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Regular Article |
From the Departments of Molecular and Medical
Genetics*
and Pediatrics,§
Oregon Health Sciences University, Portland, Oregon; Stem Cells
Incorporated,
Sunnyvale, California; 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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Several experimental models have indeed
supported the hypothesis that multipotent stem cells persist in the
adult pancreas and can give rise to a variety of differentiated
offspring. In transgenic mice expressing interferon-
under the
transcriptional control of the insulin promoter, new {beta} cells are
generated de novo throughout the life of the animal from
cells that reside within or close to the pancreatic
ducts.6,7
Another indication of the existence of
pancreatic endodermal stem cells is the emergence of hepatocytes in the
adult. The best known example is the appearance of hepatocytes in
copper-depleted rats after re-feeding of copper.8,9
In
this system, weanling rats are fed a copper-free diet for 8 weeks that
leads to complete acinar atrophy. When they are re-fed copper,
cells with multiple hepatocellular characteristics emerge from the
remaining pancreatic ducts within weeks. These cells have hepatocyte
morphology and express a variety of hepatocyte markers, such as
albumin. This work therefore suggested the presence of a pancreatic
liver progenitor cell in or close to the pancreatic
duct.10
Copper depletion alone, without re-feeding,
results in acinar atrophy and the proliferation of cells very similar
to hepatic oval cells.11
On transplantation into the
liver, these pancreatic oval cells can differentiate and display
multiple characteristics of hepatocytes.11
More recently,
specific cytokines have been identified as candidates to be involved in
this process. Transgenic mice in which the keratinocyte growth factor
gene is driven by insulin promoter consistently develop pancreatic
hepatocytes.12
The anatomical location of pancreatic stem cells has been determined to
be in or near the ducts in both {beta}-cell neogenesis and hepatocyte
generation6,10
Other work has also supported a direct role
of pancreatic ducts in these processes. Cultured pancreatic duct cells
were transplanted subcutaneously in the rat and then displayed
hepatocyte markers such as albumin and 
-fetoprotein.13
Thus, pancreatic duct epithelium itself has been considered to
represent a facultative stem cell by some
investigators.10,14
Although the earlier work described above had shown the existence of pancreas-derived cells expressing hepatocyte markers, it remains unknown whether these cells are fully functional and therefore therapeutically useful. Although pancreatic liver precursors seemed to be associated with the ducts, it remains unclear whether the ducts themselves contained the hepatocyte precursors or whether they are only anatomically close to the ducts (periductular). To address these questions, we tested adult murine pancreatic cells in the fumarylacetoacetate hydrolase (FAH) knockout liver repopulation model.15-17
Here we report that pancreatic cells from adult mice contain hepatocyte progenitor cells that can significantly repopulate the livers in fumarylacetoacetate hydrolase-deficient (FAH-) mice15,18,19 and reconstitute normal liver function. Furthermore, we provide evidence that these pancreatic liver progenitor cells are not the ducts themselves.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As transplant recipients we used the
FAH
exon 5 strain mice
previously described by this lab.18
Transplant donors were
transgenic ROSA-26 mice, a gift from P. Soriano (Fred Hutchinson Cancer
Research Center, Seattle, WA).20
All
transplantation experiments were performed with congenic mice of the
129SvJ background. All FAH mutant animals were treated with
NTBC-containing drinking water at a concentration of 7.5 mg/L (a
gift from S. Lindstedt, Gøtheborg, Sweden).21,22
This
provides an approximate dose of 1 mg kg-1 body
weight per day. For genotyping, polymerase chain reaction (PCR) was
performed with a 3 primer PCR on 200-ng tail-cut DNA as previously
described. Animal care and experiments were all in accordance with the
Guidelines of the Department of Animal Care at Oregon Health Sciences
University.
Isolation of Whole Pancreatic Cells
Whole pancreatic cells were isolated from adult (>3 months) male wild-type mice transgenic for Escherichia coli lacZ with a two step protease digestion. Briefly, the pancreas was dissected from sacrificed mice carefully avoiding injury of the liver. The harvested pancreas was immediately cut into small pieces and digested by collagenase D (Roche, Indianapolis, IN) (2.5 mg/ml, dissolved in Earle’s basic salt solution) for 25 minutes at 37°C with agitation by a magnetic stir bar. The cells were then pelleted by centrifugation, washed once with calcium-free phosphate-buffered saline (PBS), and then further digested in trypsin ethylenediaminetetraacetic acid (0.05% w/v) (Life Technologies, Inc., Rockville, MD) for 3 minutes. The proteases were neutralized by addition of 3 volumes of Dulbecco’s minimal essential medium (DMEM) with 10% bovine serum. Next, the digested cell mix was filtered through an 85-µm nylon mesh twice and the cells that came through the filter were collected. Cell number and viability were determined by Trypan blue exclusion staining under a hemocytometer.
