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Regular Articles |
From the Departments of Molecular and Medical
Genetics*
and Pediatrics,
Oregon Health Sciences University, Portland, Oregon; and the Department
of Pathology,
Texas Children’s Hospital,
Houston, Texas
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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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We have created a mouse model of the human disease hereditary tyrosinemia type I which is due to the lack of the enzyme fumarylacetoacetate hydrolase (FAH).4 FAH catalyzes the last step of the tyrosine degradation pathway, and animals lacking this enzyme develop severe hepatic dysfunction due to the accumulation of toxic upstream metabolites.5 It has been demonstrated in both humans and mice that positive selection for FAH-expressing hepatocytes occurs in an FAH-deficient liver.6,7 The drug 2-(2-nitro-4-trifluoro-methylbenzoyl)1,3-cyclohexedione (NTBC) can prevent the neonatal lethality and liver dysfunction of FAH-deficient mice.8 Mice treated with NTBC and then removed from treatment develop acute hepatocellular damage and die within 2 months. We have used this model to show the regenerative potential of hepatocytes by transplanting genetically marked wild-type cells into FAH-deficient mice. Wild-type cells transplanted into the spleen or portal vein repopulate the recipient animal to >90% within 6 to 8 weeks.6 As few as 1000 donor cells were sufficient to rescue FAH-deficient animals and restore liver function.6 Serial transplantation of limiting numbers of cells was performed and seven generations of animals were successfully repopulated.9 This experiment demonstrated that the regenerative potential of the serially repopulating hepatocytes was similar to that of hematopoietic stem cells and exceeded 100 cell doublings. This high capacity for cell division raised the question whether liver progenitor or stem cells may be responsible for the serial repopulation. Interestingly however, the only donor-derived cells in the repopulated livers were hepatocytes.9 No evidence for the emergence of biliary epithelium or any other hepatic cell type was found. Oval cells, on the other hand can give rise to both biliary epithelium and hepatocytes.10 This finding therefore was more consistent with the view that serial repopulation was carried out by differentiated hepatocytes and not progenitor cells.
To date all liver repopulation experiments reported by us and others have used unfractionated suspensions of liver cells isolated by in situ collagenase perfusion.11 These suspensions consist mostly of hepatocytes, but also contain various other hepatic cells including biliary epithelium, stellate cells, Kupffer cells, endothelial cells, and fibroblasts. Therefore two main hypotheses regarding liver repopulation can be formulated: 1) the majority of cells, ie, the differentiated hepatocytes themselves, are capable of repopulation; 2) a rarer subpopulation of cells (stem cells/progenitor cells) with high regenerative capacity is responsible. It is also possible that both hypotheses are correct, that differentiated hepatocytes are capable of short-term repopulation, and that a specific subpopulation is required for serial transplantation.
Several experimental approaches can be used to distinguish these possibilities, and three of these are reported here. First, centrifugal elutriation was used to isolate three distinct populations of adult murine hepatocytes that differed in size. The individual fractions were transplanted in competition with unfractionated hepatocytes of a genetically distinct mouse strain. Second, we wanted to test the effect of prior cell division on the ability to repopulate. The stem cell model of repopulation predicts aging and reduced capacity for cell division of differentiated hepatocytes.12 Third, we used retroviral tagging to test the clonality of liver repopulation.13
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following mouse strains were used for the competitive
transplantation experiments: the FAH
exon5
strain of mice,4
which has previously been described by
this laboratory; ROSA-26 ß-galactosidase transgenic animals obtained
from and described by P. Soriano14
; the Fanconi anemia
complementation group C (FANCC) knockout mice created by this lab and
previously described15
; and the dopamine D2 receptor
(DOPA) knockout mice kindly donated by M. Low.16
All of
these mice were of the inbred 129SvJ strain and congenic for the
purposes of transplantation. All FAHexon5
breeders and mutant animals were treated with NTBC-containing water at
a concentration of 7.5 mg/l (provided by S. Lindstedt, Gothenborg,
Sweden). NTBC is a potent inhibitor of 4-OH-phenylpyruvate dioxygenase,
the second enzyme of tyrosine catabolism.17
Treatment with
this drug prevents liver failure in FAH-mutant mice and is necessary
for the animals to survive and breed. For genotyping of the
FAHexon5 and the FANCC heterozygotes, a
3-primer polymerase chain reaction was carried out on 200 ng of
tail-cut DNA as previously described.4,15
Staining of a
small portion of tail from the ROSA-26 animals for the presence of
ß-galactosidase activity was used to genotype these animals. For the
DOPA mice, genomic DNA was digested with the restriction enzyme
EcoRV (Pharmacia, Uppsala, Sweden), and then Southern
blots were probed for the presence of the neomycin phosphotransferase
(neo) gene used in creating the knockout construct.
