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From the Hiroshima Tissue Regeneration Project,*
Hiroshima Prefecture Joint-Research Project for Regional Intensive,
Japan Science and Technology Corporation, Hiroshima Prefectural
Institute of Industrial Science and Technology, Higashihiroshima; the
Department of Second Surgery,
School of
Medicine, Hiroshima University, Higashihiroshima; and the Developmental
Biology Laboratory,
Department of Biological
Science, Graduate School of Science, Hiroshima University,
Higashihiroshima, Hiroshima, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sigal and colleagues5 actually compared the extent of autofluorescence and granularity of hepatocytes among embryos, sucklings, and adults by FACS. Hepatocytes increased their granularity and autofluorescence as the liver developed from fetus to suckling, and to adult.5 Conversely the proportion of S-phase cells progressively declined during this developmental process.5 They also showed that the adult liver contains small and mononuclear hepatocytes whose granularity and autofluorescence were comparable to fetal hepatocytes.5 Our previous studies support the results obtained by the cited authors and suggest that not only the fetal but also the adult rat liver contains a population of hepatocytes that are small in size and highly replicative.2,3
Laconi and co-workers6 described a retrorsine/partial hepatectomy rat model to assess the repopulation potential of transplanted hepatocytes in which exogenous hepatocytes can almost completely replace the host hepatocytes. The model is based on the mitoinhibitory effect of a retrorsine, pyrrolizidine alkaloid, on hepatocytes in the resident liver where transplanted hepatocytes can proliferate. The host and transplanted hepatocytes were distinguished by using dipeptidyl peptidase IV-deficient (DPPIV-) mutant Fischer rats and their wild-type counterparts (DPPIV+), respectively.6
The present study was performed to determine whether the growth potential of hepatocytes is heterogeneous and is correlated with their size, and the extent of their granularity and autofluorescence also in vivo as in vitro. The PHs, SHs, SH-R2s, and SH-R3s were prepared from DPPIV+ rats and were transplanted into the retrorsine/partial hepatectomy DPPIV- rat model to assess their replication potential as an index of growth ability. This in vivo experiment clearly demonstrated that SH-R3 hepatocytes are highly proliferative also in vivo as compared to SH-R2s and PHs. The present study strongly suggests that the relationship between the size and growth potential of hepatocytes has some biological meaning in the physiological and pathological processes taking place in the liver.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fractions of SHs and PHs were prepared as described before3 with some modifications. Disaggregated liver cells were obtained from 10-week-old Fischer male rats by the two-step collagenase perfusion method1 and were centrifuged at 50 x g for 2 minutes. The pellet was centrifuged through 45% Percoll at 50 x g for 24 minutes.3 The pellet thus obtained was further centrifuged at 50 x g for 1 minute, and the pellet and supernatant were used as a fraction of PHs and SHs, respectively.
SHs and PHs were subfractionated by a cell sorter, FACS Vantage (Becton Dickinson, Mountain View, CA) with a 100-µm nozzle.3 Fluorescence excited at 488 nm was measured through a 530-nm filter (FL1) and a 575-nm filter (FL2) with a 4-decade logarithmic amplification. To measure physical characteristics of the cells, a linear amplification was used for the forward scatter, which is a measure of cell size, and a 4-decade logarithmic amplification for the side scatter, which is a measure of cytoplasmic complexity. The optical bench was calibrated at a fixed amplitude and a photomultiplier voltage using fluorescent polystyrene beads (Fluorosbrite Calibration Grade 6-µm YG microspheres; Polysciences, Inc., Warrington, PA) and the instrument was used in the conditions in which these beads fell in the same peak channels. For all cell preparations tested, propidium iodide was added to cell suspensions at a concentration of 1 µg/ml, and the cells excluding the dye (viable cells) were sorted for further investigations. Data obtained by FACS experiments were analyzed using a CELLQuest software (Becton Dickinson). Hepatocytes which had excluded dyes of trypan blue were photographed and their diameters were determined using a NIH Image version 1.62 (NIH, Bethesda, MD).