Enrichment and Culture of Pancreatic Duct Cells
For pancreatic duct cell enrichment, a different protease digestion protocol was used. The chopped pancreas was first partially digested by collagenase D (Roche) (2.5 mg/ml, dissolved in Earle’s basic salt solution) for 20 minutes. The digestion was then filtered through a 85-µm nylon mesh. The cells retained on the mesh, enriched for ducts, were collected and then further digested under the same conditions for another 20 minutes.
Mouse pancreatic ducts were cultured by a published method23 with some modifications. Briefly, the harvested pancreas was dissected from the animal and immediately chopped into small pieces. The tissue was then partially digested by collagenase D (0.7 mg/ml, Roche) for 20 minutes. The digestion mix was sieved through a nylon mesh attached to a glass funnel to remove the predominant acinar tissue. The nylon mesh with attached duct tissue was then cut from the funnel using sterile procedures and embedded in rat-tail collagen gel.
The preparation of rat collagen was modified from a previous
report.24
Briefly, the rat tail was harvested and
sterilized in 70% ethanol. Then, the collagenous fibers of the tail
were collected, weighed, and dissolved in 0.1% acetic acid (4.8 g/L)
by stirring at 4°C for 3 to 5 days. The dissolved collagen solution
was centrifuged at 10,000 x g at 4°C to remove the
debris and sterilized by -irradiation (150 Gy). Before use, the
acidic collagen stock solution was tested for its ability to form a gel
at neutral pH. Eight times DMEM/F12 (Life Technologies, Inc.) medium
was prepared, and two parts of 8x medium was combined with one part of
serial NaOH stock solutions from 0.30 mol/L to 0.42 mol/L with 0.01
mol/L increments to make the serial neutralizing solutions. Then, one
part of each of neutralizing solutions was mixed with 4.25 parts of
acidic rat collagen stock solution. Each mixture was plated on each
well of a 24-well culture plate. The plate was transferred to a tissue
culture incubator with 5% CO2 and to equilibrate
overnight. The desired result was neutral pH (based on the medium
color) and a solid gel.
The cultures were fed with a DMEM/F12 (1:1) medium supplemented with 5% Nu-serum V (Collaborative Biomed, Bedford, MA), gentamicin (5 mg/L), and soybean trypsin inhibitor (100 mg/L; Sigma Chemical Co., St. Louis, MO), and a variety of factors including insulin (2.6 mg/L, Life Technologies, Inc.), murine epithelial growth factor (10 µg/L, Life Technologies, Inc.), dexamethasone (1 µmol/L, Sigma), and cholera toxin (100 µg/L, Life Technologies, Inc.). Half of the medium was exchanged every 3 days. After reaching confluency duct epithelial cells growing in the mesh were harvested by collagenase D (2.5 mg/ml, Roche) for 1 hour. The digestion was sieved by 85-µm size nylon mesh to collect single cells.
Pancreatic Cell Transplantation and Selection
The number and viability of harvested whole pancreatic cells, enriched duct cells, and cultured duct cells were determined by Trypan blue exclusion in a hemocytometer. The appropriate number of donor cells were resuspended in 100 µl of Dulbecco’s minimal essential media (Life Technologies, Inc.) with 10% fetal bovine serum and injected intrasplenically or directly into the portal vein of FAH mutant female recipient animals.
All FAH mutant transplant recipients were kept on NTBC until the time of transplantation. One day after transplantation they were switched to regular drinking water, not containing NTBC. The weight of experimental animals was measured weekly.
Biochemical Analysis
Samples from animals were obtained as follows. Animals were sacrificed by decapitation and blood collected by dabbing the wound onto parafilm (American National Can, Menasha, WI). For anticoagulation, the blood was immediately mixed with 10 µl of Na-heparin (Becton-Dickinson, Franklin Lakes, NJ) using a Pipetman. The red blood cells were removed by a brief centrifugation and the plasma was frozen at -80°C.
