4SCIENTIFIC REVIEW
Liver Stem/Progenitor Cells and their Transplantation:
from Fantasy to Reality as We Enter the New Millennium
David A. Shafritz*,+,‡,§ and Mariana D. Dabeva*,§
Departments of Medicine*, Cell Biology+, and Pathology‡, and the Marion Bessin Liver Research Center§
Albert Einstein College of Medicine
Bronx, NY 10461
ABSTRACT
Whether stem cells exist in solid tissues and organs has
been an area of controversy for many years. With the
advent of sophisticated and sensitive methods of cell
and molecular biology and the development of new ani-
mal models for cell transplantation, it has been possible
to identify such cells not only in the hematopoietic sys-
tem, but also in the brain, muscle, and liver. This review
highlights research conducted in our laboratory, other
laboratories of The Marion Bessin Liver Research Center
at the Albert Einstein College of Medicine, and at other
institutions during the last decade to define optimal
conditions for liver cell transplantation, identify and
characterize liver stem/progenitor cells, and transplant
these cells into suitable hosts to effectively achieve liver
repopulation and restore liver function.
INTRODUCTION
The existence, need for, and possible function of pro-
genitor or stem cells in the adult liver has been an area
of controversy for many years (Sell, 1990). The existence
of such cells was postulated initially more than 40 years
ago (Wilson and Leduc, 1958). This was based on studies
in rodents in which it appeared that cells in the distal
cholangioles of the bile ducts were responsible for
restoration of liver mass after dietary injury. At that time,
it was also established that bile duct epithelial cells and
hepatocytes are of common embryologic origin, derived
from hepatoblasts emanating from the endoderm of the
ventral foregut (Dubois, 1963; Wilson et al., 1963).
Therefore, a potential precursor/product relationship
between cells of the distal cholangioles and hepatocytes
seemed reasonable. Specific identification of these cells
has been problematic, because unique markers for liver
stem/progenitor cells have not yet been identified.
However, studies conducted during the last decade clear-
ly establish the existence of cells both within and outside
the liver that exhibit properties of stem cells and can dif-
ferentiate into mature hepatocytes and/or bile duct
epithelial cells after their transplantation and engraft-
ment into the liver.
The working hypothesis driving our research has been
that the liver contains a dormant compartment of stem
cells that under certain conditions can be activated to
proliferate and progress along the two terminally differ-
entiated epithelial lineages in this tissue, namely, the
hepatocyte and the bile duct epithelial cell. Recently, we
20 Einstein Quart. J. Biol. Med. (2002) 19:20-32.
have taken a molecular approach to clone genes whose
expression is specifically upregulated in the early fetal
liver in the hope of identifying genes that are involved in
activation, proliferation, and differentiation of these
cells (Petkov et al., 2000). We have also established sev-
eral liver regeneration models in which hepatocytes or
progenitor cells play a specific role and these models may
also be useful in defining a detailed lineage map along
which the various progenitor cells can be placed (Dabeva
and Shafritz, 1993; Dabeva et al., 1995). Finally, we have
established a rat liver cell transplantation/liver repopula-
tion model system in which we can study the proliferation
and differentiation capabilities of isolated hepatocytes,
liver stem/progenitor cells, and established liver cell lines
as well as their ability to repopulate the liver (Dabeva et
al., 1997; Laconi et al., 1998; Oren et al., 1999; Dabeva et
al., 2000a; Sandhu et al., 2001).
LIVER REGENERATION
The ability of the liver to regenerate is a property that is
unique among solid organs in mammalian species.
Following two-thirds partial hepatectomy (PH), there is
compensatory growth by the remaining liver, resulting in
restoration of the total parenchymal cell number and
mass within one to two weeks (Fausto and Webber, 1994;
Michalopoulos and DeFrancis, 1997). This process is not
really regeneration, because the lost anatomic structures
are not replaced, but the remaining tissue expands to its
original mass by proliferation of preexisting cells.
