|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 4 October 2006
doi: 10.1242/dev.02589
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Medicine, Division of Hematology and Oncology, Rhode Island Hospital and the Graduate Program in Pathobiology, Brown University Medical School, Providence, RI 02903, USA.
* Author for correspondence (e-mail: dhixson{at}lifespan.org)
Accepted 23 August 2006
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Cholangiocyte, Monoclonal antibody, Retrorsine, Partial hepatectomy, Differentiation, Bipotent, Progenitor, Fetal liver, Rat, Micromagnetic bead, Hepatoblast, Non-parenchymal cell, Two dimensional SDS-PAGE, Dipeptidyl peptidase IV, Immunofluorescence, Histochemical stain
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Upon severe
injury to the adult liver that compromises the proliferative capacity of
hepatocytes, this progenitor cell compartment is activated, initiating
expansion of oval cells that undergo differentiation into either hepatocytes
or cholangiocytes (Alison and Lovell,
2005; Sell, 1998;
Sirica, 1995). We have
previously reported that rat oval cells display phenotypes defined by a panel
of surface reactive monoclonal antibodies (MAb) that recapitulate stages in
differentiation and maturation of cholangiocytes during liver development
(Hixson et al., 2000;
Hixson et al., 1997). This
observation raised the possibility that cholangiocyte marker positive (CMP)
fetal liver epithelial cells (FLEC), like oval cells in the adult liver, would
retain a capacity for differentiation along both hepatocyte and cholangiocyte
lineages.
One approach for exploring this possibility would be to isolate CMP-FLEC
and test their differentiation potential by transplantation. In previous
studies, hepatic progenitor cells have been isolated by physical criteria, the
end result being phenotypically heterogeneous isolates enriched with cells of
particular size or density (Dabeva et al.,
1997; Hayner et al.,
1984; Hisatomi et al.,
2004; Tateno et al.,
2000). More recently, investigators interested in liver
progenitors have begun employing tactics commonly applied to the isolation of
hematopoietic stem cells, the predominant approach being use of antibodies
against cell-surface markers in combination with magnetic immunobeads or
fluorescent activated sorting by flow cytometry (FACS). Sigal et al. were one
of the first to report the enrichment of fetal rat hepatoblasts by combining
panning to remove hematopoetic cells with FACS to enrich for progenitor cells
with a high level of granularity (Sigal et
al., 1994). Enrichment for hepatic progenitors has also been
achieved by positive selection using cell-surface markers expressed by fetal
liver epithelial cells such as Thy1
(Petersen et al., 1998), Met
(Suzuki et al., 2004), Dlk
(Tanimizu et al., 2003),
E-cadherin (Nierhoff et al.,
2005; Nitou et al.,
2002) or integrin subunits
(Suzuki et al., 2002).
The majority of these studies, focus on bipotent fetal liver progenitors
present between embryonic days (ED) 12-14, a time period in rat liver
development prior to bile duct morphogenesis when Dabeva et al. reported the
presence of FLEC expressing cholangiocyte cytokeratin, CK19
(Dabeva et al., 2000). These
investigators suggested that early CMP-FLEC were hepatoblasts committed to a
cholangiocyte lineage, raising the issue of whether CMP-FLEC in ductal
structures displayed a similar restriction in differentiation potential. To
address this, we have used the retrorsine/partial hepatectomy (PH)
transplantation model of Laconi et al.
(Laconi et al., 1998) to
assess the differentiation potential of CMP-FLEC appearing between ED16 and
ED19. Our reasoning being that CMP-FLEC and their newborn equivalents are
present in larger numbers than liver progenitors at ED12-14, and that CMP-FLEC
are likely to be the progenitor cells `equivalent' of bipotent oval cells in
the adult liver. This assumption was founded on previous studies showing oval
cells are bipotent cells of ductal origin that recapitulate fetal phenotypes
displayed by CMP-FLEC and serve as progenitors of at least some chemically
induced rat hepatocellular carcinomas.
In the present report, four MAb defining surface epitopes, designated as
OC.10, OC.2, BD.2 and OC.5, have been used in combination with immunomagnetic
beads to isolate antigenically distinct subpopulations of CMP-FLEC previously
defined as follows: stage I (CMP-FLECI), OV6+; stage II
(CMP-FLECII), OV6+ (cytokeratin epitope)/OC.10+; stage III
(CMP-FLECIII), OV6+/OC10+/OC.2+; and stage IV
(CMP-FLECIV), OV6+/OC.10+/OC.2+/BD.2+/OC.5+
(Hixson et al., 2000).
Isolation schemes entailing positive and negative selection with micromagnetic
beads were designed to take advantage of the sequential appearance of MAb
defined epitopes, their continued expression throughout liver development and
presence at high levels on all mature cholangiocytes in adult rat liver.