Animal care and experiments were all in accordance with the Guidelines
of the Department of Animal Care at Oregon Health Sciences University.
Elutriation, Centrifugation, Separation, and Sizing
Elutriation was performed using a J2-M Beckman centrifuge with a JM6 rotor and large chamber (15 ml). After the initial isolation by in situ perfusion with collagenase, the cells were resuspended in 4 ml of elutriation media (5% Dulbecco’s minimum essential medium (DMEM) with 0.003% (w/v) DNase and 5 mmol/L ethylenediaminetetraacetic acid (EDTA)) at 4°C to avoid clumping. With the centrifuge spinning at 1000 rpm (4°C), flow rates were determined for separate pump settings, and then the cells were added at a flow rate of 7 ml/minute. Fractions (150 ml) were isolated from the following flow rates of 8 ml/minute to 30 ml/minute in 2-ml/min gradients. The fractions were then spun down and resuspended in 2 ml of DMEM with 10% fetal calf serum (FCS). One hundred microliters were diluted in 20 ml of Isoton fluid and sized using a Coulter Multisizer II (Hialeah, FL). Data were analyzed using MULTISIZER AccuComp 1.19 software. Isolated fractions of the appropriate size and purity were used for transplantation.
Cells were transplanted into the inbred
FAHexon5 strain of SV129 mice. For
transplantation all animals used were between 6 and 10 weeks old.
Transplantation Procedures
Parenchymal hepatocytes were isolated from congenic animals by a
two-step collagenase perfusion.18
Cell number and
viability were determined by Trypan blue exclusion in a hemocytometer.
The donor cells were resuspended in 100 µl of DMEM (GIBCO BRL,
Gaithersburg, MD) with 15% FCS and injected
intrasplenically19
into FAHexon5
recipient animals. All mutant mice were kept on NTBC until the time of
transplantation, when it was discontinued. The weights of experimental
animals were measured weekly.
Retrovirus Tagging
The G1FSvNa retrovirus construct and methods used for in vivo and ex vivo hepatocyte transduction were as previously described.6,20 Briefly, for ex vivo experiments 500,000 isolated mouse hepatocytes were plated per 100-mm dish containing DMEM with 10% FCS, 2 mmol/L glutamine, and the antibiotics penicillin and streptomycin. The cells were cultured for 36 hours, then the media were removed and replaced with 5 ml of retroviral supernatant containing 8 µg/ml polybrene. After 4 hours the supernatant was removed, and 10 ml of SUM3 media21 was added to each plate. The cells were harvested 36 to 48 hours later by trypsin treatment and intrasplenically transplanted. For in vivo experiments, 0.5-ml aliquots of supernatant were directly injected via the portal vein as previously described.6
Southern Blot Analysis
For Southern blots, liver DNA was isolated from liver that was freshly obtained or frozen at -80°C. Random 5- x 5-mm sections of tissue from the left lower lobe were used for the DNA isolation. Capillary transfer and hybridizations were performed according to standard protocols.22 For detection of the neomycin phosphotransferase gene used in generating the transgenic animals, isolated DNA was digested with BamHI (Pharmacia) and probed with a 680-bp PstI (Pharmacia) fragment isolated from a neomycin phosphotransferase cDNA. A Beckman SI Phosphoimager was used to quantitate relative band intensities.
