subsequently the slides were incubated with peroxidase-labeled
rabbit
anti-mouse
(dilution
1/50)
and
then
peroxidase-labeled
swine
anti-rabbit
immunoglobulin (dilution 1/100) (both from Dako). The red reaction product was developed
using amino-ethyl-carbazole and sections were counterstained with hematoxylin.
RESULTS.
Kinetics of the repopulation of uPA+/+SCID liver by human versus mouse hepatocytes.
Using criteria described by Sandgren (1), donor mouse hepatocytes could be distinguished
from diseased parenchyma at all examined time points (Table 2). The discriminating
characteristics of the healthy donor hepatocytes were their ample eosinophilic and somewhat
flocculent cytoplasm, whereas transgene-expressing hepatocytes were small and showed
discretely vacuolated cytoplasm (Supplementary Fig 1). Donor mouse hepatocytes were
discernable from red foci hepatocytes based on the absence of nuclear pleiomorphism (1).
These findings were confirmed by immunohistochemical staining for H2-Kb on frozen tissue,
which highlighted the H2-Kb positive donor cells against the background of H2-Kb negative
recipient hepatocytes (Supplementary Fig 3). As reported previously, human hepatocytes
were easily distinguishable from mouse hepatocytes based on their larger size and pale
cytoplasm (2) (Supplementary Fig 4).
SUPPLEMENTARY FIGURE LEGENDS.
Supplementary Figure 1. Categories of normalized human albumin plasma levels as
semi-quantitative measures of human hepatocyte engraftment efficacy in uPA+/+SCID
mice. The plasma albumin concentration was normalized for the timepoint in weeks after
transplantation at which the plasma sample was obtained (see text). Arbitrary categories of the
normalized albumin levels are shown to visually and semi-quantitatively compare
engraftment efficacy for each donor cell type. Percentage of animals sampled was used as the
dependent variable to correct for the different number of animals transplanted with each type
of cells. A. Most sacrificed animals had an absent or marginal human hepatocyte graft take. B.
In non-sacrificed animals graft take was minimal after P3 cell transplantations, compared to
all other donor cell types.
Supplementary Figure 2.
Plasma human albumin levels from animals transplanted with cryopreserved hepatocytes from
a single donor correlate with blood sampling time point after transplantation. (P2 cell donor,
n=130 blood sampled animals, r=0,501, P<0,001).
Supplementary Figure 3.
Immunohistochemistry for H2-Kb on frozen sections highlights the donor mouse hepatocytes
with membraneous positivity against the background of negative diseased liver cells, both at
the stages of single cell infiltration (left) and cluster formation (right). The donor cells are
larger in comparison with the hepatocytes of the diseased parenchyma of the recipient. The
latter also display a more vacuolated cytoplasm (original magnifications x 400).
Supplementary Figure 4.
Well-delineated clusters of human (left) and donor mouse hepatocytes (right), marked with
white arrows, are easily distinguishable from diseased recipient parenchyma by the cell sizes
and cytoplasmic features. Healthy donor mouse hepatocytes are characterized by an ample
eosinophilic and somewhat flocculent cytoplasm, whereas transgene-expressing hepatocytes
are small and show a discretely vacuolated cytoplasm. Donor mouse hepatocytes were
discernable from red foci hepatocytes based on the absence of nuclear pleiomorphism, as
described by Sandgren (1). Human hepatocytes were easily distinguishable from mouse
hepatocytes based on their larger size and pale cytoplasm (2). (Original magnifications x 200
(upper part) and x 400 (lower part)).
REFERENCES.
1.
Braun KM, Thompson AW, Sandgren EP. Hepatic microenvironment affects oval cell
localization in albumin-urokinase-type plasminogen activator transgenic mice. The American
journal of pathology 2003 Jan;162(1):195-202.
2.
Meuleman P, Libbrecht L, De Vos R, de Hemptinne B, Gevaert K, Vandekerckhove J,
et al. Morphological and biochemical characterization of a human liver in a uPA-SCID mouse
chimera. Hepatology (Baltimore, Md 2005 Apr;41(4):847-856.