Twenty µl of plasma were mixed with 80 µl of a solution of 7%
bovine serum albumin and assayed for aspartate serine
aminotransferase, bilirubin, and creatinine levels with a Kodak
Ektachem 700 chemistry analyzer (Eastman Kodak, Rochester, NY).
Quantitative serum amino acid analyses were performed on a Beckman 6300
amino acid analyzer using published methodology.25
Plasma
succinylacetone levels in plasma were measured by a 
-aminolevulinate
dehydratase inhibition assay as described.26
FAH enzyme
assays were performed at 30°C on a cytosolic fraction of homogenized
liver as described previously.27
Fumarylacetoacetate, the
substrate for the assay, is not commercially available and was prepared
enzymatically from homogentisic acid as described in the same
reference. Protein concentrations were measured with a Bio-Rad kit
(Bio-Rad, Richmond, CA).28
Histology, Immune Histology, and Electron Microscopy
Liver tissues fixed in 10% phosphate-buffered formalin, pH 7.4, were dehydrated in 100% ethanol and embedded in paraffin wax at 58°C. Four-µm sections were rehydrated and stained with hematoxylin and eosin and with a polyclonal rabbit antibody to rat FAH (graciously provided by Robert Tanguay, University of Laval, Quebec, Canada) or glutamine synthetase.29 The antibody was diluted in PBS, pH 7.4, and applied at concentrations of 1:300,000 at 37°C for 30 minutes. The glutamine synthetase antibody was used at a dilution of 1:10,000. 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 (BA-1000; Vector Laboratories, Burlingame, CA). Color development was performed with the AEC detection kit (catalogue no. 250–020; Ventana Medical Systems, Tucson, AZ).
{beta}-galactosidase staining on the harvested liver tissues was performed. The liver tissues were fixed in 2% formaldehyde and 0.2% glutaraldehyde in PBS for 30 minutes. Then the tissues were washed with PBS for two times and moved to staining medium that contained 1 mg X-gal (Research Products International, Prospect, IL) in buffer of 5 mmol/L K ferricyanide, 5 mmol/L K ferrocyanide, 2 mmol/L MgCl2, 0.02% Nonidet P-40 (all Sigma), and 40 mmol/L Hepes (pH 8.0; Life Technologies). The staining incubation was at 37°C overnight. {beta}-galactosidase histochemistry in frozen sections was performed as previously described.16 The 8- to 10-µm-thick sections of OCT-embedded liver were fixed with 1.25% glutaraldehyde in ice-cold PBS for 10 minutes and stained overnight.
For detection of male cells, in situ hybridization was performed with a digoxigenin-labeled high-copy number Y chromosome-specific repeat DNA probe as previously described.30
For electron microscopy, the cultured duct cells were harvested by collagenase D digestion. The cells were fixed in 3% glutaraldehyde buffered in 0.1 mol/L Na-cacodylate (pH 7.4). The cell samples were then postosmicated, embedded in araldite, sectioned on an AO-Reichert ultracut E microtome, and stained with uranyl acetate and lead citrate. Sections were examined in a Joel 100-CX microscope at magnifications ranging from x800 to x12,000.
Reverse Transcriptase (RT)-PCR and Northern Blots
Total cellular RNA of pancreatic duct cells were isolated from harvested pancreatic tissues and pancreatic duct cell cultures.31 RT-PCR and Northern blots were performed according to standard protocols.31 For the detection of the pancreatic duct markers carbonic anhydrase type II (CAII) and cystic fibrosis transmembrane regulator (CFTR), specific primers for both cDNA fragments were generated based on the published CAII (GenBank accession, K00811) and CFTR (GenBank accession, M69289) sequences. The primer pairs for CAII were: forward 5'-GGAGACCGGCAGTCCCCTGT-3' and reverse 5'-AGAGAGGCGGTCACACTTGT-3'. The primer pairs for CFTR were: forward 5'ACCCTTGTGGATGGGGGTTATGTGC-3' and reverse 5'-CATGGGTTCTGGGAATGGACTC-3'. RT was performed by using 2 µg of total RNA samples with random hexamers and M-MLV reverse transcriptase (Life Technologies, Inc.). The PCR conditions were: 94°C for 5 minutes; 95°C for 1 minute, 60°C for 1 minute, and 72°C for 2 minutes, for 35 cycles; 72°C for 10 minutes for ending. After RT-PCR, the DNA fragments were resolved on a 0.8% agarose gel.