To quantitate the relative ratios of ROSA to genetically unmarked wild-type cells, Southern blots were performed as described above, and the intensity of the neo signal was quantitated with a Beckman SI Phosphoimager. This signal was compared with a Southern blot standard curve generated by mixing known ratios of pure wild-type and ROSA DNA. The control samples were derived by 1) harvesting liver DNA from FAH-mutant mice transplanted with known mixes (1:9, 1:1, and 9:1) of unfractionated cells from the two strains and b) mixing pure DNA from the two strains at known ratios. In addition to loading the gels with exactly the same amount of total DNA, the neo-signal derived from the nonparenchymal cells of the recipient mutant mouse was used as an internal standard. This was possible, because the overall degree of repopulation (~95%) was similar in all experiments.
Histology and Immune Histology
For immunohistochemical analysis of isolated cells, approximately 20,000 cells were resuspended in 100 µl of DMEM and centrifuged onto poly L-lysine slides in a Cytospin III (Shandon, Pittsburgh, PA) at 500 x g for 5 minutes. The slides were fixed in 10% formalin for 10 minutes and then transferred to 80% ethanol.
The slides were incubated with a polyclonal rabbit antibody to rat FAH23 and mouse keratin 19 (CK19).24,25 The FAH antibody was diluted in phosphate-buffered saline (PBS), pH 7.4, and applied at concentrations of 1:300,000 at 37°C for 30 minutes. The CK19 antibody was used at a 1:5,000 dilution. Endogenous peroxidase activity was blocked with 3% H2O2 and methanol. Avidin and biotin pretreatment was used to prevent endogenous staining. The secondary antibody was biotinylated goat anti-rabbit IgG used at 1:250 dilution (Vector Laboratories, Burlingame, CA; BA-1000). Color development was performed with the aminoethylcarazole detection kit from Venatan Medical Systems (Tucson, AZ; catalog item 250–020).
ß-Galactosidase tissue staining consisted of washing sections of freshly harvested liver twice in PBS for 5 minutes. The liver sections were fixed by soaking in cold PBS containing 2% glutaraldehyde and 1% formaldehyde for 10 minutes and then washed twice with PBS. The sections were then stained overnight in 1 mmol/L ß-galactosidase, 3 mmol/L ferricyanide, 3 mmol/L ferrocyanide, 10 mmol/L N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 1 mmol/L MgCl2, and 1 mmol/L NP40 at 37°C.
For ß-galactosidase staining of cell suspensions, cells were fixed for 5 minutes in cold PBS with 2% glutaraldehyde and stained overnight in the same solution described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In many studies of liver regeneration, small, periportal zone 1
hepatocytes proliferate most readily. For example, after 15% partial
hepatectomy, most thymidine-labeled cells are found in this
location.26
We therefore hypothesized that hepatocytes of
different sizes may differ in their liver repopulation potential. To
address this question hepatocytes isolated from congenic 129SvJ animals
were sorted by density using centrifugal elutriation and transplanted
into FAH-mutant recipients. Centrifugal elutriation allowed the
collection of differently sized fractions of hepatocytes without any
loss of viability. The cell size profile within each fraction was then
determined by analyzing a small aliquot of each fraction in a Coulter
Multisizer II instrument with Multisizer AccuComp 1.19 software.
Analysis of unfractionated cells in 6 independent experiments revealed
the existence of three distinct populations varying in size and
contribution to the liver mass (Figure 1)
. The majority of the hepatocytes were
approximately 21 µm in diameter, with the other two fractions
consisting of hepatocytes of approximately 16 and 27 µm in diameter.
