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Regular Articles |
¶
From the Marion Bessin Liver Research Center,*
the
Division of Gastroenterology, Hepatology, and
Nutrition,
and the Departments of
Medicine,
Cell
Biology,
and Pathology,¶
Albert Einstein College of Medicine, Bronx, New York
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800 to 1000 cells; 2)
bile duct structures connected to pre-existing host bile ducts; and 3)
mixed clusters containing both hepatocytes and bile duct epithelial
cells. Liver repopulation after 6 months was moderate (5 to 10%). In
retrorsine-treated liver, transplanted cells formed large
multilobular structures containing both parenchymal and bile duct cells
and liver repopulation was extensive (60 to 80%). When the
repopulating capacity of ED 14 FLEP cells transplanted into normal
liver was compared to adult hepatocytes, three important
differences were noted: 1) FLEP cells continued to proliferate at
6 months after transplantation, whereas adult hepatocytes
ceased proliferation within the first month; 2) both the number and
size of clusters derived from FLEP cells gradually increased throughout
time but decreased throughout time with transplanted mature
hepatocytes; and 3) FLEP cells differentiated into hepatocytes when
engrafted into the liver parenchyma and into bile epithelial cells when
engrafted in the vicinity of the host bile ducts, whereas adult
hepatocytes did not form bile duct structures. Finally, after
transplantation of ED 14 FLEP cells, new clusters of
DPPIV+ cells appeared after 4 to 6 months,
suggesting reseeding of the liver by transplanted cells. This study
represents the first report with an isolated fetal liver epithelial
cell fraction in which the cells exhibit properties of
tissue-determined stem cells after their transplantation into normal
adult liver; namely, bipotency and continued proliferation long
after their transplantation.
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Introduction |
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Studies conducted in the 1960’s also established that the proliferative activity of adult hepatocytes is sufficient to repopulate the liver after two-thirds partial hepatectomy (PH) and participation by stem/progenitor cells is not required.8 However, under conditions in which the proliferative capacity of hepatocytes is impaired, progenitor or facultative stem cells are activated to proliferate and differentiate into mature hepatocytes.9-12 Epithelial cell lines have also been established from neonatal and adult liver.13 Some of these cell lines express liver-specific genes under induced experimental conditions13-15 and can also differentiate into mature hepatocytes after transplantation into the adult liver,16,17 suggesting that they retain some stem cell-like properties.8
Several recent studies have used immunoselection with specific antibodies to separate and begin to define the properties of liver stem/progenitor cells.18-20 However, the ultimate test to determine whether a particular cell has stem cell properties is to follow its proliferation and phenotypic differentiation in vivo after transplantation. Fortunately, in rodents, several excellent models have been developed to study liver repopulation after transplantation of hepatic cells, the urokinase plasminogen activator (uPA) transgenic mouse,21,22 the fumaryl acetoacetate hydrolase null mouse,23 the retrorsine (Rs)-treated rat24 and the liver x-irradiated rat.25 In the former two models, selective repopulation of the liver by transplanted cells is based on continuous destruction of host hepatocytes by toxic21 or metabolic injury,23 and in the latter two models on compromised proliferative activity of endogenous hepatocytes resulting from DNA damage,24,25 possibly coupled with augmented apoptosis.26
In the rat, an excellent model has been developed to follow the fate of transplanted liver cells, the syngeneic dipeptidyl peptidase IV (DPPIV-) mutant Fischer (F) 344 rat.27 DPPIV is an exopeptidase that is highly expressed in epithelial cells of many organs and is also weakly expressed in endothelial cells. In the liver, it is expressed in both hepatocytes and bile duct epithelial cells, in the former in a characteristic bile canalicular distribution and in the latter in a diffuse cytoplasmic expression pattern.27-30 Both of these cellular phenotypes can be readily detected and distinguished from each other by DPPIV enzyme histochemistry.31