Radioactive labelling with [3H]-thymidine and morpho-
logic studies in the 1960’s showed that many hepatocytes
are actively engaged in mitosis during liver regeneration,
and it was estimated that 70 to 90% of hepatocytes
undergo at least one round of cell division following
two-thirds PH (Grisham, 1962; Bucher and Swaffield,
1964). From these studies and others, it has been con-
cluded that the proliferative activity of adult hepatocytes
is sufficient to repopulate the liver following PH and that
the participation of stem/progenitor cells is not required
(Grisham and Thorgeirsson, 1997).
The first study demonstrating the existence of small
undifferentiated epithelial cells in the adult liver was
reported by Farber (1956), who treated rats with differ-
ent carcinogens, such as ethionine, 2-acetaminofluorine,
and 3-methyl-4-dimethylaminobenzene. In these studies,
Dr. Farber noted the proliferation of periportal epithelial
cells with scant basophilic cytoplasm and an oval shaped,
pale blue, homogeneously stained, nuclei, which he

Liver Stem/Progenitor Cells and their Transplantation: From Fantasy to Reality as We Enter the New Millennium
termed “oval cells.” However, he concluded that these
oval cells were not progenitors of hepatocytes, although
others subsequently demonstrated that under certain
pathophysiologic circumstances oval cells clearly differ-
entiate into hepatocytes or neoplastic hepatocytes
(Evarts et al., 1987; Evarts et al., 1989). In these studies,
rats were treated with 2-acetaminofluorine, which caus-
es extensive DNA damage in the liver, and were then
subjected to two-thirds PH. Under these conditions,
there is massive proliferation of oval cells in the peripor-
tal region (zone 1), associated with uptake of
[3H]-thymidine. This is followed by the appearance of
nodular masses of [3H]-thymidine labeled hepatocytes in
the mid-parenchyma (zone 2), presumably derived from
the oval cells (Evarts et al., 1987). This and other evidence
(Evarts et al., 1989) suggested that when hepatocyte
proliferation is blocked, “facultative” stem cells, as
Thorgeirsson (1993) termed them, could proliferate in
response to a regenerative stimulus. Epithelial cell lines
were established from neonatal and adult livers. Under
certain conditions, some of these relatively undifferenti-
ated cell lines expressed liver-specific genes indicative of
PH, suggesting that they were the cell culture counterpart
of oval cells and represented cultured liver progenitor or
stem-like cells (Grisham, 1980; Tsao et al., 1984).
FIGURE 1 Identification of AFP positive non-parenchymal epithelial cells in the periportal area of the normal adult rat liver. AFP mRNA expression in a
periportal non-parenchymal epithelial cell (depicted by an arrow) was detected by in situ hybridization, using a radioactively labeled AFP cDNA probe.
4SCIENTIFIC REVIEW
Our entry into the field of liver stem/progenitor cell
research was based on a chance observation made in
1989. We were studying the genetic switch between
albumin and α−fetoprotein (AFP) expression that occurs
during liver regeneration. It was observed that the albu-
min/AFP gene expression switch was regulated at the
level of transcription (Tilghman and Belayew, 1982;
Panduro–Cerda et al., 1986), and we wanted to deter-
mine whether these changes occurred simultaneously in
all cells or in separate subpopulations of cells within the
regenerating liver. This information would be critical in
establishing the specific cell types that repopulate the
liver during regeneration. For these studies, in situ molec-
ular hybridization was used to follow specific gene
expression in individual cells, a very sensitive technique,
established in our laboratory in the early 1980’s (Saber et
al., 1983a; Saber et al., 1983b). In the course of these
studies, a very surprising result was obtained that alert-
ed us to the possible existence of stem/progenitor cells in
the normal adult liver (Alpini et al., 1992; Dabeva et al.,
1993). In control untreated adult rats, we observed a
rare population of cells (1 in 20,000) that expressed high
levels of AFP mRNA (Figure 1). These cells were located in
the periportal region adjacent to mature bile ducts and
had the morphologic appearance of oval cells. They were
The Einstein Quarterly Journal of Biology and Medicine 21

Liver Stem/Progenitor Cells and their Transplantation: From Fantasy to Reality as We Enter the New Millennium
clearly distinct from the much larger mature hepatocytes
located in the parenchyma. However, there was no signif-
icant increase in the number of these cells following
two-thirds PH or carbon tetrachloride administration,
which is another method used to induce acute liver injury,
hepatic necrosis, and regeneration (Alpini et al., 1992).