To further estimate the relative quantities of CFRT mRNA in different cells we used a 4-primer RT-PCR with the housekeeping gene APRT (adenine phosphoribosyltransferase) as an internal control. The primer pairs for APRT were: forward 5'-AGCGTGCTGATACCTACCTC-3' and reverse 5'-AAGCAGTTCCTAGTGCTGCT-3'. The PCR conditions were: four primers added in a single reaction, including 40 ng of each CFTR primers and 200 ng of APRT primers in 25 µl reaction; 94°C for 5 minutes for starting; 95°C for 1 minute, 60°C for 1 minute, and 72°C for 2 minutes, for 28 cycles; 72°C for 10 minutes for ending. After RT-PCR, the DNA fragments were resolved on a 1.5% agarose gel.
Northern blots were probed with both RT-PCR generated cDNA fragments as
probes. Fifteen µg of total RNA was separated on the formaldehyde
denature gel and transferred onto Hybond-N nylon membrane. The
hybridization was performed in 50% formamide hybridization solution
the -32P-dCTP labeled probes (5 x
106
cpm/ml) at 42°C for 18 hours. The
radioactive signals were shown by the exposure on the X-ray film.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To test the hypothesis that pancreas contains a population of
liver progenitor cells, we harvested cells from whole pancreas of adult
(>3 months) male, FAH wild-type ROSA-26 mice transgenic for an
ubiquitously expressed E. coli lacZ
gene.20
A single cell suspension was transplanted into
10-week-old female FAH- mutant congenic recipients. Three to five
x 105
crude pancreatic cells were injected into
the spleens of the FAH- recipients and NTBC treatment was discontinued
immediately after transplantation to permit selection of FAH+
hepatocytes.15
Our previous work with hepatocyte
transplantation has shown that liver repopulation is only partial at 4
weeks of selection and is near-complete after 8 weeks.15
Even nontransplanted FAH mutants typically survive 6 to 8 weeks off
NTBC.22
Therefore, transplanted recipients were divided
into two groups for repopulation analysis: one group was harvested at 4
weeks after transplantation, whereas the other group was harvested
after 8 weeks. In total, 68 FAH mutants were transplanted in 12
independent experiments. During the selection period, weights of the
transplant recipients were followed weekly and all recipients lost
weight during the first 4 weeks of transplantation. In the early
harvesting group with a total of 28 recipients, nodules of FAH+
hepatocytes were found in 10 (29%) of the transplanted FAH-
recipients. In these animals, only a single 4- to 5-µm liver section
representing <0.1% of the total liver was analyzed. The FAH+ nodules
were small with 6 to 20 cells visible in cross-section, indicating that
three-dimensionally each clone contained 
10 to 50 cells (Figure 1A)
. In the 8-week-time (late)
observation group of 40 recipients, most of transplanted FAH-
recipients died with tyrosinemic symptoms for liver failure before 6
weeks, ie, before the scheduled harvesting time. However, six of 40
transplanted mice regained the originally lost weight and completely
recovered at 6 to 8 weeks after transplantation. Of these harvested at
8 weeks, four recipients (generated in three independent
transplantation experiments) had from 50 to 90% FAH+ hepatocyte
repopulation as shown by the immunohistochemistry (Figure 1B)
. The
other two recipients had developed hepatocarcinoma thus making
evaluation of liver function impossible. The carcinomas originated in
recipient tissue, not transplanted donor cells, as has been reported
previously reported in the FAH knockout model.32,33
To
confirm that the repopulating cells were indeed donor-derived, we
performed {beta}-galactosidase staining (Figure 2C)
and Y chromosome in situ
hybridization (Figure 2A)
. Because all of the transplantations were
performed by transplanting of Rosa-26 male pancreatic cells into FAH-
female recipients, the donor cells could readily be distinguished from
host cells (Figure 2)
.