In the DPPIV- mutant F344 rat, we previously reported that mature hepatocytes can fully repopulate the liver.24 This required pretreatment of the animals with Rs, a DNA-alkylating agent that disrupts cell cycle progression in hepatocytes, so that these cells cannot proliferate. Therefore, when wild-type hepatocytes are transplanted in conjunction with two-thirds PH in Rs-treated rats, transplanted cells selectively proliferate and repopulate the liver. However, in the absence of Rs treatment, transplanted hepatocytes have no proliferative advantage over endogenous hepatocytes and selective liver repopulation does not occur.24
Because the fetal liver contains highly proliferative epithelial cells32 that are progenitors of both hepatocytes and cholangiocytes,4-7 we reasoned that these cells should have a higher proliferative capacity than mature hepatocytes and might be able to selectively repopulate the liver under normal experimental conditions. Previously, we determined that embryonal day (ED) 14 rat liver contains a subpopulation of bipotent epithelial cells that can differentiate into hepatocytes or bile duct epithelial cells, depending on their engraftment site in the liver parenchyma.33 In the present study, we have followed the properties of transplanted ED 14 fetal liver epithelial progenitor (FLEP) cells and the ability of these cells to repopulate the liver throughout a period of 6 months. We have found that ED 14 FLEP cells expand very rapidly in Rs-treated animals with extensive liver repopulation (up to 60 to 80%) and form complete new liver lobules containing both hepatocytes and bile ducts. However, significant liver repopulation by transplanted ED 14 FLEP cells (5 to 10%) also occurs in the normal liver. Most interestingly, the number and size of DPPIV+ cell clusters increases progressively throughout time, clusters of both unipotent (hepatocytic or bile ductular) and bipotent (mixed hepatocytic and bile ductular) phenotype are observed, some transplanted cells are still proliferating 6 months after transplantation, and there is progressive reseeding of the liver with transplanted cells throughout time. Thus, in contrast to adult hepatocytes, which can repopulate the liver only when there is substantial selection pressure, early fetal liver epithelial cells can repopulate the liver in a normal hepatocellular environment.
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Materials and Methods |
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Rs and diaminobenzidine, glycine-proline-4-methoxy-ß-naphtylamide, 4-methoxy-ß-naphtylamide, glycine-proline p-nitroaniline, sodium nitrite, ammonium sulfamate, N-1(-naphthyl)ethylenediamine dihydrochloride were purchased from Sigma Chemical Co (St. Louis, MO). Rat endothelial cell antigen, RECA-1, clone HIS 52, was from Accurate Chemical and Scientific Corporation (Westbury, NY). DPPIV monoclonal antibody (monoclonal mouse anti-rat CD26), clone OX61, was from Harlan Sera-Lab Limited (Loughborough, England). Vectastain Elite ABC kit was from Vector Laboratories, Burlingame, CA. Rabbit anti-rat red blood cell IgG was from Rockland (Gilbertsville, PA). Radioactive 35S-UTP (SJ603) and cytokeratin (CK)-19 antibody (RPN 1165) were obtained from Amersham Life Science Products (Arlington Heights, IL). Autoradiographic emulsion, type NBT2, was purchased from Eastman Kodak Company (New Haven, CT).
Animals and Animal Treatment
Timed, pregnant Fischer (F) 344 rats were purchased from Taconic Farms (Germantown, NY). Mutant DPPIV-deficient (DPPIV-) F344 rats were obtained from the Special Animal Core of the Liver Research Center, Albert Einstein College of Medicine. All studies with animals were conducted under protocols approved by the Animal Care Use Committee of the Albert Einstein College of Medicine and were in accordance with National Institutes of Health Guidelines. Rs treatment of the animals was as described previously.24 For studies in normal rats, animals of 150 to 180 g were used as cell transplantation recipients.
Isolation and Transplantation of FLEP Cells
FLEP cells were isolated on ED 14, 16, or 18 from normal DPPIV+ pregnant rats by a modification of the procedure of Sigal and colleagues,34 as described previously.33 Freshly isolated FLEP (4.0 x 105) cells were transplanted through the portal vein immediately after two-thirds PH. FLEP cells were transplanted into normal and Rs-treated, male or female DPPIV- F344 rats. The livers were removed and the tissue analyzed 1, 2, 4, and 6 months after cell transplantation. In most experiments, four animals were used for each time point. To compare the extent of liver repopulation by adult hepatocytes and fetal hepatoblasts, equal numbers of cells (4.0 x 105) were injected into the portal vein of normal animals in conjunction with PH and livers were removed 1, 2, 4, and 6 months after cell transplantation.