Testing for bipotency, CMP-FLEC isolated from dipeptidyl peptidase IV (DPPIV)
positive (DPPIV+) rats were transplanted following PH into DPPIV-host rats
previously treated with retrorsine (Gordon
et al., 2000a). Results showed CMP-FLEC expressing as many as
seven cholangiocyte markers retained a high capacity for differentiation along
a hepatocyte lineage. Additionally, we present evidence suggesting that
ED16-19 CMP-FLEC have a higher capacity for engraftment and/or expansion in
retrorsine/PH-treated adult liver than CMP negative fetal hepatoblasts and
non-parenchymal cells, thus raising the possibility that liver epithelial
cells with similar phenotypes may constitute an important progenitor
population in newborn and adult liver.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Retrorsine treatment and Retrorsine/PH/DPPV+ liver transplantation model
Host DPPIV-rats 6-8 weeks of age were treated with retrorsine as previously
described (Gordon et al.,
2000b). At 4-5 weeks after the second retrorsine injection,
5x105 DPPIV+ fetal liver ductal progenitor cells were
transplanted into the liver of a DPPIV-host rat via the spleen immediately
following a two-thirds PH. DPPIV-host rats were transplanted with
subpopulations isolated from ED16 fetal livers
(Table 1,
Fig. 1A,B) and sacrificed at 48
hours, 1, 3 and 6 months, and 1 year post transplant; ED18/19 fetal livers
(Table 1,
Fig. 1C) were sacrificed at 48
hours, 2 weeks, 3, 6 and 9 months, and 1 year post transplant. Livers were
excised following collection of blood by heart puncture. Tissues were immersed
in OCT freezing medium or directly frozen in hexane cooled by an acetone/dry
ice bath and stored at -80°C.
|
). MAb OV6 was obtained from Stewart Sell at Ordway Research
Institute, Wadsworth Center, NYS Department of Health. This MAb recognizes an
epitope on the cytokeratin 19, a cholangiocyte cytokeratin
(Bisgaard et al., 1993;
Dunsford et al., 1989). MAbs
specific for AFP and CYP450 2E1 was purchased from Santa Cruz Biotechnology
(Santa Cruz, CA).
Isolation and transplantation of CMP-FLEC
Fetal livers were excised from dams and placed in ice-cold HBSS. Cells
resuspended in pre-digest buffer (Hanks with 0.15 mM EGTA) were minced and
incubated for 10 minutes at 37°C, shaking at 120 rpm. After settling on
ice for 2-5 minutes, the supernatant was removed and liver fragments incubated
in 30 ml digestion buffer (Hanks containing 2.5 mM CaCl2, 0.1%
collagenase B (Roche) and 6 mg/100 ml DNase I) for 10 minutes at 37°C,
shaking at 120 rpm. After dissociation by several passages through decreasing
size pipettes, digestion was stopped by 6-10 ml hepatoblast medium
(Rogler, 1997) and the cell
suspension was filtered through a MiniMac filter (Miltenyi). Fetal liver cells
resuspended in 5 ml hepatoblast medium, were incubated at 4°C rocking for
30 minutes with cell-surface reactive MAb at 25-50
µg/5x105 cells. After three washes in HBSS, cells were
resuspended in sterile MiniMac running buffer (PBS containing 5% BSA, and 2 mM
EDTA) in a minimum volume of 1 ml per
5x105-1x106 cells. Cells were then incubated
for 30 minutes at 4°C with Miltenyi rat anti mouse IgG (subtype specific)
or rat anti mouse IgM microbeads at a ratio of 20 µl micro magnetic
beads/107 cells followed by three washes with Mini Mac running
buffer. Labeled cells resuspended in Mini Mac running buffer were isolated
with an autoMACS automated micromagnetic cell sorter using autoMACS programs
(Brandt et al., 1996;
Griwatz et al., 1995) for
double column positive selection and depletion. Depending on the cell
fractionation strategy being used, this cell separation protocol was repeated
using a second cell-surface reactive MAb. Aliquots of cell isolates prepared
by positive and negative (depletion) selection were tested for Trypan Blue dye
exclusion to determine viability. Isolates with viabilities greater than 80%
were used for transplantation. Isolates were analyzed by FACS to determine
percentage of cells reactive with positive selection MAb. Cells were also
stored at -80°C as cytospins for indirect immunofluorescence (IIF)
analysis or as pellets for subsequent analysis by 2D gel electrophoresis or
enzyme assays.
FACS analysis
Isolated fetal liver subpopulations with bound MAb-micromagnetic bead
complexes were washed in PBS and resuspended in FACS buffer (PBS containing
0.1% sodium azide and 1% BSA). Cells were then incubated for 30 minutes at
4°C with anti-isotypic FITC (Sigma)-labeled antibodies to allow
visualization of the bound MAb by binding to sites on the primary antibody
unoccupied by the anti Ig magnetic beads carrying only one anti Ig
molecule/bead. In some cases, bead bound cells were re-incubated with primary
antibody used for selection followed by secondary antibody. Both methods
produced equivalent results. Fluorochrome-conjugated isotype antibodies of the
wrong specificity (anti Fc or mu chain specific secondary antibodies with IgM
or IgG primary antibodies, respectively) were used as controls. FACS was
performed as previously described (Laurie
et al., 2005).
