Proc. Natl. Acad. Sci. USA
Vol. 92, pp. 5860-5864, June 1995
Integration of transplanted hepatocytes into host liver plates
demonstrated with dipeptidyl peptidase IV-deficient rats
(liver cell plate/bile canaliculus/gap junction/acute liver failure/gene therapy)
SANJEEV GUPTA, PANKAJ RAJVANSHI, AND CHANG-DON LEE
Marion Bessin Liver Research Center, Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461-1602
Communicated by Rudi Schmid, University of California, San Francisco, February 21, 1995
tion, we transplanted normal hepatocytes into syngeneic rats
deficient in dipeptidyl peptidase IV activity. When isolated
hepatocytes were injected into splenic pulp, cells promptly
migrated into hepatic sinusoids. To examine whether trans-
planted hepatocytes entered liver plates and integrated with
host hepatocytes, we analyzed sharing of hepatocyte-specific
gap junctions and bile canaliculi. Colocalization studies
showed gapjunctions uniting adjacent transplanted and host
hepatocytes in liver plates. Visualization of bile canalicular
domains in transplanted and host hepatocytes with dipeptidyl
peptidase IV and ATPase activities, respectively, demon-
strated hybrid bile canaliculi, which excreted a fluorescent
conjugated bile acid analogue. These results indicate that
transplanted hepatocytes swiftly overcome mechanical barri-
ers in hepatic sinusoids to enter liver plates andjoin host cells.
Integration into liver parenchyma should physiologically reg-
ulate the function or disposition of transplanted hepatocytes
and benefit applications such as gene therapy.
To analyze mechanisms of liver repopula-
Liver repopulation has significant potential for ex vivo gene
therapy (1). The potential of ex vivo gene therapy was recently
demonstrated in familial hypercholesterolemia, although the fate
of transplanted hepatocytes in the liver was undefined (2, 3).
Hepatocyte transplantation to salvage an acutely injured liver has
similarly gained interest, although optimal results will require
massive liver repopulation (1). Cells can be safely delivered into
hepatic sinusoids; however, as the portal vascular bed has limited
capacity, augmentation ofthe transplanted hepatocyte mass may
require either cell proliferation or repeated cell transplantation.
Such strategies should be facilitated by integration of trans-
planted hepatocytes into liver plates. Indefinite persistence of
cells in hepatic sinusoids mayimpede bloodflowor limitrepeated
cell transplantation. Although portal hypertension induced by
transplanting cells into hepatic sinusoids is short-lasting (4),
whether microcirculatory alterations accompany normalization
of portal pressures is unknown. Moreover, nonavailability of
cell-cell contact or autocrine signals to hepatocytes in hepatic
sinusoids could limit proliferation or other biological processes.
On the other hand, integration of transplanted hepatocytes into
liver plates should restore position-specific hepatic gene expres-
sion and lineage-specific cellular life history (5). However, despite
recent localization of genetically marked transplanted hepato-
cytes in liver (6-8), whether cells integrated into liver plates was
Based upon indefinite persistence of transplanted hepato-
cytes in liver (7, 8), we predicted that cells should enter liver
plates, although this would require circumvention of the
sinusoidal endothelium and space of Disse. To test this hy-
pothesis, systems were employed that used mutant Fischer 344
rats lacking dipeptidyl peptidase IV (DPPIV) activity (9),
which serves also as a marker of bile canaliculi (10). Cell
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integrations could be demonstrated by formation of specific
hybrid organelles, such as bile canaliculi or gapjunctions (11, 12).
MATERIALS AND METHODS
Animals. Donor Fischer 344 rats were commercially ob-
tained (Harlan-Sprague-Dawley). Twelve syngeneic DPPIV-
recipients, -200 g each, were provided by the Special Animals
Core of the Liver Research Center at the Albert Einstein
College of Medicine. Hepatocyte isolation by collagenase
perfusion and injection of 2 x 107 cells into the splenic pulp
of each DPPIV- rat were as described (4). Animal use was
approved by the Animal Care and Use Committee at the
Albert Einstein College of Medicine. During 8 months of
studies, recipients were killed at intervals for tissue analysis.