AFP was originally discovered in the 1960’s by the Russian
scientist Dr. Garri Abelev (Abelev, 1971). This protein is
very similar in structure to albumin and is indeed located
in tandem with albumin on the same chromosome, chro-
mosome 5 in the mouse (D’Eustachio et al., 1981).
Expression of AFP occurs first in the yolk sac and then in
the fetal hepatoblasts. Its expression decreases as albu-
min expression increases in late gestation (Sala-Trepat et
al., 1979; Tilghman and Belayew, 1982), and it is turned
off almost completely after birth only to reappear during
liver carcinogenesis. Thus, it is the classic onco-fetal pro-
tein. Our speculation was that the rare AFP expressing
cells in the periportal area of the adult liver most proba-
bly represent a vestigal population of stem/progenitor
cells (i.e., fetal hepatoblasts) or were a direct descendent
of these cells.
FIGURE 2 Incorporation of transplanted hepatocytes into the parenchymal structure of the liver with formation of hybrid bile canaliculi between trans-
planted cells and host hepatocytes (depicted by arrows).
22 EINSTEIN QUARTERLY, Copyright © 2002
4SCIENTIFIC REVIEW
ACTIVATION OF HEPATOCYTE PROGENITOR CELLS IN
THE ADULT LIVER
To study the properties of liver progenitor cells further,
we induced their proliferation by treating rats with
D-galactosamine, an agent that is taken up by hepato-
cytes and incorporated into UDP-galactosamine and
depletes the cell of UTP disrupting energy metabolism.
All RNA and protein synthesis is inhibited, there is exten-
sive liver necrosis, and at the same time proliferation of
hepatocytes is temporarily blocked. Under these condi-
tions, D-galactosamine induces proliferation of small
epithelial cells (oval cells) in the periportal region
(Dabeva and Shafritz, 1993). These cells (positive for AFP)
expand rapidly, move out into the hepatic parenchyma,
and go through a morphologic and phenotypic transi-
tion into hepatocytes with induction of albumin and
glucose-6-phosphatase gene expression and loss of AFP
gene expression (Dabeva and Shafritz, 1993). After D-
galactosamine administration, liver mass is fully restored
within 10 to 14 days, so that when hepatocytes are
blocked in their proliferative capacity, liver regeneration
occurs through activation of liver progenitor cells

Liver Stem/Progenitor Cells and their Transplantation: From Fantasy to Reality as We Enter the New Millennium
(Dabeva and Shafritz, 1993). Similar results were obtained
by Fausto and coworkers (Lemire et al., 1991), although
they concluded that mature bile duct cells were the source
of the proliferating cells, rather than a separate com-
partment of less well-differentiated liver progenitor cells.
LIVER CELL TRANSPLANTATION MODEL
To ultimately prove that the cells activated in D-galac-
tosamine treated liver are indeed progenitor cells, the
best approach would be to isolate these cells, transplant
them into a suitably treated host liver, and monitor recip-
ients for proliferation and differentiation of transplanted
cells into mature liver epithelial cell phenotypes (i.e.,
hepatocytes and bile duct epithelial cells). To accomplish
this goal, we utilized an inbred strain of Fischer rats
(F344) in which a mutant substrain is deficient in expres-
sion of a specific apical membrane protein, dipeptidyl
peptidase IV (DPPIV). Other than absence of DPPIV
enzyme activity, these animals exhibit a normal pheno-
4SCIENTIFIC REVIEW
FIGURE 3 Spontaneous liver repopulation
of uPA transgenic mouse liver by endoge-
nous hepatocytes that have deleted the uPA
transgene. Left, liver from a five week old
non-transgenic mouse; Center, liver from a
hemizygous uPA transgenic mouse; Right,
liver from a homozygous uPA transgenic
mouse.