Histochemical Detection and Enzyme Assay for DPPIV
The histochemical detection of DPPIV-positive transplanted cells in the liver of mutant DPPIV- F344 rats was performed on frozen liver sections, as described previously.31 DPPIV enzyme activity was determined in homogenates of liver tissue by a modification of the procedure of Nagatsu and colleagues.35 The substrate used for the enzyme assay was glycyl-proline p-nitroanilide (Sigma), which is cleaved by DPPIV, releasing p-nitroaniline. The latter is subjected to diazotization and converted into an azo dye with maximum optical density at 540 nm. The readings were taken against a control sample processed in the presence of 10 mmol/L Diprotin A (Sigma), a specific inhibitor of DPPIV. A standard linear curve was prepared with 0.05 to 1.0 µm p-nitroaniline, dissolved in 2% methanol. In brief, 0.1 ml of 3 mmol/L substrate solution (prepared in 1% Triton X-100) was added to 0.1 ml of 0.1 to 1% liver homogenate, prepared in 0.1 mol/L glycine buffer, pH 8.7, containing 1% Triton X-100. The samples were incubated for 30 minutes at 37°C. The reaction was stopped by adding 0.8 ml of 5% HClO4 and centrifuged for 10 minutes at 4000 rpm in the cold. One half ml of clear supernatant was transferred to a new tube and 0.5 ml of 0.2% sodium nitrite was added for diazotization. After 10 minutes incubation in the cold, 0.5 ml of 0.5% of ammonium sulfamate was added to decompose excess sodium nitrite and the samples were incubated for an additional 2 minutes at room temperature. One ml of a 0.05% solution of N-(1-naphthyl)ethylenediamine (Sigma) in 95% ethanol was then added and the samples were incubated for 30 minutes at 37°C to form the azo dye salt. Readings were taken at 540 nm against a control processed the same way, but to which 10 mmol/L Dipronin A was added.
Dual in Situ Hybridization and Immunohistochemistry Labeling for DPPIV and Histone 3 mRNA Expression
Dual in situ hybridization/immunohistochemistry labeling for DPPIV and histone 3 mRNA was performed on frozen sections, as described previously.36 First, the sections were processed with a monoclonal antibody against DPPIV (anti-rat CD26) and peroxidase activity was revealed by diaminobenzidine. In situ hybridization was then performed, using a 35S-labeled histone-3 antisense riboprobe to detect cells in S phase, as previously reported.36
Determination of Liver Repopulation by Transplanted FLEP Cells
Analysis of liver repopulation was conducted by two independent methods: 1) scanning slides histochemically stained for DPPIV enzyme activity with a high-resolution Polaroid CS-600 scanner (Polaroid Corp., Cambridge, MA) and measuring the red stained areas (DPPIV+) versus the total area of the liver section, using Adobe Photoshop; and 2) biochemical determination of DPPIV enzyme activity in liver homogenate, as described above. DPPIV enzyme activity of the livers after transplantation versus the enzyme activity of normal liver, taken as 100, was used to calculate the percent liver repopulation.
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Results |
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In our previous studies of liver repopulation with adult
hepatocytes, pretreatment of recipients with Rs was used to augment
proliferation of transplanted cells. Therefore, we first transplanted
ED 14 FLEP cells under the Rs/PH protocol to gauge their proliferative
potential under maximally induced host conditions. As shown in Figure 1
, there was substantial proliferation of
transplanted ED 14 FLEP cells within 1 month, as evidenced by circular
clusters containing 50 to 100 DPPIV+ cells in
5-µm sections (Figure 1A)
, increasing to >250
DPPIV+ cells at 2 months (Figure 1B)
, 500 to 1000
DPPIV+ cells at 4 months (Figure 1C)
and >1000
DPPIV+ cells at 6 months (Figure 1D)
. At 4 and 6
months, transplanted cell clusters became confluent (Figure 1, C and D)
, encompassing multiple lobules. We also observed
DPPIV+ cholangiocytes that formed mature
DPPIV+ bile ducts. DPPIV+
bile duct structures were evident as early as 1 month after ED 14 FLEP
cell transplantation, but became more numerous and fully developed at
later time points (Figure 1, C and D
, arrows).
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In the absence of Rs pretreatment, clusters of
DPPIV+ cells were still observed at 1 month after
ED 14 FLEP cell transplantation but were less numerous and contained
fewer cells than observed in Rs-treated animals (Figure 2A)
. However, the number and size of
DPPIV+ cell clusters increased throughout time
(Figure 2; B, C, and D
) and at 4 to 6 months after ED 14 FLEP cell
transplantation, mature bile duct structures represented a prominent
feature (Figure 2D)
. In both Rs-treated and normal rats, PH was
required for proliferation of transplanted ED 14 FLEP cells.