Isolation of DPPIV+ donor cells from DPPIV-host rat livers
Livers of host rats were digested by perfusing the liver with collagenase
as previously described (Yang et al.,
1993). Isolated hepatocytes suspended in HBSS containing 10% fetal
bovine serum and 25-50 µg per 5x105 cells MAb 236.3
specific for DPPIV (Thompson et al.,
1991) were incubated at 4°C for 30 minutes, washed in HBSS,
and resuspended in HBSS containing 20 µl micro magnetic
beads/107 cells. After incubation with shaking at 4°C for 30
minutes, DPPIV+ donor derived hepatocytes were isolated with an autoMACS
automated micromagnetic bead cell sorter using a double column positive
selection program (Brandt et al.,
1996; Griwatz et al.,
1995). Purity of hepatocyte isolates was determined by IIF
labeling of acetone fixed cytospins. Only donor and host hepatocyte isolates
containing more than 90% DPPIV+ or DPPIV-hepatocytes, respectively, were used
for 2D gel analysis.
DPPIV enzyme assay on tissue extracts
DPPIV enzyme assays on detergent extracts of fresh or frozen/thawed liver
tissue taken from DPPIV-host rats transplanted with DPPIV+ donor cells were
performed as described previously (Piazza
et al., 1989) using gly-pro-MNA (Sigma) as substrate. Specific
enzyme activity was expressed as the µM of product/mg protein/minute. The
µM of product was determined from a standard curve of absorbance at 525 nM
versus [MNA]. Specificity was demonstrated by the ability of synthetic
tripeptide Val-Pro-Leu to inhibit enzymatic activity. Autocleavage was
determined by eliminating detergent extracts from the reaction solution.
IIF staining of donor colonies
Serial frozen tissue sections were fixed in ice-cold acetone and labeled by
a previously described IIF protocol
(Erickson et al., 2006).
Secondary antibodies used were AlexaFluor 488 goat anti-mouse IgG (H+L)
(Molecular Probes) or AlexaFluor 594 goat anti-mouse IgM (mu chain specific)
(Molecular Probes). Double label IIF was performed by sequential incubations
with a mixture of two primary antibodies followed by a mixture of appropriate
secondary antibodies or, alternatively, by sequential incubation with the
first primary MAb and an AlexaFluor 488- or 594-conjugated anti mouse Ig
secondary followed by second primary MAb and AlexaFluor 594- or 488-conjugated
secondary, respectively. Sequential staining was then repeated with the order
of primary MAbs reversed to control for interference between primary MAbs of
interest.
Histochemical staining protocols
Histochemical reactions were performed on acetone fixed frozen sections.
Detection of canalicular ATPase was performed as previously described
(Wachstein and Meisel, 1957)
using ATP, lead nitrate and magnesium sulfate. Staining of tissue sections for
glutamyl transpeptidase was carried out according to the method of
Rutenburg et al. (Rutenberg et al.,
1969), using -glutamy-4-methoxy-naphthylamide as substrate.
DPPIV activity was detected by incubating sections with
glycyl-L-proline-4-methoxy-2-naphthylamide (Sigma) and Fast Blue BB Salt
(Sigma) as described previously (Lojda,
1979), and glucose-6-phosphotase activity was detected by
histochemical staining performed as previously described
(Maly and Sasse, 1983).
Morphometry
The size distribution of donor derived DPPIV+ colonies of hepatocytes was
determined in the Molecular Pathology Core of the Center for Cancer Research
Development at Rhode Island Hospital using Image-Pro Plus 5.0 software.
Cross-sectional areas were determined by applying Image Pro Plus area
algorithm to colony outlined using the auto-trace feature. Statistical
analysis of area of donor hepatocyte colonies was performed using Kruskal
Wallis ANOVA followed by the Mann-Whitney-Bonferroni test for multiple
comparisons.
Two-dimensional gel analysis
Donor hepatocytes harvested from host rat livers at one-year post
transplant were used for 2D gel analysis. Cell extract was prepared by
solubilizing 5x106 cells in 0.5% NP-40 for 1 hour at 4°C
and removing insoluble material at 30,000 g for 20 minutes.
ZOOM isoelectric focusing (IEF) strips (Invitrogen, Carlsbad, CA) were
rehydrated according to manufacturers protocol in ZOOM IPG runner cassette
previously loaded with 155 µl sample rehydration buffer (Invitrogen)
containing 7.5 µg of cell extract. IEF strips were then equilibrated in
NuPAGE LDS reducing sample buffer for 15 minutes and alkylated 15 minutes at
room temperature in reducing NuPAGE SDS sample buffer containing 125 mM
iodoacetamide. Isoelectric focusing was performed according to manufacturers
instructions (Invitrogen). For second dimension separation, IEF strips were
sealed onto the top of NuPAGE Novex 4-12% Bis-Tris Zoom gels with 0.5% agarose
at 55-65°C. SDS-PAGE was then performed at 200 V for 40-50 minutes
according to manufacturer's protocol. Proteins in the 2D gel were visualized
by silver staining using Silver Quest silver staining kit (Invitrogen).
Comparative analysis of 2D gels
Protein expression patterns were compared with PD Quest 2D analysis
software using no more than five landmark protein spots. The `closest
neighbor' setting for computer generated matching was used to overlay gels
being compared.