Further characterization of these fractions consistently showed that
the 21-µm fraction provided 60 to 75% of all of the cells in the
suspension. The other two fractions of isolated hepatocytes displayed
yields of 5 to 15% for the 16-µm-diameter population and 15 to 25%
for the 27-µm-diameter fraction. The use of different medium flow
rates through the elutriator permitted the isolation of relatively pure
populations of each of these three sizes (>80%, Table 1
). The profiles from each enriched
fraction used in one competitive transplantation and the profile of an
unfractionated population of hepatocytes are depicted in Figure 1
.
Quantitative data regarding the size distribution of elutriated cells
are given in Table 1
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Elutriated cells were always transplanted in competition with
unfractionated cells from a second animal carrying a neo
marker in a different chromosomal location. Both populations were from
donor animals of the same sex and age. The unfractionated cells were
always isolated first, and therefore the time from isolation to
transplantation was shorter for elutriated cells, providing them with a
potential mild survival advantage. All mice used (recipients and
donors) were from the same inbred mouse strain, 129SvJ, and thus
differed only in their neo marker integration site. Only
heterozygotes were used, and there is no evidence that any of the
targeted gene disruptions (D2 receptor, FANCC, and ROSA-26) have an
effect on hepatocyte division in the heterozygous state. Cells from the
ROSA-26 strain were usually used for elutriation. However, to be
certain that the marker insertions had no effect on repopulation
capacity, FANCC hepatocytes were used for elutriation in some
experiments and competed with unsorted ROSA-26 cells. The different
neo marker integration sites permitted the accurate
measurement of the contribution of the different populations to overall
repopulation by quantitative Southern blot analysis (Figure 2)
. In addition, the
ß-galactosidase-expressing ROSA-26 cells provided a rapid visual
estimate of the degree of repopulation contributed by "blue cells"
(Figure 3)
. The size-enriched populations
of cells were transplanted at ratios of 1:1, 1:4, or 1:5 to the
unsorted cells to more easily determine their capacity for
repopulation. In all experiments, the repopulation ability of the
isolated fractions never exceeded 45% when transplanted at a 1:1 ratio
(Table 2)
. Therefore none of the enriched
fractions had a repopulation capacity substantially superior to
unfractionated cells. Overall, the 21-µm fraction appeared to compete
the best of the three fractions, showing an average percent
repopulation of ~35% in 1:1 and 21% in 1:4 transplants. The extent
of repopulation contributed by the 27-µm fraction was only slightly
lower, averaging ~33% in 1:1 and 15% in 1:4 transplants. However,
the differences between these two groups were not significant when
analyzed using the Student’s t-test
(P = 0.85 for the 1:1 transplants). In contrast,
the fraction containing the isolated 16-µm cells competed least well.
Small cells never contributed more than 8% when transplanted at a 1:1
ratio. The differences in the 1:1 competitive repopulation assays were
statistically significant (P < 0.0001 for the
16-µm:21-µm comparison; P = 0.01 for the
16-µm:27-µm comparison). The total number of cells transplanted was
also varied. There was some variation in the measured degree of
repopulation even in animals that received the exact identical mix of
cells. For example one of the mice that received a 1:1 mix of 27-µm
cells and unfractionated competitors had only 17% repopulation,
whereas the other two had 40% and 42%, respectively (Table 2)
. The
most likely explanation for the observed variation is the area of
tissue sampling. For the elutriation experiments, we isolated DNA from
randomly selected small chunks of liver.
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. The cells
were also negative for CK-19 staining, indicating the absence of duct
cells in the preparations.
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Our previous serial transplantation experiments had generated animals whose livers were repopulated with liver cells that had undergone many rounds of cell division.9 The vast majority of cells in these repopulated livers were fully differentiated hepatocytes of donor origin. We hypothesized that the repopulation ability of these "aged" hepatocytes may differ from cells derived from young donor animals, which had divided many fewer times.
We therefore performed competitive repopulation experiments with
hepatocytes from FAHexon5 animals that had
been repopulated with donor cells from heterozygote D2 receptor
knockout mice. Animals from both round 1 (average number of cell
divisions ~15) and round 2 of serial transplantation (average number
of cell divisions ~30) were used. The cells were transplanted in
competition with hepatocytes isolated from 2-month-old naive ROSA-26
mice. In other experiments we used serially transplanted ROSA-26
hepatocytes in competition with normal congenic wild-type hepatocytes.