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Liver repopulation by ED 14 FLEP cells versus adult hepatocytes transplanted into normal liver showed vastly different results, because there was a progressive increase in the number and size of DPPIV+ clusters throughout time with ED 14 FLEP cells, but no increase in the number or size of clusters after the first month with adult hepatocytes. In addition, DPPIV+ bile duct structures were observed only very rarely after transplantation of adult hepatocytes.
To quantitate liver repopulation in the normal rat using FLEP cells
compared with adult hepatocytes, we determined the number of
clusters/cm2, the number of cells/cluster, the
estimated number of cell divisions/cluster, and the percentage of liver
repopulation. As shown in Table 1
, with
ED 14 FLEP cells, there was a progressive increase in the number of
clusters/cm2
throughout time, with a threefold
increase between 1 and 2 months, a threefold increase between 2 and 4
months, and a fivefold increase between 4 and 6 months. The number of
cells/cluster also increased, as did the calculated average number of
cell divisions/cluster (assuming that each cluster was derived from a
single cell, that there was no loss of cells from these clusters, and
that the general shape of the clusters is spherical). It should be
noted, however, that in the 6-month specimens, small clusters were not
included in the calculation of the number of cells/cluster. These small
clusters were often multiple and located in the vicinity of large
clusters (see below). The percentage of liver repopulation by ED 14
FLEP cells also increased throughout time, with the greatest increase
occurring between 4 to 6 months, reaching a maximum of 5 to 10%. In
selected areas, liver repopulation was as high as 20 to 30%.
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. The number of
clusters/cm2
at 1 month after cell
transplantation was much higher with adult hepatocytes compared to ED
14 FLEP cells (70 versus 1.7). However, the average size of
clusters produced by hepatocytes at 1 month after cell transplantation
was much smaller than with ED 14 FLEP cells, the number of
clusters/cm2
did not increase throughout time
(they actually decreased by 40%), the number of cell divisions per
cluster (3 to 4) also did not increase and the overall percentage of
liver repopulation was nil after the first month. Phenotypic Characterization of DPPIV+ Cell Clusters at 6 Months after ED 14 FLEP Cell Transplantation
To further characterize individual clusters produced from ED 14 FLEP cells, 50 serial sections were prepared from a normal rat 6 months after cell transplantation. Individual clusters were mapped by color digital photography of sequential fields of the 25th section, preparation of a large composite print from the sequential photomicrographs and assignment of a number to each cluster. One hundred and five clusters were examined through the complete series of serial sections. In most instances, the entire cluster could be viewed from top to bottom, although some clusters spanned beyond the 50 sections.
Table 2
shows the proportions of
unipotent (hepatocytic or bile ductular) and bipotent (hepatocytic/bile
ductular) cells in the fraction used for ED 14 FLEP cell
transplantation, as determined by 
-fetoprotein (AFP) and CK-19
expression,33
and the proportions of hepatocytic, bile
ductular and mixed clusters (containing both hepatocytes and mature
bile ducts) at 6 months after cell transplantation, as determined by
histological analysis. In addition to the three types of hepatic
epithelial cell clusters, we also observed clusters of transplanted
endothelial cells. Examples of each cell cluster type are shown in
Figure 3
; A, hepatocytic; B, bile
ductular; C, mixed, and D, endothelial. The vast majority of cells in
the ED 14 FLEP cell fraction used for transplantation were unipotent,
88% hepatocytic and 9% bile ductular; only 3% were bipotential
(Table 2)
. However, at 6 months after cell transplantation, the
majority of clusters were of mixed phenotype (56%), comprised of large
numbers of hepatocytes together with mature, multilobulated bile ducts
in one or more regions (Figure 3C)
. By analysis of serial sections,
most new bile ducts could be traced back to portal regions. In some
instances, duct prolifes were present at one or both extreme ends of a
large hepatocytic cluster. Virtually all of the large clusters (ie,
those containing >500 cells and spanning >30 sections) were of mixed
phenotype. Pure hepatocytic clusters (31%, example shown in Figure 3A
)
and bile duct cell clusters (5%, example shown in Figure 3B
) generally
spanned 10 to 25 serial sections for hepatocytes and 6 to 12 serial
sections for bile ducts. The former were generally located in the
parenchyma and the latter in conjunction with portal regions.