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). MAb
OC.2 and an anti TfR MAb were used for depletion or selection of hematopoietic
cells. As previously reported (Hixson et
al., 1997; Hixson and Fowler,
1997), OC.2 is expressed on hematopoietic cells in a myeloid
lineage and appears on CMP-FLEC starting at stage III. In fetal liver, TfR has
been detected in previous investigations at high levels on erythroid
progenitors, immature granulocytes and monocytes but only at low levels on
most primitive hematopoietic stem cells
(Sposi et al., 2000). Our
emphasis on myeloid cells was derived from previous studies by Willenbring and
Grompe (Willenbring and Grompe,
2004) showing fusion between hepatocytes and cells committed to a
myelomonocytic lineage was the dominant source of bone-marrow derived liver
epithelial cells. Unfractionated fetal liver cells, shown by Dabeva et al.
(Dabeva et al., 2000) to
efficiently engraft in retrorsine/PH treated host livers, were used as a
positive control. Flow charts of different positive and negative selection schemes used to prepare isolates in Table 1 are shown in Fig. 1. Isolates shown in Table 1 were either depleted of (Table 1, isolates I,T,U) or enriched for (Table 1, isolates B,D,G,M,P,R,V) CMP-FLEC. As OV6+/OC.10-cells (CMP-FLECI) were rarely found even at ED16, isolates E, J and N were also essentially free of CMP-FLEC. As indicated in Table 1, fetal liver isolates depleted of CMP-FLEC varied in their content of hematopoietic cells and hepatoblasts. This is exemplified by isolate U, which should contain most of the hepatoblasts in ED18/19 fetal liver; isolates I and T, which should contain primarily hematopoietic cells in erythroid and myeloid lineages at ED16 and ED18/19, respectively; and isolate E which should contain all of the ED16 hepatoblasts and most of the nonparenchymal cells.
When isolates enriched for CMP-FLEC from a single developmental stage were analyzed by IIF or FACS analysis, 90-95% of the cells showed reactivity with the antibody used for positive selection. This is illustrated by representative cytospins of total and OC.2+ ED16 fetal liver isolates (Table 1, Fig. 2A,B; isolates A, F and B, respectively), total and OC.10+ ED18/19 fetal liver isolates (Table 1, Fig. 2C,D; isolates O and R, V respectively) and by representative FACS analysis of CMP-FLECIV isolated by single step selection with a mixture of MAb BD.2 and MAb OC.5 (Table 1, isolate P; Fig. 2E,F). FACS analysis showed that 94% of cells in the bound fraction (Fig. 2E) but only 2% of the unbound fraction (Fig. 2F, isolate Q) were positive for BD.2 and OC.5. Similar purity levels were achieved for CMP-FLECII isolated from ED16 fetal liver by two different protocols (Table 1, isolates D and M): the first involving depletion with MAb OC.2 followed by positive selection with MAb OC.10 (Table 1, Fig. 1A, isolate D); the second involving positive selection with MAb OC.10 after depletion with MAb OC.2 and anti-TfR, the latter step included to remove erythroid cells (Table 1, Fig. 1B, isolate M). Highly enriched CMP-FLECIII were obtained from ED18/19 fetal liver by a four-step protocol consisting of: depletion with MAb BD.2 and OC.5 to remove CMP-FLECIV (Table 1, Fig. 1C, isolate P); positive selection with MAb OC.10 (Table 1, Fig. 1C, isolate R); cleavage of the magnetic microbead anti IgM conjugate on the surface of OC.10+ cells with Miltenyi multisort release reagent removing the magnetic bead; and positive selection with MAb OC.2 (Table 1, Fig. 1C, isolate V). For comparative purposes, an isolate enriched for CMP-FLECII and CMP-FLECIII was also prepared from ED16 fetal liver in a single step positive selection with MAb OC.10 (Table 1, Fig. 1B, isolate G) to rule out adverse effects of a multi-step isolation protocol. Similar levels of engraftment were observed at 3 months with isolates G and R, as well as with corresponding CMP-FLEC depleted fetal liver fractions.
|
|
).
Donor hepatocyte colonies derived from CMP-FLECIII and CMP-FLECIV increase in size with increasing time after transplantation
To provide an estimate of the rate of colony expansion, the cross-sectional
area of discrete DPPIV+ colonies in frozen sections from host livers
transplanted with CMP-FLECII/III and CMP-FLECIV
(Table 1,
Fig. 1C, isolates R and P,
respectively) was determined using Image-Pro Plus 5.0 software
(Fig. 5). As coalescence of
colonies was seen as early as three months and was very common at 6 and 9
months post transplant (Fig.
6A, isolate P), DPPIV+ colonies were followed through several
serial sections and only those determined not to be a product of merging
colonies were chosen for analysis (Fig.
6B, isolate B). Although this approach clearly underestimated the
average area of donor CMP-FLEC, morphometric analysis could be performed only
on discrete colonies. Colonies representative of those used to determine
colony size (cross sectional area) at 3 and 9 months after transplantation are
shown in Figs 3 and
6, respectively. As shown in
Fig. 5, the average area of
colonies derived from CMP-FLECII/III (isolate R) and
CMP-FLECIV (isolate P) increased two- to fourfold during a 6 month
period starting at 3 months after transplantation, suggesting that donor
DPPIV+ CMP-FLEC were actively proliferating.