LiverNonparenchymal Epithelial Cells. FNRL (Fischer 344
neonatal rat liver) cells served as controls (13). FNRL cells
respond to hepatic stimulator substance (14) and react with H-4
and OV-6 monoclonal antibodies, which recognize hepatocyte
and oval-cell antigens, respectively (15). When transformed,
FNRL cells produce tumors of hepatocyte, biliary, or other
lineages (16). The FNRL cells should have produced DPPIV+
bile canaliculi after differentiation into hepatocytes in vivo.
Synthesis and Use of a Fluorescent Conjugated Bile Acid
Analogue. To test biliary function, a fluorescent compound,
NO-choloyl-Ne-NBD-lysine (CNBG) was synthesized in the lab-
oratory of Alan Hofmann by Claudio D. Schteingart and
Huong-Tu Ton-Nu. Briefly, the method involved coupling of
4-chloro-7-nitrobenz-2-oxa-1,3-diazole (NBD) to Nactholoylly-
sine (17). A derivative of the naturally conjugated bile salt
choloylglycine, the compound has a fluorophore (NBD) coupled
by a five-atom tether to the a-carbon of glycine. The compound
should have a pKa of 3.9, on the basis of the ionization behavior
of choloylglycine (18), and enters hepatocytes by a Na+-
dependent mechanism (19), presumably involving the Ntcp trans-
porter (20). The protonated form of CNBG was dissolved in
phosphate-buffered saline, pH 7.4 (PBS). The compound was
empirically infused i.v. into a hepatocyte recipient at 4 ,tmol/min
per kg ofbody mass (6 ,umol, or 3 mg, over 10 min), although the
Vmax for CNBG transport is unknown. To detect CNBG
excretion in bile canaliculi and for other analysis, tissues were
frozen in cold methylbutane and stored at -80°C. Fluores-
cence was examined under a fluorescein isothiocyanate filter
in cryostat sections secured on the microscope stage and
DPPIV activity was then colocalized in tissues in situ.
Histological Studies. Cryostat sections, 5 ,um thick, were
fixed for 10 min in acetone/chloroform, 1:1 (vol/vol), at 4°C
and incubated with a DPPIV substrate to demonstrate enzy-
matic activity (21). Biliary ATPase activity was detected by
incubating sections with ATP (22). To colocalize DPPIV and
ATPase activities, cryostat sections were reacted first for
DPPIV activity, washed with PBS, and immediately processed
for ATPase activity. Glucose-6-phosphatase activity was de-
tected in unfixed cryostat sections (23). The 32-kDa major
Abbreviations: Cx, connexin; DPPIV, dipeptidyl peptidase IV.
Proc. Natl. Acad. Sci. USA 92 (1995)
hepatic gapjunction protein, connexin 32 (Cx32), was localized
by an anti-rat antibody (7C6, aCx32) (24). For immunostain-
ing, cryostat sections were blocked with sheep serum and
incubated with aCx32 (1:100 dilution), and antibody binding
was detected with a peroxidase-based system (BioGenex Lab-
oratories, San Ramon, CA).
Demonstration ofLiver Repopulation. Utilization ofDPPIV-
recipients allowed the use of strategies described elsewhere
(9). Optimal localization of transplanted cells required that all
donor hepatocytes were DPPIV+, and recipients, DPPIV-
(Fig. 1 A and B).
Although the organization of hepatic lobules is complex,
hepatocytes are arranged in liver plates surrounded by sinu-
soids containing blood and littoral cells, while bile drains into
canaliculi between adjacent hepatocytes. In view of the inti-
mate anatomic relationship between adjacent hepatocytes in
normal liver, we reasoned that if transplanted cells were to
integrate into liver plates, this should lead to the appearance
of hybrid or shared organelles, such as bile canaliculi and gap
junctions. Cell translocations into liver plates could be dem-
onstrated by delineation of sinusoidal and bile canalicular
domains of host or transplanted hepatocytes. To demonstrate
shared gap junctions in the intercellular domains of trans-
planted and host hepatocytes in liver cell plates, we immuno-
stained tissue sections previously reacted for DPPIV activity.