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. These values were
compared to wild-type controls and untransplanted FAH mutant mice off
NTBC (Table 1)
. Only the alanine aminotransferase (ALT) and
tyrosine levels were mildly elevated in the FAH mutant mice repopulated
with pancreas-derived cells compared to wild type. This result is
similar to those obtained with transplantation of hepatocytes, in which
repopulation of <80% also results in mildly abnormal ALT and
tyrosine.16
Importantly, the plasma level of
succinylacetone, the hallmark metabolite for tyrosinemia was completely
normalized in pancreatic cell-transplanted mutants. Furthermore, the
normal plasma levels of all amino acids represents a global measure of
many hepatic enzymes including the urea cycle (glutamine, citrulline,
arginine) and glucose metabolism (alanine). The expression pattern of
glutamine synthetase was also analyzed by immunohistochemistry in the
repopulated livers, and found to be limited to zone 3 hepatocytes
adjacent to hepatic veins as in wild-type controls (Figure 1C)
. This
indicates that the normal zonular organization of the liver lobule was
re-established with the repopulation.29
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Serial Transplantation of Hepatocytes Derived from Pancreatic Cells
To address whether the pancreatic cell-derived FAH+ hepatocytes
retain their regenerative capacity, we serially retransplanted 1
x 105
cells from two repopulated livers into
additional FAH- recipients. With the first of these donors, two
additional rounds of serial transplantation were successful, resulting
in phenotypic correction of two generations of FAH mutants. FAH-
immunohistochemistry and {beta}-galactosidase staining were used to
measure the degree of repopulation, which exceeded 50% in all cases
(data not shown). The number of cell divisions required to achieve 50%
repopulation is estimated to be 10.16
Therefore at
least 20 cell divisions are needed to obtain the sequential
repopulation observed. The second donor was used for only one
additional round of serial transplantation, again resulting in a high
percentage (>50%) of repopulation in the secondary recipients. These
results show that pancreas-derived hepatocytes have a regenerative
capacity similar to liver-derived hepatocytes.
Transplantation of Cell Suspensions Enriched for Pancreatic Ducts
Previous reports suggested that both pancreatic liver stem cells
and the cells responsible for {beta}-cell neogenesis reside in the
pancreatic duct system.10,14
We therefore hypothesized
that pancreatic cell suspensions enriched for duct cells would be
superior for liver repopulation compared to cell suspensions containing
all pancreatic cell types. We designed two methods to enrich for
transplantation of duct cells: First, we concentrated ducts from
freshly isolated pancreas by performing partial collagenase digestion
and then filtering through a nylon mesh to capture the undigested duct
fragments for further digestions into a single cell suspension. Second,
we established high-density primary cultures of duct cells (see below).
For the first approach, two steps of collagenase digestions were used
to enrich the pancreatic duct cells. The chopped whole pancreas was
first digested mildly by collagenase D to remove the acinar cells. The
undigested part contained mainly of the duct tissues and was collected
by being retained on the filter after being passed through a nylon
mesh. Figure 3A
shows the morphology of
the intact ducts collected by this method before the second digestion
step. The enrichment for duct cells was verified by performing Northern
blots and RT-PCR for two specific markers, the CFTR and CAII (Figure 4)
.
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,
only three of 34 (9%) of the animals analyzed had any evidence for
hepatocyte repopulation. Thus, contrary to our expectation, enrichment
for duct cells did not enhance liver repopulation.
|
We next established an improved method for culturing primary
pancreatic duct cells. In a modification of a previously published
technique,23,34
ducts collected on a nylon mesh were not
removed before embedding in collagen, but rather were placed into the
gel together with the mesh. This led to a rapid outgrowth of duct
epithelium at high density and enhanced the yields of duct epithelial
cell fivefold to 10-fold compared with the previous method. The duct
cells in the collagen gel became confluent on the mesh lattice 2 to 3
weeks after plating, yielding 10 million pure pancreatic duct
epithelial cells from a single pancreas. The phenotypic identity of the
cultured cells was verified by two methods. First, the cultured cells
were shown to have typical duct epithelium morphology by phase
microscopy (Figure 3B)
and electron microscopy (Figure 3C)
. Second, the
expression of the marker genes CFTR and CAII was demonstrated by RT-PCR
and Northern blot (Figure 4, A and B)
. Both markers were highly
expressed. For transplantation, confluent duct epithelium was harvested
by incubation with collagenase D followed by
trypsin/ethylenediaminetetraacetic acid and 5 x
105
viable cells were injected into the spleen of
FAH mutant recipients. A total of 28 FAH mutant mice were transplanted.