CMP-FLEC have a higher capacity for engraftment and expansion in the adult liver than hepatoblasts and other non-parenchymal cells
Surprisingly, when fetal liver isolates were depleted of CMP-FLEC, level of
engraftment dropped dramatically. While DPPIV+ hepatocyte colonies derived
from CMP-FLECII, CMP-FLECIII and CMP-FLECIV
were present in a high percentage of frozen sections stained histochemically
for DPPIV activity (50-70% nine months post transplant), examination of over
500 histochemically stained sections from five different regions of liver from
host rats receiving isolates with a high content of fetal hepatoblasts
(Table 1, isolates E,J,N,U;
Fig. 1B,C), yielded only a few
DPPIV+ donor colonies (Fig. 3I,
isolate U). These colonies were never large in size, and usually appeared as
clusters of fewer than eight cells displaying canalicular DPPIV activity. By
contrast, no DPPIV+ colonies were detected in animals receiving isolates
deficient in both hepatoblasts and CMP-FLEC
(Table 1, isolates I and T;
Fig. 1B,C).
|
CMP-FLEC lose cholangiocyte markers and differentiate into functionally mature hepatocytes following transplantation into retrorsine/PH treated DPPIV-host rat livers
Extensive phenotypic analysis using histochemical and IIF protocols was
carried out to determine the degree of functional differentiation displayed by
cells within donor-derived colonies. Results summarized in
Table 2 show that by 3-9 months
after transplantation, donor cells within DPPIV+ colonies derived from
CMP-FLECII, CMP-FLECIII and CMP-FLECIV lost
stage-specific cholangiocyte markers used for selection, remained negative for
later stage cholangiocyte markers and acquired an array of hepatocyte specific
markers not expressed by the original CMP-FLEC isolate prior to
transplantation that included H.4, H.1 and CYP2E1
(Fig. 8A,B,C respectively), and
CEACAM1, leucine aminopeptidase, glucose-6-phosphatase, H.2 and canalicular
ATPase (Table 2).
|
|
|
86%. Similar results were
obtained when spot match comparisons were made between 2D protein expression
patterns of donor DPPIV+ (Fig.
9) and host DPPIV-hepatocytes isolated from retrorsine/PH treated
rats at 1 year after transplantation of CMP-FLECIV
(Fig. 9D,E), a population of
CMP-FLEC expressing seven different MAb defined cholangiocyte markers (OV6,
OC10, OC.2, BD.2, OC.5, GGT, BD.1). Comparison of the 2D gel profiles from
DPPIV-host (Fig. 9D) and DPPIV+
donor hepatocytes (Fig. 9E)
yielded an 82% spot match (Table
3). These findings were consistent with differentiation of
CMP-FLECII and CMP-FLECIV into functionally mature
hepatocytes following transplantation into a retrorsine/PH-treated DPPIV-host
liver. The high degree of similarity observed between donor and host
hepatocytes was contrasted by the low percentage of matching spots found in a
comparison of 2D gel profiles from CMP-FLECII and host hepatocytes
at 1 year post-transplant (Table
3, 21% spot match) or in a comparison of 2D gel profiles from
donor hepatocyte and CMP-FLECII from which they were derived
(Table 3, 25% spot match).
|
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Based on a report by Faris et al., we also know that adult
liver contains significant levels of cholangiocytes with stage II phenotypes
(Faris et al., 2001).
Together, these findings suggest the possibility of isolating bipotent
progenitors suitable for cellular therapy of liver disease from newborn and
adult rats a possibility being examined in ongoing investigations.
|
). Whether there are significant differences among
various CMP-FLEC isolates from ED16 or ED18/19 fetal liver in rate and extent
of repopulation or efficiency of engraftment also remains unknown. To quantify
differences in these parameters, each cell isolate would have to be titrated
to determine efficiency of engraftment and colony size as a function of donor
cell number and time after injection. Although such studies could be
informative if carried out with sufficient numbers of animals to establish
statistical significance, they are beyond the primary goal of the present
investigation, which is to establish if cholangiocyte marker positive fetal
liver cells are committed to a cholangiocyte lineage.
|
|
). Results from phenotypic analysis in
Table 2 and
Fig. 8, showed that following
engraftment into host liver, CMP-FLEC organized into hepatic cords
morphologically indistinguishable from those in the host liver became
polarized, as evidenced by formation of well-defined bile canaliculae, and
lost cholangiocyte and acquired hepatocyte markers. When the extent of
hepatocytic differentiation was evaluated by 2D gel analysis, spot-match
comparison of more than 600 proteins showed that protein expression profiles
of CMP-FLEC derived hepatocytes were virtually identical to those of host
hepatocytes but differed significantly from expression profiles of the donor
CMP-FLEC (22% spot match). Together, these results constitute strong evidence
for differentiation of CMP-FLEC into functionally mature hepatocytes.
|
). We have
previously demonstrated the presence of immature cholangiocytes in 8-day
newborn rat liver with stage II phenotypes (OV6+/OC.10+/OC.2-), a finding
consistent with immaturity of the biliary system during the perinatal period
and persistence of AFP-positive cells for up to 4 weeks post partum
(Omori et al., 1997).