Dual studies to simultaneously distinguish between gap junc-
tions and transplanted hepatocytes did not require additional
manipulations. However, to demonstrate hybrid bile canaliculi
in transplanted and host hepatocytes, we developed additional
experimental strategies. Briefly, sequential reactions for DP-
PIV and ATPase activities visualized donor and host hepato-
cytes containing differently colored bile canaliculi (Fig. 1 C
Entry of Transplanted Hepatocytes into Liver Plates. We
injected cells into spleen to reproducibly and safely deposit
them into hepatic sinusoids (4, 25). Intrasplenic hepatocyte
transplantation may confer additional advantages, such as
tolerance to heterologous antigens and superior cellular gene
expression (26, 27). When hepatocytes were transplanted into
DPPIV- recipients, transplanted cells could be unequivocally
localized in the liver (Fig. 2). Initially, transplanted cells were
situated in portal spaces but entered host liver plates in <1
week. Hepatocyte recipients were killed at intervals and
survival of transplanted DPPIV+ hepatocytes was demon-
strated for up to the 8-month duration of the studies, in
agreement with previous findings (7, 8).
Demonstration ofHybrid Gap Junctions. Hepatic gap junc-
tions are recognized in thin sections as septilaminar linear
structures formed by apposed membraneswith an extracellular
gap (12). Hepatic Cx32 is several times more abundant than
Cx26, although they colocalize in gap junction plaques. There-
fore,we used aCx32 antibody to visualize hepatic gapjunctions
(Fig. 3). Reaction of tissues with aCx32 visualized gap junc-
tions in the expected domains of adjacent hepatocytes but not
in biliary cells. This strategy provided an excellent demonstra-
tion of hybrid gap junctions uniting adjacent transplanted and
host hepatocytes (Fig. 3).
Demonstration of Functionally Intact Hybrid Bile Canal-
iculi. Bile canaliculi are
-0.75 ,um in diameter and are
composed of membranes from adjacent cells (28). The cana-
liculi exhibit contractile responses, a variety of ectoenzymes,
and energy-dependent transport systems (11). These mecha-
nisms allowed us to utilize differential expression of DPPIV
and ATPase activities to demonstrate integration of host and
transplanted hepatocytes (Fig. 4). We found hybrid bile canal-
iculi containing components of DPPIV+ donor and ATPase+
showing absence of DPPIV activity. (C and D) Sequential staining for DPPIV, followed by ATPase activity. (C) Donor liver shows red DPPIV+
bile canaliculi, whereas bile ducts exhibit ATPase activity (in brown). (D) DPPIV- liver shows only ATPase activity in bile canaliculi. (x 170.)
(A) DPPIV staining of a normal F344 donor liver. All hepatocytes but not bile ducts are stained (P, portal area). (B) DPPIV- rat liver
Medical Sciences:Guptaet aL
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canaliculi. (Methyl green counterstain; X340.)
(A) DPPIV+ cells in vascular spaces 2 hr after transplantation. (B) Transplanted cells in liverplates after 7daysshow well-defined bile
host hepatocytes in liver plates. Hybrid bile canaliculi could
have developed onlywhen transplanted hepatocytes physically
joined host hepatocytes in liver plates. Use of hybrid bile
canaliculi as an indicator showed that transplanted hepatocytes
were fully integrated in liver plates within 7 days after trans-
plantation, with indefinite survival in hosts up to the 8-month
duration ofthe experiment. In contrast, whenFNRL cellswere
transplanted into DPPIV- recipients, no bile canaliculi
formed and DPPIV staining was negative (data not shown),
which suggested absence of differentiation into hepatocytes.