These experiments showed (Table 3)
that
hepatocytes from naïve animals and serially transplanted
hepatocytes (from either one or two rounds) competed equally well.
Impressively, cells from a round 7 serial transplantation recipient (at
least 60 cell divisions) competed effectively with unexpanded cells.
Therefore, neither fresh, previously untransplanted hepatocytes nor
serially transplanted hepatocytes had a selective advantage in
therapeutic repopulation in our model.
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In the hematopoietic system, retroviral marking has been used
extensively to ask questions about the clonality of bone marrow
repopulation and the relative frequency of cells capable of long-term
reconstitution of the organ.13
We therefore decided to use
this same approach to liver repopulation. When Moloney murine leukemia
virus-based retroviral vectors transduce cells, their genome is
integrated into the host chromosome, but the site of integration is
random.27
Therefore, each proviral integration site
represents a clonal marker. Predominance of a single clone or a few
clones in a population can be detected as an integration site-specific
junction fragment on Southern blot analysis. We have previously shown
that FAHexon5 hepatocytes corrected via
in vivo or ex vivo gene therapy selectively
repopulate mutant liver.6,20
We applied the same viral
vector and gene transfer techniques for retroviral marking. First, we
transduced FAH-deficient hepatocytes for 5 hours in vitro
with the previously described vector 36 hours after plating. These
cells were then harvested 24 hours later, and 200,000 infected cells
were intrasplenically transplanted into each recipient. After the
selection and repopulation period of 8 weeks, hepatocytes were isolated
from surviving recipients, and either 10,000 or 100,000 cells were then
serially transplanted into secondary recipients. An aliquot of each
cell suspension was processed for DNA isolation and subjected to
Southern blot analysis. The G1FSvN proviral DNA could be detected at an
average copy number of 1 per hepatocyte genome in all repopulated
livers. In most of the independently performed experiments, no proviral
junction fragments were detected when either 10,000 or 100,000 cells
were serially transplanted (Table 4)
.
Monoclonal repopulation, however, was observed in one instance. In this
experiment the primary recipient was transplanted with 200,000 ex
vivo transduced cells, and 10,000 cells were serially
transplanted. Southern blot analysis of the primary recipient failed to
show predominance of a clonal population. It is interesting, however,
that secondary recipients displayed a single proviral integration site,
indicating monoclonal repopulation (Figure 5)
. Continued serial transplantation of
this clonal population for two additional rounds resulted in complete
rescue of liver function and normal histology in the recipients (data
not shown). This result, although obtained only in one instance,
demonstrates that liver repopulation can be effected by a single cell
derived from adult mouse liver.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Competitive repopulation with sorted fractions of liver cells has not been previously reported. We chose size sorting because small, periportal hepatocytes had been previously reported to proliferate preferentially in some experimental settings.26 Our results clearly show that, contrary to our expectations, small, mononucleated hepatocytes repopulated significantly less well than larger hepatocytes. The elutriated 16-µm population contained ~15% of larger cells. It is therefore even possible that small hepatocytes don’t repopulate at all and that the entire repopulation achieved by this fraction was due to contaminating larger cells. The reason for the lower repopulation capacity of this fraction currently is not known. Liver repopulation is a complex process that involves homing to the liver, entry through the sinusoids, migration and integration into the parenchyma, and subsequent cell division. Therefore, the data presented here cannot simply be interpreted to indicate a lower capacity for cell division in the small cells. They may home less efficiently or not possess the ability to integrate into the parenchyma. Additional experiments directed at measuring these parameters will be needed to determine the precise mechanism of the lower repopulation efficiency. Although the largest cells (27 µm) repopulated slightly less well than 21-µm hepatocytes, the difference between the two groups was small and not significant statistically. The most abundant size fraction constituting approximately two-thirds of all cells isolated by collagenase perfusion had the best repopulation properties. Thus, cell sorting before transplantation is not required at least for short-term repopulation.