FIGURE 4 Repopulation of uPA transgenic
mouse liver with transplanted normal mouse
hepatocytes containing a lacZ marker trans-
gene. Livers from a nontransgenic control
(top, left), a lacZ positive control (top, cen-
ter), a control mouse lacking the Alb-uPA
transgene and transplanted with lacZ cells
(top, right), and three livers from Alb-uPA
transgenic mice receiving lacZ cells (bottom).
All tissues were stained with X-gal, which
produces a blue color in lacZ positive cells.
Red areas (R) represent endogenous cells
that have lost uPA transgene expression and
white areas (W) represent residual endoge-
nous liver still expressing the uPA transgene.
type and are otherwise healthy. They were developed as
a liver cell transplantation model by Hixson, Faris, Doyle,
and coworkers in the early 1990’s (Thompson et al.,
1991). Using wild type (DPPIV+) F344 hepatocytes as
donor cells transplanted into DPPIV- F344 rats, Gupta and
coworkers (Gupta et al., 1995) and our laboratory
(Dabeva et al., 1997) observed that transplanted hepato-
cytes became fully integrated into the liver parenchymal
cord structure and form hybrid bile canaliculi with host
hepatocytes (Figure 2). This is consistent with studies con-
ducted earlier in The Marion Bessin Liver Research Center
(Gupta et al., 1990; Ponder et al., 1991), although it is still
not clear precisely how the hepatocyte, a rather large
cell, crosses the vascular space, penetrating through the
liver sinusoids, and becomes incorporated into the
parenchyma. Epithelial progenitor cells were isolated
from the liver and pancreas (the pancreas is also derived
from the gut endoderm) and, after transplantation, both
of these cell types differentiate into hepatocytes.
However, with hepatocytes or epithelial progenitor cells
from either the liver or pancreas only limited prolifera-
The Einstein Quarterly Journal of Biology and Medicine 23

Liver Stem/Progenitor Cells and their Transplantation: From Fantasy to Reality as We Enter the New Millennium
DAY 2
WEEK 3
WEEK 6
FIGURE 5 Liver repopulation in the FAH null mouse after transplanta-
tion of FAH+ hepatocytes. FAH is detected in expanding clusters of
transplanted FAH+ hepatocytes by enzyme histochemistry two days, three
weeks, and six weeks after transplantation of wild-type hepatocytes.
tion of the transplanted cells occurs (at most two to
three cell divisions with hepatocytes and five to six cell
divisions with progenitor cells).
NEWER MODELS OF LIVER REPOPULATION
As mentioned above, until recently it was believed that
hepatocytes are terminally differentiated liver epithelial
24 EINSTEIN QUARTERLY, Copyright © 2002
4SCIENTIFIC REVIEW
FIGURE 6 Liver repopulation by DPPIV+ hepatocytes in DPPIV- mutant
Fischer 344 rats in the retrorsine/partial hepatectomy model. A) Liver
from a DPPIV- rat before hepatocyte transplantation. B) Liver from the
same DPPIV- rat nine months after transplantation of DPPIV+
hepatocytes.
cells that can undergo one or two rounds of cell division
during liver regeneration. Following this process, hepa-
tocytes exit from the cell cycle, enter and remain in Go
with a very long half-life and eventually undergo senes-
cence. This is one of the main reasons why it has been
difficult to propagate primary hepatocytes in cell culture.