Transplanted hepatocytes retained glucose-6-phosphatase
activity within host liver plates, indicating preservation of
hepatic gene expression (data not shown). To demonstrate
excretory function in hybrid bile canaliculi, we used a fluo-
rescent bile salt analogue. When the fluorescent bile salt was
administered several weeks after transplantation of syngeneic
DPPIV+ hepatocytes, excretion of the infused bile salt was
apparent in bile canaliculi throughout the liver, including
hybrid bile canaliculi formed by transplanted cells (Fig. 5).
This study provides unequivocal evidence for entry of trans-
planted hepatocytes into liver plates and integration with host
hepatocytes. The ability of transplanted hepatocytes to tra-
verse the sinusoidal endothelium and space of Disse is re-
markable. Direct access to the space of Disse is regulated by
endothelial cell fenestrae "150 nm in diameter, although this
dimension depends upon the contractile state ofthe perifenes-
tral cytoskeleton (11). Delineation of the precise mechanisms
will require further studies; however, differences in the size of
hepatocytes (20-35 ,um) and fenestral openings indicate that
aCx32 antibody. (C) Recipient liver showing gap junctions in a group of transplanted cells. (D) Higher-power view of gap junctions joining a
transplanted hepatocyte (red bile canaliculi) and host hepatocytes. (A, B, and D, x850; C, x 170.)
(A) Normal rat liver showing circumscribed gap junctions in intercellular hepatocyte domains. (B) Negative control with omission of
Proc. Natl. Acad. Sci. USA 92(1995)
Proc. Natl. Acad. Sci. USA 92 (1995)
are red. Note ATPase activity in bile canaliculi, as well as bile ducts seen intop center. Higher-magnification views are shown(BandC)of areas
marked with arrows. (x170.) (B and C) Interconnected networks ofbile canaliculidraininghost andtransplanted hepatocytes.In someareas, brown
and red colors overlap, indicating contributions from transplanted and host cells. Elsewhere, bile canalicular networks extend fromtransplanted
cells into host hepatocytes. (x850.)
(A) Hybrid bile canaliculi in hepatocyte recipients after DPPIV andATPase reactions. Bile canalicularcomponentsintransplanted cells
remodeling must occur during entry of transplanted cells into
Maintenance of polarity in differentiated epithelial cells is
critical for specialized functions. Restoration of normal hep-
atocyte polarity-as defined by the sinusoidal domain abutting
the space of Disse, the intercellular domain containing junc-
tional complexes with interspersed gap junction channels, and
the apical or canalicular domain between adjacent cells-
required integration into liver plates and fusion of cell mem-
brane organelles between juxtaposed transplanted and host
cells. The gap junction channels may occupy -3% of the
hepatocyte cell membrane with 12 identical subunits, 6 of
which, termed a connexon or hemichannel, are contributedby
each cell (12). We hypothesized that hybrid gap junctions
would require connexons from transplanted and adjacent host
hepatocytes. Light microscopy was adequate for visualizing
gap junctions in hepatocytes because, although individual
channels may measure only 15-18 nm, gap junctional plaques
could exceed 1 ,um in diameter (12). Expression ofgapjunction
proteins in the liver is cell type specific. Bile duct cells and
littoral cells express Cx43 (29), whereas hepatocytes express
Cx32 and/or Cx26 as major gap junction proteins (30). In the
rat liver, the Cx32 gap junction protein is -10-fold more
abundant than Cx26. Also, Cx32protein is uniformly expressed
Methods). A bright fluorescent portal area surrounded with a lattice of bile canaliculi is visible after excretion of the bile acid analogue. (B) Same
area of the liver as inA after in situ fixation with acetone/chloroform and reaction for DPPIV activity. Transplanted hepatocytes are present in
upper left corner and elsewhere.