It is important, however, to make some qualifications regarding the interpretation of these results. First, our experiments addressed only one round of repopulation, representing approximately 15 cell divisions. It is possible that serial transplantation of sorted populations that addresses the issue of long-term regenerative capacity may yield different results. Second, it is possible that size fractionation alone is not capable of enriching for the most regenerative liver cells and that a subpopulation with this property exists within the 21-µm fraction. Third, we excluded nonparenchymal cells from the current experiments. It is conceivable that liver progenitor cells reside within this fraction.
The results obtained with competitive repopulation between naïve and serially transplanted liver cells were surprising. Before the experiments, we had considered two possible scenarios: first, serial transplantation may enrich for more highly regenerative cells and these populations would therefore outcompete "normal" hepatocytes; second, multiple cell divisions may age the cells so that naïve hepatocytes would dominate. Our data supported neither hypothesis. Serial transplantation neither enhanced nor diminished the repopulation capacity of the cells to any significant degree. One interpretation is that virtually all cells in the transplanted fraction (ie, all hepatocytes) have stem cell-like regenerative capacity. Alternatively, the ratio between stem/progenitor cells and differentiated cells may be kept very constant by a regulatory mechanism and may not be altered by serial transplantation. Repopulated livers would reestablish the same ratio as naïve livers. In either case the data support the notion that therapeutic repopulation by unsorted adult liver cell suspensions does not impair the regenerative capacity of the repopulated liver. This is promising for the use of this procedure in a clinical setting. In addition, this result indicates that cell division itself does not limit the regenerative capacity of hepatocytes and that it may be possible to expand hepatocytes in vitro manyfold before use in transplantation. In this regard, however, it is important to caution that mouse cells have telomeres which are longer than those of human cells, so our results may not be directly applicable to humans.33 We have not yet measured the telomere length in serially transplanted cells or determined whether hepatocytes express telomerase activity in some settings. The lack of age effect on repopulation ability observed here appears inconsistent with the delayed response to partial hepatectomy observed in older animals.34,35 It is possible that the altered response in these studies was due to age effects on nonhepatocytes involved in liver regeneration (stellate cells for example). Alternatively, serial transplantation may not accurately mimic in situ aging of cells. Competitive repopulation with hepatocytes harvested from old and young animals will be needed to address this question directly.
The retroviral marking studies reported here were performed with relatively high numbers of marked transplanted cells. Most experiments produced the consistent result that only polyclonal repopulation was observed when 10,000 or 100,000 marked cells were serially transplanted. This indicates that the serially transplantable population of liver cells is considerably more common than 1:10,000. This applies to cells retrovirally transduced in tissue culture as well as to those marked in vivo. Interestingly, we observed one example of initial polyclonal repopulation becoming monoclonal on the second round of serial transplantation. Although this observation cannot be interpreted in terms of the frequency of serially repopulating cells, it illustrates the fact that a single adult liver cell has the regenerative capacity to serially reconstitute several generations of mouse livers. The retroviral marking studies reported here need to be refined in the future by reducing the number of marked cells to 100 to 1,000 until clonality can be detected. Because we used a Moloney murine leukemia virus-based vector, it can be argued that we preferentially labeled cells that divide rapidly after partial hepatectomy or after plating in tissue culture. Future experiments will therefore have to include marking of quiescent cells with lentiviral vectors.
Taken together the results reported here indicate that the ability to produce one round of liver repopulation resides in the majority of adult mouse hepatocytes and not a rare stem cell population.
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Acknowledgements |
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Footnotes |
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Supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK 51592 (to M. G. and M. F.) and NIDDK National Research Service Award DK009429 (to K. O.).
Accepted for publication August 24, 1999.
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-interferon in mice with a targeted disruption of the Fanconi anemia C gene. Blood 1996, 88:49-58This article has been cited by other articles:
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