Hepatocyte Transplantation
Replicative potential of hepatocytes prepared from DPPIV+ male Fischer rats was determined as an index of growth ability by transplanting them into DPPIV- Fisher female rats following the protocol described by Laconi.6 The DPPIV- rats weighing 130 to 140 g were given two intraperitoneal injections of retrorsine of 30 mg/kg body weight, 2 weeks apart. Four weeks after the second injection, the animals were subjected to two-thirds partial hepatectomy. Hepatocytes prepared as above from DPPIV+ male rats were transplanted into the above DPPIV- rats via the portal vein. The number of transplanted cells was 2 x 105 cells for PHs and SHs, and 1.5 x 105 cells for SH-R2s and SH-R3s. Each of these four fractions of hepatocytes was transplanted to five individual rats. All animals were harvested at 21 days after transplantation and the liver was examined for detecting the transplanted hepatocytes.7,8 Cryosections (10-µm thick) were prepared from the liver, fixed in ice-cold acetone for 5 minutes, air-dried, and washed for 5 minutes in ice-cold 95% ethanol. The sections were air-dried and incubated for 40 to 60 minutes in a substrate reagent consisting of 0.5 mg/ml gly-pro-methoxy-ß-naphthylamide (Sigma Chemical Co., St. Louis, MO), 1 mg/ml Fast Blue BB (Sigma Chemical Co.), 100 mmol/L Tris-maleate, pH 6.5, and 100 mmol/L NaCl. Then the sections were washed with phosphate-buffered saline and fixed in 10% formaldehyde. All tissue sections were counterstained with hematoxylin. An identical transplantation of DPPIV+ SHs was made as a control experiment, in which the host mutant animals did not receive the retrorsine exposure, but received partial hepatectomy. Only small colonies containing two or three transplanted SHs were formed in this transplantation experiment, indicating the usability of this retrorsine-hepatectomy animal model to assess the in vivo growth potential of hepatocytes.
Morphometric Analysis
Transplanted hepatocytes homed to the liver, started to replicate there, and formed small colonies at an early phase after transplantation. We made semiserial sections with a thickness of 10 µm at intervals of 100 µm from liver specimens. Different parts of a colony were seen in several different serial sections when the diameter of the colony was >100 µm, which corresponds to 7,850 µm2 in area. We selected the section in which the longest diameter for the colony concerned was seen, and calculated the colony area using this diameter. For a colony whose diameter was <100 µm, we assumed that the diameter measured in a section is the longest diameter of the colony and calculate the colony area using this diameter. We only measured the colonies that consisted of more than eight transplanted hepatocytes that were oval or round in shape. To quantitate the area of these colonies, 40 portal areas were examined in each animal at 21 days after transplantation. The volume of a colony was calculated from the area of the colony assuming that the colony was in a form of sphere and that the cross-section was made along the maximum diameter of the sphere. To calculate the mean diameter of cells in colonies formed from transplanted hepatocytes, the area and cell number of 10 colonies were measured. The mean cell number in a colony was calculated using parameters of the area or volume of the colony, and the mean cell diameter of hepatocytes.
Detection of Liver-Specific Markers
Cryosections with a thickness of 5 µm were prepared from the
liver transplanted with hepatocytes, fixed in -20°C acetone for 5
minutes, and used for immunohistochemical detection of liver-related
proteins. Primary antibodies used were: rabbit anti-rat albumin
(Cappel, Durham, NC), mouse monoclonal antibody against cytokeratin 19
(CK19, Amersham), and rabbit anti-rat 
-fetoprotein antiserum
(-FP, a gift from Dr. T. Mitaka, Sapporo Medical University).
The antibodies were visualized by a Vectastain ABC kit (Vector
Laboratories, Burlingame, CA) using diaminobenzidine as a
substrate.
Engraftment Index of Transplanted Hepatocytes
PHs and SHs were prepared from DPPIV+ rats and 2 x 105 cells of each of them were transplanted into a retrorsine-treated and hepatectomized mutant DPPIV- rat liver via the portal vein. We made liver cryosections after weighing liver mass at 48 hours after transplantation. The approximate numbers of total hepatocytes were calculated from the liver weight using the value of (115 ± 15) x 106 hepatocytes/g liver.9 We calculated a ratio of transplanted to host hepatocytes by counting their numbers on tissue sections. engraftment index was calculated by the ratio of transplanted to host hepatocytes, total number of hepatocytes of the liver, and the number of transplanted hepatocytes.
Statistical Analysis
The area of colonies formed from transplanted hepatocytes was measured with a NIH image version 1.62 software and the data were analyzed with Stad view version 5.0 software (SAS Institute Inc., Cary, NC). Results were expressed as mean ± SE, and the significance of difference was analyzed by Student’s t-test when data were normally distributed and otherwise by Mann-Whitney rank sum test. A P value of <0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SHs and PHs were prepared and placed on nonadhesive dishes. Their
diameter was determined as a measure of cell size. SHs showed a
diameter of 16.3 to 28.1 µm, and PHs showed a diameter of 22.9 to
34.2 µm, the average being 21.5 ± 0.4 µm and 26.5 ± 0.4
µm, respectively, as reported previously.3
We showed
that SHs could be subfractionated into SH-R2s and SH-R3s by
FACS.3
The former showed a greater extent of granularity
and autofluorescence than the latter (Figure 1A)
. In contrast, PHs produced only one
population (PH-R2s) that corresponded to the SH-R2s with respect to
their size, and the extent of granularity and autofluorescence. SHs
were fractionated into SH-R2s and SH-R3s by FACS (Figure 1B)
. Mean
diameter of freshly isolated SH-R2s and SH-R3s was 22.5 ± 0.13
µm (n = 5) and 17.7 ± 0.10 µm
(n = 5), respectively. The difference between
SH-R2s and SH-R3s was statistically significant
(P < 0.0001, Student’s t-test).