(A) Fluorescence microscopy of liver from a hepatocyte recipient after infusion of a fluorescent bile acid analogue (see Materials and
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5864Medical Sciences: Gupta et at
throughoutthehepatic lobule, whereas Cx26proteinispref-
erentially expressedinperiportal hepatocyteswith agradient
across thehepaticlobule,which couldpotentiallybehelpfulfor
examiningcellular differentiation intransplanted cells.
Formation ofhybridbile canaliculi intransplanted hepato-
cytesbut not innonhepatocytesindicates that this feature
could also be used as aparameterof cell differentiation.
retain DPPIV and ATPase activities, althoughin a diffuse
patternconsistent withdisruptionof bile canaliculi.Hepato-
cytesin the intact liver also maintain DPPIV and ATPase
activitiesdespite perturbationssuch aspartial hepatectomyor
carbon tetrachloride-induced liverinjury (ref. 31,andS.G.,
unpublished observations). Therefore,ourstrategyto colocal-
ize bile canaliculi intransplantedand hosthepatocytes was
effective andprovedto be more convenient than cumbersome
Integrationoftransplanted hepatocytesin the liverparen-
chymaisparticularly significantfor massive liverrepopulation.
Untilrecently,themagnitudeof liverrepopulation by hepa-
tocyte transplantationhas been limited to 1-2% of the host
liver(7, 8). However,in studies to bereported elsewhere, we
have been able in one session totransplant hepatocytes
representing upto 10-15% of thehepaticmass.Rapidtrans-
location oftransplanted hepatocytesfrom the sinusoids into
the liver cellplateshould facilitatetestingadditionalstrategies
for massive liverrepopulation,such asby repeatedcell trans-
plantation. Integrationoftransplanted hepatocytesin liver
plateswill allow studies to define theirproliferative potential
in thehepatic lobule, which couldrepresentan alternative
strategyfor massive liverrepopulation. Transplanted hepato-
cytes expresscellulargenesmoreeffectivelyin the liver than
inectopicsites such asspleen, peritoneal cavity,and dorsal fat
pad (27). Although regulationofhepatic gene expressionis
complex,with contributions from extracellular matrixcompo-
nents,cell-cellinteractions, exposuretospecifichormones or
growth factors,andpositionin thehepaticlobule(5), integra-
tion oftransplantedcells into the liverplateshould maximize
thepotentialfor cellulargene expressionandproliferation.
Oursystemsdescribed here will beparticularlyrelevant for
studies of differentiation inhepatic progenitorcells.Although
evidence for activation ofprogenitor liver cells has been
provided, suitablebioassays arenecessary toanalyze the
differentiation and fate of theseprogenitorsin vivo.Develop-
ment ofhybrid bile canaliculiappears to bespecific for
hepatocytes,since we were unable to demonstratehybridbile
canaliculi when FNRL cells weretransplanted, presumably
due to lack of their differentiation intohepatocytes. One
reason for interest inprogenitorcells would be theircapacity
forgreater proliferation,which should be facilitatedbyinte-
grationofhepatocytesinto liverparenchyma. Gap junction
channels could contribute to cellproliferation by facilitating
cellularsignal transduction.Incorporation of transplanted
cells into the liverplatewill facilitateanalysis of whether
hepatocytes stream fromportal towardpericentral areas,
which willimprove anunderstandingof their natural life
history. Finally,unencumberedentryand assimilation ofhepa-
tocytesinto liver cellplates suggestthatparticlesizemay only
partlydictate fenestralregulationoftraffickinginto thespace
ofDisse, speciallywhenbiological agentsare involved. Our
findingsshould also be relevant for workersinvestigating in
We thank Dr. Alan F. Hofmann (University of California, San
Diego) and Dr. Elliot L. Hertzberg (Albert Einstein College of
Medicine) for providing the fluorescent bile acid analogue and the
aCx32 antibody, respectively. This work was partly supported by
National Institutes of Health Grants DK-K08-01909 and ROI
DK46952 to S.G. and P30-41296 to the Marion Bessin Liver Research
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