Experiments are in progress to determine whether these early cholangiocytes
retain the bipotentiality of fetal counterparts. We have also successfully
isolated cholangiocytes with stage III phenotypes from 4-week-old rats (Hixson
and Brilliant, unpublished), but whether these cells retain a capacity for
hepatocyte differentiation remains to be determined. The next logical step in
studying CMP-FLEC subpopulations will be to determine their ability to grow in
vitro in primary and long term culture, an ability previously demonstrated for
adult oval cells (Braun et al.,
1987; Hixson et al.,
1997; Radaeva and Steinberg,
1995; Yang et al.,
1993) and cholangiocytes
(Boylan et al., 2001;
McKay, 2000). Additionally, we
have previously shown CDE6 cells, a continuous line of oval cells isolated
from the liver of a rat maintained on a choline-deficient diet with ethionine,
can differentiate into hepatocytes following transplantation into the liver
(Hixson et al., 1997) showing
that oval cells maintain their bipotency after extended maintenance in
vitro.
Previous analysis of fetal, newborn and adult rat liver indicated that
OC.10 expression was restricted to ductal structures
(Hixson et al., 1997), leading
us to conclude that OC.10+ CMP-FLEC isolates were derived from epithelial
cells that compose the intrahepatic ducts. The ability of these immature
cholangiocytes to undergo maturation and morphogenesis into mature ductal
structures in the adult liver was revealed by the presence of DPPIV+ ducts
proximal to portal areas in host rats transplanted with OC.10+ CMP-FLEC.
However, the frequency of these DPPIV+ ducts was relatively low, raising the
possibility that the micro-environment in retrorsine/PH-treated liver was
favoring differentiation of CMP-FLEC along a hepatocyte lineage. Arguing
against this was the discovery of clusters of hepatocyte-like cells with
canalicular structures strongly positive for DPPIV in the spleen and pancreas
of retrorsine/PH rats injected intrasplenically with CMP-FLEC (Simper-Ronan
and Hixson, unpublished), findings supporting our previous suggestion that
hepatocyte differentiation is a default pathway for bipotent progenitors that
escape the influence of the portal mesenchyme
(Hixson et al., 1992).
In summary, our results show that CMP-FLEC retain bipotentiality as they progress towards a mature ductal phenotype and accumulate ductal cell lineage markers. We found that fetal liver isolates enriched for CMP-FLEC were able to repopulate up to 70% of host liver. By contrast, depletion of CMP-FLEC greatly reduced the extent of liver repopulation, suggesting that CMP-FLEC rather than hepatoblasts were the primary effectors of donor cell-mediated liver regeneration. The close similarity in 2D SDS-PAGE protein expression patterns of DPPIV-host hepatocytes and DPPIV+ hepatocytes derived from donor CMP-FLEC provided strong evidence for the differentiation of CMP-FLEC into functionally mature hepatocytes.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Alison, M. R. and Lovell, M. J. (2005). Liver cancer: the role of stem cells. Cell Prolif. 38,407 -421.[CrossRef][Medline]
Bisgaard, H. C., Parmelee, D. C., Dunsford, H. A., Sechi, S. and Thorgeirsson, S. S. (1993). Keratin 14 protein in cultured nonparenchymal rat hepatic epithelial cells: characterization of keratin 14 and keratin 19 as antigens for the commonly used mouse monoclonal antibody OV-6. Mol. Carcinog. 7,60 -66.[Medline]
Boylan, J. M., Anand, P. and Gruppuso, P. A.
(2001). Ribosomal protein S6 phosphorylation and function during
late gestation liver development in the rat. J. Biol.
Chem. 276,44457
-44463.
Brandt, B., Junker, R., Griwatz, C., Heidl, S., Brinkmann, O.,
Semjonow, A., Assmann, G. and Zanker, K. S. (1996). Isolation
of prostate-derived single cells and cell clusters from human peripheral
blood. Cancer Res. 56,4556
-4561.
Braun, L., Goyette, M., Yaswen, P., Thompson, N. L. and Fausto,
N. (1987). Growth in culture and tumorigenicity after
transfection with the ras oncogene of liver epithelial cells from
carcinogen-treated rats. Cancer Res.
47,4116
-4124.
Dabeva, M. D., Hwang, S. G., Vasa, S. R., Hurston, E., Novikoff,
P. M., Hixson, D. C., Gupta, S. and Shafritz, D. A. (1997).
Differentiation of pancreatic epithelial progenitor cells into hepatocytes
following transplantation into rat liver. Proc. Natl. Acad. Sci.
USA 94,7356
-7361.
Dabeva, M. D., Petkov, P. M., Sandhu, J., Oren, R., Laconi, E.,
Hurston, E. and Shafritz, D. A. (2000). Proliferation and
differentiation of fetal liver epithelial progenitor cells after
transplantation into adult rat liver. Am. J. Pathol.
156,2017
-2031.
Dunsford, H. A., Karnasuta, C., Hunt, J. M. and Sell, S.
(1989). Different lineages of chemically induced hepatocellular
carcinoma in rats defined by monoclonal antibodies. Cancer
Res. 49,4894
-4900.
Erickson, B. M., Thompson, N. L. and Hixson, D. C. (2006). Tightly regulated induction of the adhesion molecule necl-5/CD155 during rat liver regeneration and acute liver injury. Hepatology 43,325 -334.[CrossRef][Medline]
Faris, R. A., Monfils, B. A., Dunsford, H. A. and Hixson, D.