However, in the early to mid 1990’s, some very unexpected
and rather surprising findings were observed in two
genetically modified mouse model systems. Sangren et
al. (1991) developed a transgenic mouse model to study
protease function by inserting the urokinase plasmino-
gen activator (uPA) gene into the liver under the control
of the albumin promoter. uPA is a potent protease. In the
alb-uPA transgenic mouse, uPA is synthesized exclusively
in the liver and is secreted into the blood stream, where
it was being studied as a thrombolytic agent. However,
some of the protease remained in the liver and caused
severe inflammation. The liver undergoes continuous
injury from uPA expression leading to extensive necrosis
and most animals died. However, in two uPA kindreds,

Liver Stem/Progenitor Cells and their Transplantation: From Fantasy to Reality as We Enter the New Millennium
some animals survived. In these animals, large clusters of
normal liver cells were observed and in some instances
almost the entire liver was replaced by these normal
hepatocytes (Figure 3) (Sangren et al., 1991). It was sub-
sequently shown that the normal cells had deleted the
uPA transgene and then proliferated to restore the liver
mass. These investigators then transplanted single cell
suspensions of genetically marked wild-type hepatocytes
into uPA transgenic mice and these cells also repopulat-
ed the liver (Figure 4) (Rhim et al., 1994). It was
calculated that during liver repopulation, each trans-
planted cell underwent 12 to 14 divisions.
A second model, the fumaryl acetoacetate hydrolase
(FAH) null mouse (Overturf et al., 1996), was subse-
quently developed to study the human disorder,
hereditary tyrosinemia type 1 (HT1). In this model, tyrosine
metabolism is blocked at the last step in tyrosine catabo-
lism, conversion of fumarylacetoacetate to fumarate,
acetoacetate, and succinate. This leads to the accumula-
tion of the upstream metabolic intermediates, including
maleylacetoacetate and fumarylacetoacetate, which are
toxic and cause continuous liver injury, chronic liver dis-
ease, cirrhosis, and hepatocellular carcinoma. Similar to
findings in uPA transgenic mice, humans with HT1 occa-
sionally show clusters of normal hepatocytes in an
otherwise extensively diseased liver (Kvittingen et al.,
1994). In the FAH null mouse model, transplantation of
wild-type hepatocytes also leads to extensive liver repop-
ulation and restoration of normal liver architecture and
function (Figure 5) (Overturf et al., 1996). In the FAH null
model, as few as 10,000 wild-type hepatocytes can be
serially transplanted through seven generations of mice
with total liver repopulation in each mouse (Overturf et
al., 1997). If one considers all hepatocytes to have equal
proliferative capacity in these serial transplantation stud-
ies, it can be concluded that hepatocytes have the ability
to undergo up to 100 cell divisions or more, equivalent to
4SCIENTIFIC REVIEW
FIGURE 7 Detection of normal serum albu-
min levels in Nagase analbuminemic rats
after transplantation of wild-type hepato-
cytes. SDS gel electrophoresis; lane 1, serum
albumin standard; lanes 2 and 3, serum from
control Nagase analbuminemic rats; lanes 4-
7, serum from different rats four weeks after
receiving transplanted hepatocytes; and
lane 8, serum from a control wild-type rat.
the most robust of stem cells, and that the innate regen-
erative power of fully mature parenchymal cells in the
liver is essentially infinite.
RETRORSINE/PARTIAL HEPATECTOMY MODEL OF LIVER
REPOPULATION IN THE RAT
A key factor favoring liver repopulation by transplanted
cells in both uPA transgenic and FAH null mice is an enor-
mous selection pressure for repopulation by normal
hepatocytes. However, both of these conditions are peri-
natal lethal and rarely will comparable conditions be met
in humans. To develop an alternative approach for liver
repopulation, which might have a more general applica-
tion, we collaborated with Dr. Ezio Laconi from Cagliari,
Italy, who had developed a strategy to create an envi-
ronment in the liver that would favor repopulation of
tissue mass with transplanted cells (Laconi et al., 1995).