Endothelial cell clusters (7% of total, example shown in Figure 3D
)
were initially thought to represent FLEP cells that had proliferated
but remained undifferentiated. However, dual immunohistochemistry
revealed that they were positive for both DPPIV and anti-rat RECA-1
antibody, which is specific for a surface protein of endothelial
cells37
(data not shown).
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From the above analysis, it was not possible to distinguish
whether the mixed clusters are derived from bipotent progenitor cells
or from small aggregates of transplanted cells containing both
unipotent hepatocytic and bile ductular progenitors. However, two
observations with serial sections suggested that a substantial number
are truly bipotent. In some areas, we observed multiple small to
mid-sized clusters of hepatocytes close to a portal region (Figure 4)
. On following these clusters through
the serial sections, initially they were negative for
DPPIV+ bile duct cells (Figure 4, A and B)
.
However, when some came into contact with the portal space,
DPPIV+ bile duct cells were noted (Figure 4C)
.
Continuing further through the sections, when the transplanted
hepatocytic clusters were no longer in contact with the portal region
(which was still present in the section), DPPIV+
bile duct cells were no longer present (Figure 4, D and E)
. We
interpret these results as evidence that some cells in proliferated
clusters within the hepatic parenchyma seem to have the ability to
cross the limiting plate and differentiate into bile duct epithelial
cells. In other instances, we observed mature
DPPIV+ bile duct structures in portal regions in
which a few cells in the adjacent parenchyma in continuity with the
duct exhibited a differentiated hepatocytic morphology (Figure 5)
. These clusters, which are primarily
of biliary phenotype, also seem to be derived from bipotent cells.
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A most interesting observation during the 6-month period after
transplantation of FLEP cells into the normal adult rat liver was a
50-fold increase in the number of clusters/cm2
(Table 1)
. This increase was most accentuated between 4 to 6 months. On
analysis of serial sections, most of the new clusters were located in
regions adjacent to large mixed clusters and were of either
hepatocytic, mixed, or ductal phenotype (Figure 6)
. These secondary clusters were often
multiple and occasionally showed bile ductular elements when they came
into contact with pre-existing portal ducts, again suggesting
bipotency.
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To further evaluate the repopulation potential and phenotypic
properties of FLEP cells at different times during liver development,
studies were conducted in normal animals with FLEP cells isolated from
ED 16 and ED 18 fetal rat liver. With ED 16 FLEP cells, 2
clusters/cm2
were observed at 1 or 2 months after
cell transplantation and 3 to 4 clusters/cm2
were present at 4 months. The clusters observed were of small to medium
size and were predominantly hepatocytic (Figure 7, A and B)
. However, several
medium-sized clusters with a mixed phenotype were also identified
(Figure 7C)
. The ductular elements in these mixed clusters were smaller
and less well developed than those observed with ED 14 FLEP cells. A
significant number of pure bile duct clusters was also observed (Figure 7D)
. With ED 18 FLEP cells, sparsely scattered, very small hepatocytic
clusters were observed at 1 month after cell transplantation (Figure 7E)
, comparable in size with clusters obtained after transplantation of
adult hepatocytes. At 4 months after transplantation of ED 18 FLEP
cells, DPPIV+ transplanted cell clusters were
still present but were not increased significantly in size or number
(Figure 7F)
.
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Histochemical analysis of DPPIV expression in
DPPIV- rats transplanted with
DPPIV+ ED 14 FLEP cells showed a progressive
increase in the size and number of DPPIV+ cell
clusters in both Rs-treated and normal rats in the 6-month period after
cell transplantation. To quantitate liver repopulation in the whole
liver, we developed an assay for DPPIV enzyme activity in liver
homogenate. As shown in Figure 8
, DPPIV
enzyme activity produced by transplanted ED 14 FLEP cells increased
progressively throughout time in both Rs-treated (Figure 8A)
and normal
rats (Figure 8B)
. These results were consistent with the percentage of
liver repopulation determined by histochemical analysis (see Table 1
).
By both methods, the greatest increase in liver repopulation in normal
rats transplanted with ED 14 FLEP cells occurred between 4 and 6
months, which most likely reflects reseeding of the liver by
transplanted ED 14 FLEP cells.
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, in both Rs-treated and normal liver at
4 and 6 months after ED 14 FLEP cell transplantation,
DPPIV+ cells in S phase were readily identified.