These size characteristics were in good agreement with the previously
reported ones.3
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PHs and SHs were prepared from DPPIV+ rats
and 2 x 105
cells of each of them were
transplanted into a retrorsine-treated and hepatectomized mutant
DPPIV- rat liver via the portal vein.
Megalocytes appeared in the retrorsine-treated liver from 7 to 14 days
after partial hepatectomy (see, for example, Figure 5
), indicating that
the retrorsine actually produced its effect on the liver in the present
study as reported previously.6
Their engraftment index
determined at 48 hours after transplantation was 11.0 ± 6.4%
(n = 3) for PHs and 8.9 ± 2.2%
(n = 3) for SHs.
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. Size of
colonies formed from SHs, PHs, and SH-R3s was apparently heterogeneous
as compared with that from SH-R2s, which was confirmed by quantitative
analysis of colony areas as described in Figure 3
. Apparently, colonies of SHs and SH-R3s
were larger in their areas than those of PHs and SH-R2s, respectively.
To quantitatively compare the colony size formed from each of
transplanted hepatocytes, colony areas were measured in 40 portal areas
of each recipient animal (Figure 3)
. The mean colony area was
calculated from these measurements (Table 1)
. The colony area of SHs and SH-R3s was
1.5-fold to twofold larger than that of PHs and SH-R2s,
respectively. The differences between PHs and SHs, and between SH-R2s
and SH-R3s, were statistically significant (P <
0.0001, Mann-Whitney sum rank test). To obtain a more practical index
of the growth potential of these hepatocytes we calculated the mean
colony volume assuming that the colonies were clonally expanded and
that each colony formed a sphere. The diameter of such a hypothetical
sphere was measured from the semiserial sections with the thickness of
10 µm prepared from liver specimens at intervals of 100 µm as
described in Materials and Methods section and as shown in Table 1
.
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. The number per area formed from SHs
and SH-R3s was twofold and 2.2-fold larger than that from PHs and
SH-R2s, respectively. Likewise, the number per volume was
threefold and 3.2-fold larger, respectively. The average cell division
number of transplanted PHs, SHs, SH-R2s, and SH-R3s during 21 days was
calculated as 7 to 8, 9 to 10, 8 to 9, and 10 to11, respectively (Table 2)
. Thus, we concluded that the growth potential of hepatocytes is also
heterogeneous in vivo and is correlated with their size.
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The cells in the colonies of SH-R2s and SH-R3s were characterized
with respect to their expression of three lineage-specific markers of
liver cells: albumin as a universal marker of hepatocytes, -FP as a
phenotype of immature or neoplastic hepatocytes, and CK19 as a marker
of bile duct epithelial cells. The cells in colonies of SH-R2s and
SH-R3s expressed albumin at a high level comparable to the surrounding
host hepatocytes (Figure 4, C and D)
.
There was no expression of -FP (Figure 4, E and F)
and CK19 (Figure 4, G and H)
in the cells in colonies formed from both SH-R2s and
SH-R3s. These results indicated that both SH-R2s and SH-R3s replicated
retaining phenotypes of hepatocytes.
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on the same liver section where the
SH-R3-derived colony was seen (Figure 5A)
. We measured the size of the
hepatocytes in colonies of the host-derived SH-like progenitor cells
and in colonies formed from SH-R3s at 7 days after transplantation.
Their diameter was 12.8 ± 0.46 µm for the former and 12.0
± 0.08 µm for the latter, indicating that these two populations of
hepatocytes were indistinguishable in their size
(P = 0.1285). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study was performed to investigate whether smaller
hepatocytes exhibit a higher growth potential than larger ones also
in vivo. SHs and PHs were isolated from
DPPIV+ rats and were transplanted into
retrorsine-treated and partial hepatectomized
DPPIV- rats. De Roos and
colleagues15
investigated the engraftment index by
transplanting BrdU-labeled rat hepatocytes into normal rat liver via
the portal vein. Transplanted cells were primarily lost at 24 hours,
and 7% of the injected cells were engrafted.15
These
hepatocytes migrated into the liver parenchyma from the portal venules
at 48 hours.15
Gupta and co-workers16
reported that hepatocytes transplanted via spleen were translocated
from sinusoids into liver plates between 16 and 20 hours after
transplantation and were suggested to be engrafted into the liver
parenchyma later than 24 hours. The engraftment index obtained in the
present study at 48 hours after transplantation was 8.9 ± 2.2%
for SHs and 11.0 ± 6.4% for PHs, which was comparable to that
reported by De Roos et al.15
We noticed that the number of
engrafted single hepatocytes observed at 48 hours was smaller that of
colonies formed from them at 21 days after transplantation. This
difference might be explained as initially observed single hepatocytes
in the periportal regions (zone 1) grew to form colonies, some of which
split and formed new colonies in the mid-regions (zone 2) as Laconi et
al6
suggested.