Rats were treated with retrorsine, a pyrrolizidine alka-
loid that is taken up by the liver and metabolized in
hepatocytes to its biologically active form, which then
alkylates cellular DNA and prevents hepatocytes from
proliferating by disrupting their progression through the
cell cycle (Samuel and Jago, 1975). In contrast to D-galac-
tosamine treatment, which is transient, the mitoinhibitory
effect of retrorsine is long lasting. When retrorsine treat-
ed rats are subjected to PH, endogenous hepatocytes are
unable to proliferate. By combining retrorsine/PH treat-
ment with the DPPIV cell transplantation model and
following the proliferation and differentiation of trans-
planted wild-type hepatocytes, we were able to fully
repopulate the retrorsine-treated liver with adult hepa-
tocytes (Figure 6) (Laconi et al., 1998). The liver mass
returned to near normal size and the transplanted cells
were fully active, biochemically, and physiologically. The
latter was demonstrated with conversion of a genetically
albumin-deficient rat to a normal phenotype, expressing a
normal serum albumin level (Figure 7) (Oren et al., 1999).
The Einstein Quarterly Journal of Biology and Medicine 25

Liver Stem/Progenitor Cells and their Transplantation: From Fantasy to Reality as We Enter the New Millennium
OTHER MODELS OF LIVER REPOPULATION
C. Rogler and colleagues at the Albert Einstein College of
Medicine have developed an additional model of liver
repopulation in which recipient mice can accept
xenographs. This required cross breeding of uPA trans-
genic mice (with constant liver injury) with Rag2-/- mice
which are T and B cell deficient and are immunotolerant
to transplanted cells even across species barriers. In
uPA+/+/Rag2-/- mice, transplanted woodchuck hepatocytes
can repopulate the host liver (Peterson et al., 1998) and
human hepatocytes can also proliferate in the liver after
transplantation into these mice (Dandri et al., 1999).
Irradiation has also been used to block proliferation of
endogenous hepatocytes and allows transplanted wild-
type hepatocytes to fully repopulate the liver (Guha et
al., 1999). Apoptosis of DNA damaged endogenous
hepatocytes probably plays a role in long-term liver
repopulation in both the retrorsine and irradiation mod-
els. In this regard, injection of Fas antibody has also been
used to induce apoptosis in the host liver and to allow
proliferation of transplanted hepatocytes transduced
with an adenovirus containing the anti-apoptotic gene,
bcl-2 (Mignon et al., 1998).
LIMITATIONS OF HEPATOCYTE TRANSPLANTATION
PROTOCOLS FOR LIVER REPOPULATION
With these results in hand, why do laboratories continue
to pursue the liver stem cell? The reasons are many.
First, in order for mature hepatocytes to repopulate the
liver, there needs to be a substantial selection advantage
for transplanted cells in conjunction with extensive
ongoing liver damage, parenchymal loss, or inability of
endogenous hepatocytes to proliferate. However, most
conditions for which liver cell transplantation might rep-
resent useful therapy do not meet these conditions. In
addition, if stem/progenitor cells continue to proliferate
long after their transplantation, these cells would not
need great selection pressure to ultimately repopulate
the liver. Second, as mentioned previously, differentiated
hepatocytes cannot be propagated effectively in cell cul-
ture. Hopefully, this will not be true for liver
stem/progenitor cells, and it will be possible to use cul-
tured liver stem/progenitor cells or cell lines for liver
repopulation. Third, although transplanted hepatocytes
remain at high levels and for a long time in rodent liver
(studies have been reported for up to one year or half
the lifetime of a rat), similar results may not occur with
mature hepatocytes in humans. This view is supported
by studies in patients with familial hypercholesterolemia
and congenital hyperbilirubinemia (Crigler-Najjar
Syndrome, type I) in which from the data reported it
would seem that transplanted hepatocytes do not prolif-
erate significantly after their transplantation (Grossman
et al., 1995; Fox et al., 1998). This limitation is critical,
since portal hypertension, hepatic infarction, and
26 EINSTEIN QUARTERLY, Copyright © 2002
4SCIENTIFIC REVIEW
ischemic necrosis become major complications when the
amount of liver cells transplanted exceeds one to two
percent of the hepatic parenchymal mass. Finally, it has
been difficult to introduce foreign genes into hepato-
cytes in vitro and effective gene therapy might be more
readily achieved with liver stem/progenitor cells. A par-
tial list of potential advantages of liver stem/progenitor
cells for liver repopulation, compared to mature hepato-
cytes, is given in Table I.