These results demonstrate the long-term proliferative capacity of
transplanted rat ED 14 FLEP cells and support our other evidence for
continuous repopulation of the liver by these cells.
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In the present study, there was extensive proliferation and liver repopulation with ED 14 cells in Rs-treated rats (60 to 80% in 6 months), but more importantly, significant repopulation also occurred in normal rats (5 to 10% in 6 months). In both Rs-treated and normal rats, PH was required to obtain significant repopulation by transplanted ED 14 FLEP cells, probably related to increased seeding or engraftment efficiency after PH. Although the seeding efficiency (determined by the number of clusters observed at 1 month) was 5- to 10-fold higher with adult hepatocytes compared to ED 14 FLEP cells, long-term repopulation did not occur with adult hepatocytes in normal rats. From these studies, we conclude that ED 14 FLEP cells have a clear proliferative advantage after transplantation to the liver compared to mature hepatocytes.
Stem Cell Properties of ED 14 FLEP Cells
Stem cells are generally considered to exhibit the following properties: 1) self replication or renewal, 2) differentiation into two or more specific cell phenotypes, and 3) long-term repopulation of the host under appropriate circumstances.38 Because there are no specific markers for liver stem cells, it was not possible to demonstrate self-renewal of undifferentiated liver stem cells in our model system. However, we have obtained considerable evidence for bipotency of transplanted ED 14 FLEP cells and long-term liver repopulation. 1) Although only 3% of cells in the transplanted fraction were dually marked for AFP and CK-19, >50% of the clusters observed after 6 months contained both hepatocytes and mature bile ducts (termed "mixed" clusters), suggesting that bipotent progenitor cells preferentially engrafted compared to unipotent progenitors. 2) Mixed clusters were the largest, indicating that they have greater proliferative activity than clusters containing only hepatocytes or bile duct cells. 3) Many mixed clusters also contained mature bile ducts in different regions, often separated by large distances. In other instances, newly synthesized bile ducts were located in a single portal region at one edge of a very large cluster. In both cases, it would seem that the original transplanted cells engrafted at or near a junction between the portal space and parenchymal cords (ie, the limiting plate) and then began to proliferate in both directions. Thus, ED 14 FLEP cells have the flexibility to differentiate into both hepatic epithelial cell types and cues from the lobular zone in which they are engrafted seem to direct their differentiation.
Other observations on serial sections further support the bipotency of transplanted ED 14 FLEP cells. First, when some small to moderately sized cell clusters with an apparent hepatocytic phenotype contacted a specific portal region, a few transplanted cells differentiated into mature bile duct cells. Secondly, we observed very well-differentiated bile ducts with a small number of mature hepatocytes emanating from one region of the duct. These findings are consistent with a recent report by Paku and colleagues39 in the rat 2-AAF/PH model in which proliferating epithelial progenitor cells within the biliary compartment have been shown to migrate across the ductular basement membrane into the surrounding hepatic parenchyma. Therefore, it would seem that bidirectional flow of hepatic epithelial cells occurs across the ductular basement membrane and that these cells may then differentiate into hepatocytes or bile duct cells depending on local factors in the cellular compartment in which they reside.
A third observation that became apparent at 4 and 6 months after cell transplantation was that many large clusters of mixed phenotype contained small secondary clusters in their immediate vicinity of hepatocytic, mixed, or ductal phenotype. This suggested that transplanted cells separated from their original clusters and continued to proliferate and differentiate along both the hepatocytic and ductal lineages. Nonetheless, although the above evidence seems compelling, formal proof that large mixed clusters are derived from bipotent cells will require viral genetic marking studies.
Long-term liver repopulation has been achieved previously with mature
hepatocytes in both rats and mice.22-26
However, in all
of these studies, strong selection pressure was needed to favor the
proliferation and/or survival of transplanted hepatocytes, and mature
bile ducts were not produced, although this could reflect selective
injury to host hepatocytes in these models. In addition, serial
transplantation and repopulation has been achieved with mature
hepatocytes in the fumaryl acetoacetate hydrolase null mouse,
indicating that these highly differentiated cells exhibit far more
proliferative capacity than previously imagined.40
Although the level of repopulation with ED 14 FLEP cells was much less
in the present study, it occurred in a nonselective environment, under
conditions in which mature hepatocytes do not repopulate the liver. We
also noted a reduced proliferative capacity and repopulation by ED 16
and ED 18 FLEP cells. The property of forming secondary clusters or
reseeding of the liver also appears to be unique to ED 14 FLEP cells.