The colonies formed from transplanted cells at 21 days were examined in
all residual livers of the recipients. Our present in vivo
assay on growth potential of subfractionated hepatocytes showed that
SH-R3s were most proliferative, followed by SHs, SH-R2s, and PHs in
this sequence. This result was consistent with the previous in
vitro study.3
Therefore, we concluded that the growth
potential of hepatocytes is heterogeneous and is correlated with their
size, and the extent of their granularity and autofluorescence in
vivo as in vitro. It was suggested that the sex of host
rats affects the growth of transplanted hepatocytes in the
retrorsine/hepatectomy model.6
The transplants grew faster
in the male hosts than in the female. The present study used female
rats as hosts. A previous retrorsine/hepatectomy model using female
hosts showed that the transplanted hepatocytes underwent 12 to 13
cell divisions for 2 months,6
which was comparable to that
obtained for PHs in the present study. Therefore, it can be said that
our in vivo assay on the growth potential was done properly.
Thus, we convincingly concluded that SHs is of higher growth ability
than PHs, and SH-R3s than SH-R2s. Overturf and
colleagues17
performed experiments to relate the
regenerative capacity of mouse hepatocytes with the hepatocyte’s size
in their serial transplantation study. The donor hepatocytes were
fractionated into small (16 µm)-, medium (21 µm)-, and large (27
µm)-sized cells using centrifugal elutriation, and separately
transplanted into recipient mice. Their results were apparently against
ours. The growth ability during the first round of transplantation was
higher when the size was larger. The reason of this discrepancy is not
clear at present, but might reside in the difference of species between
rats and mice. In addition, it should be noted that the in
vivo growth potential assessed by the colony formation of the
transplanted hepatocytes actually represents the repopulation
potential, which does not necessarily represent their replication
potential, because there are several steps for the cells before the
colony formation such as streaming in the blood, homing through
sinusoids, engraftment into the liver plate, proliferation, and the
colony formation.16
Many known and unknown factors might
affect each of these steps, depending on the species of animals and the
model of transplantation experiments.
Previously, we characterized the phenotypes of cells in colonies formed
in vitro by SHs and PHs and found some cells became both
CK19- and 1-antitrypsin-positive,3
suggesting the
phenotypic plasticity of hepatocytes in vitro. We asked if
SH-R3s also show such plasticity in vivo by examining the
expression of marker proteins specific to hepatocytes, immature
hepatocytes, and bile duct epithelial cells. The cells in colonies
formed from transplanted SH-R3s were positive to albumin, but negative
to both CK19 and -FP, which differed from the results of the
in vitro study. It seems that there is a regulation in
vivo under which hepatocytes stably express hepatocytic
phenotypes, but not express the biliary ones.
Recently, the presence of a population of SH-like progenitor cells was demonstrated in the retrorsine-exposed rat.11 These SHs emerged after the liver was partially hepatectomized, expanded, and replaced the entire liver mass. These cells expressed phenotypes characteristic of fetal hepatoblasts, oval cells, and fully differentiated hepatocytes. The average diameter of these SH-like progenitor cells was estimated to be 13.1 µm at 3 days after the operation.11 In the present study we also noticed an emergence of the colony made of such host-derived SHs in the SH-R3s transplanted liver. These host-derived and transplant-derived colonies of SHs co-existed in the same liver. The diameter of hepatocytes in colonies derived from SH-R3s and the host-derived SH colonies was similar, 12.0 and 12.8 µm, respectively. It is tempting to speculate that SH-R3s that we isolated from the normal liver play an important role(s) to repair the tissues when the liver is severely damaged. In this situation SH-R3s are activated and act as the SH-like progenitor cells that were identified by Gordon et al.11 The identity between our SH-R3s and the SH-like progenitor cells is currently under investigation.
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Acknowledgements |
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Footnotes |
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Supported by a grant from the Hiroshima Tissue Regeneration Project.
Shigeru Katayama and Chise Tateno contributed equally to this work.
Accepted for publication September 25, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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