C. (1991). Antigenic relationship between oval cells and a
subpopulation of hepatic foci, nodules, and carcinomas induced by the
"resistant hepatocyte" model system. Cancer
Res. 51,1308
-1317.
Faris, R. A., Konkin, T. and Halpert, G. (2001). Liver stem cells: a potential source of hepatocytes for the treatment of human liver disease. Artif. Organs 25,513 -521.[CrossRef][Medline]
Gordon, G. J., Coleman, W. and Grisham, J. W.
(2000a). Temporal analysis of hepatocyte differentiation by small
hepatocyte-like progenitor cells during liver regeneration in
retrorsine-exposed rats. Am. J. Pathol.
157,771
-786.
Gordon, G. J., Coleman, W. B., Hixson, D. C. and Grisham, J.
W. (2000b). Liver regeneration in rats with
retrorsine-induced hepatocellular injury proceeds through a novel cellular
response. Am. J. Pathol.
156,607
-619.
Griwatz, C., Brandt, B., Assmann, G. and Zanker, K. S. (1995). An immunological enrichment method for epithelial cells from peripheral blood. J. Immunol. Methods 183,251 -265.[CrossRef][Medline]
Hayner, N. T., Braun, L., Yaswen, P., Brooks, M. and Fausto,
N. (1984). Isozyme profiles of oval cells, parenchymal cells,
and biliary cells isolated by centrifugal elutriation from normal and
preneoplastic livers. Cancer Res.
44,332
-338.
Hisatomi, Y., Okumura, K., Nakamura, K., Matsumoto, S., Satoh, A., Nagano, K., Yamamoto, T. and Endo, F. (2004). Flow cytometric isolation of endodermal progenitors from mouse salivary gland differentiate into hepatic and pancreatic lineages. Hepatology 39,667 -675.[CrossRef][Medline]
Hixson, D. C. and Fowler, L. C. (1997). Development and phenotypic heterogeneity of intrahepatic biliary epithelial cells. In Biliary and Pancreatic Ductal Epithelium (ed. A. E. Sirica and D. S. Longnecker), pp. 1-40. New York: Marcel Dekker.
Hixson, D. C., Faris, R. A., Yang, L. and Novikoff, P. (1992). Antigenic clues to liver development, renewal and carcinogenesis. In The Role of Cell Types in Hepatocarcinogenesis (ed. A. E. Sirica), pp.151 -182. Boca Raton: CRC Press.
Hixson, D. C., Chapman, L., McBride, A., Faris, R. and Yang,
L. (1997). Antigenic phenotypes common to rat oval cells,
primary hepatocellular carcinomas and developing bile ducts.
Carcinogenesis 18,1169
-1175.
Hixson, D. C., Brown, J., McBride, A. C. and Affigne, S. (2000). Differentiation status of rat ductal cells and ethionine-induced hepatic carcinomas defined with surface-reactive monoclonal antibodies. Exp. Mol. Pathol. 68,152 -169.[CrossRef][Medline]
Laconi, E., Oren, R. and Mukhopadhyay, D. K., Hurston, E.,
Laconi, S., Pani, P., Dabeva, M. D. and Shafritz, D. A.
(1998). Long-term, near total liver replacement by
transplantation of isolated hepatocytes in rats treated with retrorsine.
Am. J. Pathol. 153,319
-329.
Laurie, N. A., Comegys, M. M., Carreiro, M. P., Brown, J. F.,
Flanagan, D. L., Brilliant, K. E. and Hixson, D. C. (2005).
Carcinoembryonic antigen-related cell adhesion molecule 1a-4L suppression of
rat hepatocellular carcinomas. Cancer Res.
65,11010
-11017.
Lenzi, R., Liu, M. H., Tarsetti, F., Slott, P. A., Alpini, G., Zhai, W. R., Paronetto, F., Lenzen, R. and Tavoloni, N. (1992). Histogenesis of bile duct-like cells proliferating during ethionine hepatocarcinogenesis. Evidence for a biliary epithelial nature of oval cells. Lab. Invest. 66,390 -402.[Medline]
Lojda, Z. (1979). Studies on dipeptidyl (amino) Peptidase IV (glycyl-proline naphthylamidase). II. Blood Vessels. Histochemistry 59,153 -166.[CrossRef][Medline]
Maly, I. P. and Sasse, D. (1983). A technical note on the histochemical demonstration of G6Pase activity. Histochemistry 78,409 -411.[CrossRef][Medline]
McKay, R. (2000). Stem cells - hype and hope. Nature 406,361 -364.[CrossRef][Medline]
Nierhoff, D., Ogawa, A., Oertel, M., Chen, Y. Q. and Shafritz, D. A. (2005). Purification and characterization of mouse fetal liver epithelial cells with high in vivo repopulation capacity. Hepatology 42,130 -139.[CrossRef][Medline]
Nitou, M., Sugiyama, Y., Ishikawa, K. and Shiojiri, N. (2002). Purification of fetal mouse hepatoblasts by magnetic beads coated with monoclonal anti-e-cadherin antibodies and their in vitro culture. Exp. Cell Res. 279,330 -343.[CrossRef][Medline]
Omori, M., Evarts, R. P., Omori, N., Hu, Z., Marsden, E. R. and
Thorgeirsson, S. S. (1997). Expression of 
-Fetoprotein
and stem cell factor/c-kit system in bile duct ligated young rats.