The number of clusters increased 50-fold during the 6-month period
after cell transplantation, and from serial section analysis, we
estimate that the entire liver contained 10,000 to 20,000 clusters.
Finally, direct evidence for long-term proliferative activity of
repopulating FLEP cells was obtained by demonstrating histone 3 mRNA
expression in DPPIV+ cells 6 months after their
transplantation into the normal liver.
Factors Driving Liver Repopulation by ED 14 FLEP Cells
The mechanism and factors leading to preferential accumulation and repopulation of the liver by ED 14 FLEP cells are not known. Acute reduction in liver mass stimulates an immediate proliferative response in the remaining liver, first in hepatocytes and then in nonparenchymal cells.8 During this period, both endogenous hepatocytes and transplanted cells are stimulated to proliferate. However, once the liver mass returns to normal (within 10 to 14 days), there is no apparent stimulus for continued proliferation of transplanted (or endogenous) cells. Consistent with this notion, the size of transplanted cell clusters derived from adult hepatocytes did not increase after 1 month. However, with transplanted ED 14 FLEP cells, proliferation continued progressively during the entire 6-month experimental period. Why ED 14 FLEP cells continued to proliferate in the host liver after the apparent stimulus for liver growth had ceased remains unclear. The mechanism by which PH enhances engraftment of ED 14 FLEP cells is also not known, but this is probably related to the induction of specific hormones, cytokines, and growth factors, as well as other changes in the liver microenvironment and extracellular matrix that occur during liver regeneration.41-43
In the field of hematopoietic cell transplantation, making space for transplanted cells to repopulate the host has been a long-held concept; however, recent studies have suggested that this may not be necessary under some circumstances.44,45 Our present studies in the liver suggest that making space for transplanted cells is not necessary for expansion of ED 14 FLEP cells once they have engrafted. Whether transplanted ED 14 FLEP cells release signals that cause a dropout of neighboring hepatocytes and their replacement by newly proliferated transplanted cells is an intriguing question that needs to be explored.
Plasticity of Transplanted Cells
Recent studies have shown that crude bone marrow cells or purified hematopoietic stem cells can differentiate into hepatocytes on engraftment into the liver46-50 and into brain or muscle phenotypes when transplanted into these respective tissues.51-53 An interesting question is whether hematopoietic cells in our transplanted cell fraction might contribute to liver repopulation. It is also possible that some transplanted hematopoietic or fetal liver stem/progenitor cells initially remain dormant within the liver and become activated throughout time. However, we consider this to be unlikely, because we did not observe the late appearance of DPPIV+ cells in the liver in the absence of PH or activation of dormant ED 14 FLEP cells if PH was performed weeks to months after cell transplantation.
Other Models for Normal Liver Repopulation
Most previous attempts to repopulate the normal rat liver have transplanted large numbers of adult hepatocytes (up to 1 x 108 cells) into the spleen or have used repeated transplantation of cells.54-56 Other studies have used ED 18 and older fetal liver tissue transplanted to the spleen or on solid support matrices implanted intraperitoneally,57-60 as well as mature hepatocytes attached to microcarrier beads.61 In addition, isolated fetal hepatocytes from late gestation when transplanted intraportally into Nagase analbuminemic rats gave partial correction of serum albumin when atrophy of nontransplanted lobes was subsequently induced by portal branch ligation.62 All of these approaches, however, have limited practical application.
Utility of ED 14 FLEP Cells
The advantages of using early fetal liver stem/progenitor cells for cell transplantation therapy are the small size of the cells (10 to 12 mµ), so that they might disperse more broadly throughout the liver parenchyma, their lesser tendency to obstruct the liver sinusoids and produce portal hypertension, and their ability to repopulate an essentially normal liver. These cells also replace parenchymal hepatocytes and form new bile ducts for an extended period after their transplantation. The present study demonstrates, in vivo, the tissue-determined stem cell potential of early rat FLEP cells and suggests that such cells may be useful for therapeutic liver repopulation, as well as to serve as a vehicle for ex vivo gene therapy.
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Acknowledgements |
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Footnotes |
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Supported in part by National Institutes of Health grants RO1 DK17609, RO1 DK56496, and P30 DK41296 (all to D. A. S.) and the Gail I. Zuckerman for Research in Chronic Liver Diseases of Children (to M. D. D.).