None of the animals survived the complete selection period of 8 weeks,
and none of the 14 animals harvested at 4 weeks for histological
analysis had any evidence of donor cell engraftment in the liver. Thus,
cultured primary pancreatic duct epithelium was not able to undergo
hepatocyte differentiation in our in vivo repopulation
assay.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the past, it was not possible to determine whether pancreatic
hepatocytes simply represented transdifferentiation and metaplasia
under pathological conditions or if they were true hepatocytes, derived
from an undifferentiated progenitor cell (stem cell). In the
copper-depleted rats and keratinocyte growth factor transgenic mice,
pancreatic hepatocytes display many characteristics of immature liver
cells, such as expression of -fetoprotein, also seen in
hepatocarcinoma.11,12
The experiments reported here
demonstrate unambiguously that the pancreas of the normal adult mouse
contains undifferentiated progenitors of fully functional hepatocytes.
These transplantable cells can phenotypically ameliorate the
tyrosinemic liver disease of FAH mutant mice and their progeny are
indistinguishable from liver resident hepatocytes. As in previous work
resulting in partially repopulated tyrosinemic mice, liver functions
that reflect cell autonomous properties of FAH mutant hepatocytes (ie,
hepatocarcinoma, release of transaminases, release of tyrosine) were
still slightly abnormal here also.16,32,33
This
observation does not indicate incomplete differentiation of
pancreas-derived hepatocytes, but rather reflects the cell autonomous
nature of liver disease in FAH deficiency.33
The data support the concept of a pancreatic stem cell able to differentiate into the hepatic lineage. The same cell may also be able differentiate into different pancreatic lineages and thus could be multipotent. The frequency and exact phenotypic identity of this stem cell were not ascertained in the experiments reported here. However, we did seek to determine whether pancreatic ducts harbor the stem cells as previously suggested. Our results indicated that differentiated ducts themselves were not directly responsible for liver repopulation, but did not exclude the possibility that the repopulating cell was anatomically close to the ducts. Mild collagenase digestion may separate the pancreatic hepatocyte progenitors from the ducts, with which it may be anatomically associated in situ. Such transitional cells located between the ducts and the parenchyma may represent the stem cell fraction. Others have recently reported {beta}-cell neogenesis from cultured pancreatic duct cells.14,36 Despite this claim, however, the authors did not demonstrate that the {beta} cells originated from ducts themselves rather than periductular cells. Thus, it is still possible that the pancreatic hepatocyte progenitors described here and {beta}-cell progenitors reported by others might be identical. Alternatively, two distinct progenitor populations may exist.
It is unlikely that pancreatic liver stem cells would find a therapeutic application for the treatment of liver diseases. The pancreas resides in an anatomically inaccessible location, making it a poor source of cells for liver repopulation. Furthermore, the efficiency of liver repopulation by pancreatic cells was inferior to what can be achieved with liver-derived hepatocytes. Despite transplantation of a large number of donor cells, only a small fraction of animals had significant degrees of liver replacement. The significance of the work reported here lies in the description of an in vivo transplantation assay for pancreatic stem cells. Our data clearly indicate that the liver repopulating cells reside in a single cell suspension. Therefore, fractionation of the cell suspension into subpopulations should be possible by standard cell sorting methods such as fluorescence-activated cell sorting for cell surface markers. Using such methodology the purification and isolation of pancreatic liver precursors should be possible. Because of the common embryological origin of liver and pancreas, it is possible that this liver stem cell may also have the potential to give rise to other endodermal cell types. Therefore liver repopulation by pancreatic donor cells may serve as a surrogate assay for pluripotent stem cells and could potentially be used to purify stem cells capable of {beta}-cell neogenesis.
The findings reported here are also relevant to the issue of liver regeneration. It has been controversial for some time whether stem cells are sometimes involved in reconstitution of the adult liver.37,38 Our own work has shown that hepatocytes themselves are highly efficient in liver repopulation.17 The results obtained here clearly indicate that therapeutic liver repopulation is also possible with undifferentiated progenitors. Whether transplantation of stem cells has practical advantages compared to transplantation of hepatocytes currently remains unknown, but it is conceivable that they could be useful for the treatment of liver disease that is not limited to hepatocytes.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health NIDDK grant D-51592 (to M. G.) and Mel Howard Research Fellowship from American Liver Foundation (to X. W.).
Accepted for publication October 4, 2000.
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