Hepatology 25,1115
-1122.[CrossRef][Medline]
Petersen, B. E., Goff, J. P., Greenberger, J. S. and Michalopoulos, G. K. (1998). Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat. Hepatology 27,433 -445.[CrossRef][Medline]
Piazza, G. A., Callanan, H. M., Mowery, J. and Hixson, D. C. (1989). Evidence for a role of dipeptidyl peptidase IV in fibronectin-mediated interactions of hepatocytes with extracellular matrix. Biochem. J. 262,327 -334.[Medline]
Radaeva, S. and Steinberg, P. (1995). Phenotype
and differentiation patterns of the oval cell lines OC/CDE6 and OC/CDE22
derived from the livers of carcinogen-treated rats. Cancer
Res. 55,1028
-1038.
Rogler, L. (1997). Selective bipotential differentiation of mouse embryonic hepatoblasts in vitro. Am. J. Pathol. 150,591 -602.[Abstract]
Rutenberg, A. M., Kim, H., Fischbein, J. W., Hanker, J. S.,
Wasserkrug, H. L. and Seligman, A. M. (1969). Histochemical
and ultrastructural demonstration of -glutamyl transpeptidase activity.
J. Histochem. Cytochem.
17,517
-526.[Abstract]
Sandhu, J. S., Petkov, P. M., Dabeva, M. D. and Shafritz, D.
A. (2001). Stem cell properties and repopulation of the rat
liver by fetal liver epithelial progenitor cells. Am. J.
Pathol. 159,1323
-1334.
Sell, S. (1998). Comparison of liver progenitor cells in human atypical ductular reactions with those seen in experimental models of liver injury. Hepatology 27,317 -331.[CrossRef][Medline]
Sell, S. (2001). Heterogeneity and plasticity of hepatocyte lineage cells. Hepatology 33,738 -750.[CrossRef][Medline]
Sigal, S. H., Brill, S., Reid, L. M., Zvibel, I., Gupta, S., Hixson, D., Faris, R. and Holst, P. A. (1994). Characterization and enrichment of fetal rat hepatoblasts by immunoadsorption ("panning") and fluorescence-activated cell sorting. Hepatology 19,999 -1006.[CrossRef][Medline]
Sirica, A. E. (1995). Ductular hepatocytes. Histol. Histopathol. 10,433 -456.[Medline]
Sposi, N. M., Cianetti, L., Tritarelli, E., Pelosi, E., Militi, S., Barberi, T., Gabbianelli, M., Saulle, E., Kuhn, L., Peschle, C. et al. (2000). Mechanisms of differential transferrin receptor expression in normal hematopoiesis. Eur. J. Biochem. 267,6762 -6774.[Medline]
Suzuki, A., Zheng, Y., Kaneko, S., Onodera, M., Fukao, K.,
Nakauchi, H. and Taniguchi, H. (2002). Clonal identification
and characterization of self-renewing pluripotent stem cells in the developing
liver. J. Cell Biol.
156,173
-184.
Suzuki, A., Zheng, Y. W., Fukao, K., Nakauchi, H. and Taniguchi, H. (2004). Liver repopulation by c-Met-positive stem/progenitor cells isolated from the developing rat liver. Hepatogastroenterology 51,423 -426.[Medline]
Tanimizu, N., Nishikawa, M., Saito, H., Tsujimura, T. and
Miyajima, A. (2003). Isolation of hepatoblasts based on the
expression of Dlk/Pref-1. J. Cell Sci.
116,1775
-1786.
Tateno, C., Takai-Kajihara, K., Yamasaki, C., Sato, H. and Yoshizato, K. (2000). Heterogeneity of growth potential of adult rat hepatocytes in vitro. Hepatology 31, 65-74.[CrossRef][Medline]
Thompson, N. L., Hixson, D. C., Callanan, H., Panzica, M., Flanagan, D., Faris, R. A., Hong, W., Hartel-Schenck, S. and Doyle, D. (1991). A Fischer rat substrain deficient in dipeptidyl peptidase IV activity makes normal steady state RNA levels and an altered protein. Biochem. J. 273,497 -502.[Medline]
Wachstein, M. and Meisel, E. (1957). Histochemistry of hepatic phosphatases of a physiologic pH; with special reference to the demonstration of bile canaliculi. Am. J. Clin. Pathol. 27,13 -23.[Medline]
Willenbring, H. and Grompe, M. (2004). Delineating the hepatocyte's hematopoietic fusion partner. Cell Cycle 3,1489 -1491.[Medline]
Yang, L., Faris, R. A. and Hixson, D. C. (1993). Long-term culture and characteristics of normal rat liver bile duct epithelial cells. Gastroenterology 104,840 -852.[Medline]
This article has been cited by other articles:
|
|
 |
|
  N. Brezillon, D. Kremsdorf, and M. C. Weiss Cell therapy for the diseased liver: from stem cell biology to novel models for hepatotropic human pathogens Dis. Model. Mech., September 1, 2008; 1(2-3): 113 - 130. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||
