Supporting Material & Methods
1
Histochemistry
Masson’s trichrome, H&E and Sirius Red staining of paraffin-embedded liver sections was
performed, using standard techniques. To determine glucose-6-phosphatase activity, unfixed
liver cryosections were incubated for 10-12 min in substrate reagent: 10 mM D-Glucose-6phosphate, 270 mM sucrose, 2.4 mM Pb(NO3)2 and 40 mM Tris/maleate buffer (pH 6.5). After
post-fixation in 4% paraformaldehyde solution, cryosections were immersed in 0.22% (NH4)2S
diluted in 0.1 M Tris maleate/0.1 M NaCl and counterstained with Hematoxylin.
Immunohistochemistry
Five μm sections of paraffin-embedded liver tissue were deparaffinized in xylene and rehydrated,
followed by microwave antigen retrieval for 17 or 30 min in 10 mM citrate buffer (except for αSMA detection). Mouse anti-α-SMA (Sigma-Aldrich), rabbit anti-EGFP (Novus), mouse antiCD26 (Santa Cruz), sheep anti-albumin (Bethyl), or rabbit anti-glutamine synthetase (SigmaAldrich) were used as primary antibodies, followed by biotin-conjugated horse anti-mouse IgG
(Vector) (followed by the Vectastain ABC procedure), peroxidase-conjugated donkey anti-rabbit
IgG (GE Healthcare), alkaline phosphatase-conjugated horse anti-mouse IgG (Vector), biotinconjugated rabbit anti-sheep IgG (Rockland) or biotin-conjugated horse anti rabbit-IgG (Vector)
(followed by ABC procedure) as secondary antibodies. Antibody staining was detected with
Vector NovaRed/DAB/Vector Black substrate kits and tissue sections were counterstained with
Hematoxylin or Nuclear Fast Red.
Five μm cryosections were stained with mouse anti-CD26 (Santa Cruz) and mouse anti-α-SMA
or mouse anti-CK-19 (Progen), followed by TRITC-conjugated rabbit anti-mouse IgG1
(Rockland) and biotin-conjugated goat anti-mouse IgG2 (Nordic Immunological). For α-SMA or
CK-19 detection, cryosections were finally incubated with Streptavidin-CyTM2 (Jackson
ImmunoResearch).
Supporting Material & Methods
2
Cryosections were stained with biotin-conjugated mouse anti-CD26 (Cedarlane) and mouse antiKi-67 (BD Biosciences) as primary antibodies and were developed by Streptavidin-CyTM2
(Jackson) and TRITC-conjugated rabbit anti-mouse IgG1.
Cryosections were stained with mouse anti-CD26 (Santa Cruz) and rabbit anti-EpCAM (abcam)
as primary antibodies and were developed by donkey anti-mouse-IgG-Cy2 and donkey antirabbit-Cy3 (Jackson).
Supporting Figure 1:
Repopulation and functional incorporation of differentiated donor cells transplanted without PH
into rats with progressing fibrosis/cirrhosis. 1.4-1.6 x 108 ED15 fetal liver cells were transplanted
into DPPIV– rats at 3 months after starting TAA administration, which was continued thereafter
(200 mg/kg, twice weekly). (A,B) Patterns of DPPIV expression (A) and G6Pase activity (B) in
two adjacent liver sections from one recipient rat at 4 months after cell transplantation. These
liver sections demonstrate that DPPIV+ cells differentiated into hepatocytic cells, become
incorporated into the fibrotic liver structure and exhibit G6Pase expression. (C) Quantitative RTPCR analysis for mRNA levels of selected genes specific for hepatocytic functions in recipient
livers at 4 months after FLSPC transplantation into rats with advanced fibrosis/cirrhosis. Values
are mean ± SEM of liver samples from FLSPC-transplanted rats (n=3) and are expressed as fold
differences in gene expression compared to liver samples derived from age-matched nontransplanted TAA-treated rats (n=3). One representative example from 3 replicate experiments is
shown.
Supporting Figure 2:
Supporting Material & Methods
3
Liver tissue replacement without partial hepatectomy after cell infusion of enriched ED15
FLSPCs into a recipient rat with progressing fibrosis/cirrhosis. After isolation of unfractionated
fetal liver cells, negative selection was performed by incubating ~3.5 x 10 8 cells with a
monoclonal mouse antibody specific for rat erythrocytes and erythroid progenitors (BD
Biosciences) at 4°C for 30 min. Cells were washed twice in Hanks’ balanced salt solution
(HBSS) containing 0.3% BSA, 0.8 mM MgCl2, 10 mM HEPES and incubated with rat antimouse IgM bound to magnetic microbeads (Miltenyi) at 4°C for 30 min. The cell suspension was
washed and passed through LS-columns placed in a MidiMACSTM Separator (Miltenyi).
Erythroid cells were retained in the magnetic field (column); non-erythroid cells enriched with
epithelial stem/progenitor cells passed through the columns. (A,B) Cytospins of unfractionated
vs. cytospins of enriched ED15 FLSPCs were stained with mouse anti-E-cadherin antibody (BD
Biosciences), followed by CyTm3-conjugated donkey anti-mouse IgG (Jackson) as secondary
antibody. Unfractionated fetal liver cells contained 2.6% E-cadherin+ epithelial cells (A). After
enrichment with immunomagnetic beads, this increased to 11.4% (4.4-fold enrichment) in the
erythroid-depleted fraction (B). (C) Enriched fetal liver cells (~8 x 107) from DPPIV+ F344 rats
were infused into a DPPIV– F344 rat with progressing fibrosis/cirrhosis without partial
hepatectomy. At one month after cell infusion, the rat was sacrificed and liver sections were
stained using enzyme histochemistry for DPPIV to detect expanding DPPIV+ cell clusters. The
majority of the liver was successfully engrafted by transplanted fetal liver cells, which formed
DPPIV+ cell clusters; some of them were already encompassing entire fibrotic lobules. The
image contains 4 merged adjacent microscopic fields. Original magnification x400 (A,B) and
x50 (C).