LIVER REPOPULATION WITH FETAL LIVER STEM/PRO-
GENITOR CELLS
As eluded to earlier, classical embryological studies have
traced the proliferation and differentiation of progeni-
tor or determined stem cells into the hepatocytic and
biliary epithelial lineages during normal liver develop-
ment in both rodents and humans (Shiojiri et al., 1991;
for review, Grisham and Thorgeirsson, 1997). This process
begins at embryonic day (ED) 8.5 in the mouse with pro-
liferation of epithelial cells of the ventral foregut and
migration of these cells into the septum transversum,
where they come into contact with mesenchymal cells.
These cells express AFP and then albumin, and subse-
quently, the liver bud forms at ED11. At this time, the
morphology of the undifferentiated small epithelial pro-
genitor cell changes to that of the characteristic
hepatoblast , which begins to differentiate into hepato-
cytes and biliary epithelial cells between ED15 and ED16.
Thus, the fetal liver contains epithelial cells that are in
different stages of lineage progression, some of which
might still contain the full potential of liver stem cells.
In the early to mid 1990’s, Reid, Gupta, and coworkers
isolated and characterized rat fetal liver epithelial cells
using immunoselection by “panning” and fluorescence
activated cell sorting (FACS) with the hope of identifying
unique oval cell markers in hepatoblasts, but these mark-
ers were shared with hematopoietic cells (Sigal et al.,
1994). They subsequently transplanted unfractionated
ED14 fetal liver cells into the spleen of DPPIV- rats and
observed DPPIV+ and glucose-6-phosphatase+ cells in the
spleen, as well as small numbers of DPPIV+ cells with a
diffuse (undifferentiated) pattern of DPPIV expression in
the liver (Sigal et al., 1995). Additional characterization of
ED12 to ED14 fetal liver cells in our laboratory indicated
that there are at least three distinct subpopulations of
hepatic epithelial progenitor cells within the fetal liver
at this time unipotent progenitor cells expressing either
albumin/AFP (hepatocytic) or cytokeratin-19 (biliary
epithelial) markers and bipotent stem/progenitor cells
simultaneously expressing albumin/AFP and cytokeratin-
19 (Dabeva et al., 2000b).
Under protocols developed in our laboratory, ED14 fetal
liver stem/progenitor cells proliferate extensively after
they are transplanted. This can be achieved either in the
absence or presence of retrorsine treatment, although
 4SCIENTIFIC REVIEW
Liver Stem/Progenitor Cells and their Transplantation: From Fantasy to Reality as We Enter the New Millennium

FIGURE 8 Production of mixed clusters containing both mature hepatocytes and bile duct structures in the liver after transplantation of bipotent ED14
fetal rat liver stem/progenitor cells. The arrows point to transplanted cells forming mature bile ducts structures joining with a preexisting host duct with-
in a large cluster of proliferated cells exhibiting a well-differentiated hepatocytic phenotype that have become fully integrated into the parenchymal
structure.