Accepted for publication June 25, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-fetoprotein mRNA expression in the retrorsine model for hepatocytes transplantation. Cancer Res 1998, 58:5825-5834This article has been cited by other articles:
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  D. Haridass, Q. Yuan, P. D. Becker, T. Cantz, M. Iken, M. Rothe, N. Narain, M. Bock, M. Norder, N. Legrand, et al. Repopulation Efficiencies of Adult Hepatocytes, Fetal Liver Progenitor Cells, and Embryonic Stem Cell-Derived Hepatic Cells in Albumin-Promoter-Enhancer Urokinase-Type Plasminogen Activator Mice Am. J. Pathol., October 1, 2009; 175(4): 1483 - 1492. [Abstract] [Full Text] [PDF]
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 P Sancho-Bru, M Najimi, M Caruso, K Pauwelyn, T Cantz, S Forbes, T Roskams, M Ott, U Gehling, E Sokal, et al. Stem and progenitor cells for liver repopulation: can we standardise the process from bench to bedside? Gut, April 1, 2009; 58(4): 594 - 603. [Abstract] [Full Text] [PDF]
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 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]
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 R. Miki, N. Tatsumi, K. Matsumoto, and Y. Yokouchi New primary culture systems to study the differentiation and proliferation of mouse fetal hepatoblasts Am J Physiol Gastrointest Liver Physiol, February 1, 2008; 294(2): G529 - G539. [Abstract] [Full Text] [PDF]
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N. Kamo, K. Yasuchika, H. Fujii, T. Hoppo, T. Machimoto, T. Ishii, N. Fujita, T. Tsuruo, J. K. Yamashita, H. Kubo, et al. Two populations of Thy1-positive mesenchymal cells regulate in vitro maturation of hepatic progenitor cells Am J Physiol Gastrointest Liver Physiol, February 1, 2007; 292(2): G526 - G534. [Abstract] [Full Text] [PDF]
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 R. Simper-Ronan, K. Brilliant, D. Flanagan, M. Carreiro, H. Callanan, E. Sabo, and D. C. Hixson Cholangiocyte marker-positive and -negative fetal liver cells differ significantly in their ability to regenerate the livers of adult rats exposed to retrorsine Development, November 1, 2006; 133(21): 4269 - 4279. [Abstract] [Full Text] [PDF]
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 S. Eventov-Friedman, H. Katchman, E. Shezen, A. Aronovich, D. Tchorsh, B. Dekel, E. Freud, and Y. Reisner Embryonic pig liver, pancreas, and lung as a source for transplantation: Optimal organogenesis without teratoma depends on distinct time windows PNAS, February 22, 2005; 102(8): 2928 - 2933. [Abstract] [Full Text] [PDF]
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H. Strick-Marchand, S. Morosan, P. Charneau, D. Kremsdorf, and M. C. Weiss Bipotential mouse embryonic liver stem cell lines contribute to liver regeneration and differentiate as bile ducts and hepatocytes PNAS, June 1, 2004; 101(22): 8360 - 8365. [Abstract] [Full Text] [PDF]
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T. Cantz, D. M. Zuckerman, M. R. Burda, M. Dandri, B. Goricke, S. Thalhammer, W. M. Heckl, M. P. Manns, J. Petersen, and M. Ott Quantitative Gene Expression Analysis Reveals Transition of Fetal Liver Progenitor Cells to Mature Hepatocytes after Transplantation in uPA/RAG-2 Mice Am. J. Pathol., January 1, 2003; 162(1): 37 - 45. [Abstract] [Full Text] [PDF]
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J.-E. Allain, I. Dagher, D. Mahieu-Caputo, N. Loux, M. Andreoletti, K. Westerman, P. Briand, D. Franco, P. Leboulch, and A. Weber Immortalization of a primate bipotent epithelial liver stem cell PNAS, March 19, 2002; 99(6): 3639 - 3644. [Abstract] [Full Text] [PDF]
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 H. Malhi, A. N. Irani, S. Gagandeep, and S. Gupta Isolation of human progenitor liver epithelial cells with extensive replication capacity and differentiation into mature hepatocytes J. Cell Sci., January 7, 2002; 115(13): 2679 - 2688. [Abstract] [Full Text] [PDF]
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