TABLE 1 POTENTIAL ADVANTAGES OF LIVER STEM/PROGENITOR CELLS COMPARED

TO MATURE HEPATOCYTES FOR LIVER REPOPULATION

1. Less selection pressure required to proliferate after transplantation

2. Propagatable in culture and possibility to develop liver stem/progenitor cell lines
3. Proliferation may continue for a long time after transplantation
4. May be responsive to hormones and growth factors
5. Possibility to regenerate complete liver lobules
6. May remain viable for a long time and maintain the restored liver mass better
7. Possible use for ex vivo gene therapy

The Einstein Quarterly Journal of Biology and Medicine 27


Liver Stem/Progenitor Cells and their Transplantation: From Fantasy to Reality as We Enter the New Millennium
repopulation occurs more rapidly and is more extensive
in retrorsine treated hosts (Dabeva et al., 2000b; Sandhu
et al., 2001). In both cases, PH is required for effective liver
repopulation. With transplanted fetal liver stem/progen-
itor cells, we observe formation of new hepatic cords
and bile ducts (Figure 8) and in the case of retrorsine
treatment complete new liver lobules (Sandhu et al.,
2001). Using double labeling with immunohistochem-
istry and in situ hybridization, we have shown that
transplanted stem/progenitor cells undergo active DNA
synthesis for an extended period of four to six months.
In the absence of retrorsine treatment, mature hepato-
cytes show proliferative activity for only two to four
weeks following their transplantation (during the
regenerative phase after PH). Under these circum-
stances, we observe at most three to four cell divisions,
and there is no significant liver repopulation. In contrast,
with ED12 to ED14 fetal liver stem/progenitor cells in the
absence of retrorsine treatment, we have observed up to
nine to ten cell divisions (determined by counting the
number of cells in very large clusters at six months after
cell transplantation) and have been able to achieve up
to ten percent liver repopulation (Sandhu et al., 2001).
Thus, transplantation with fetal liver stem/progenitor
cells clearly has advantages over mature hepatocytes.
TRANSPLANTATION OF HEMATOPOIETIC STEM CELLS
INTO THE LIVER
Tissue determined stem/progenitor cells have been iden-
tified in the bone marrow, liver, and brain (Fuchs and
Serge, 2000; Gage, 1998). Bone marrow and brain
stem/progenitor cells can change their phenotype when
transplanted into another host tissue (Farrari et al.,
1998; Bjornson et al., 1999; Kopin et al., 1999; for sum-
mary of current studies, see Figure 9 and Vogel, 2000).
In this regard, transplanted bone marrow cells that
engraft into the liver differentiate into hepatocytes.
Petersen et al. (1999) transplanted unfractionated bone
marrow cells from male rats (containing a Y chromo-
some) into lethally irradiated female recipients and
showed the presence of Y chromosome containing
hepatocytes in the recipient. In separate studies, Theise
et al. (2000a) reported that lethally irradiated female
mice that received either unfractionated bone marrow
cells or FACS-sorted CD34+/lin- cells from male donors
showed Y chromosome positive cells in the liver by fluo-
rescence in situ hybridization (FISH). In the latter studies,
cells with the morphologic appearance of hepatocytes
and both a Y chromosome and evidence for synthesis of
albumin were still present at eight months following
bone marrow transplantation. However, in neither case
was there extensive liver repopulation. Most recently,
Theise et al. (2000b) and Alison et al. (2000) have report-
ed the presence of Y chromosome positive hepatocytes
in the liver of human female recipients of male bone
marrow cells and in human male recipients of orthotopic
liver transplants from female donors.
28 EINSTEIN QUARTERLY, Copyright © 2002
4SCIENTIFIC REVIEW
ROLE OF THE LIVER MICROENVIRONMENT
An interesting aspect of liver repopulation is that the
host site in which the transplanted cells are engrafted
seems to play a role in their phenotypic behavior. When
hepatocytes are transplanted into the dorsal fat pad,
spleen, or peritoneal cavity retain expression of some
hepatocyte specific genes, but this is usually quite limit-
ed. However, when hepatocytes are transplanted to the
liver, cells with a robust hepatocytic phenotype are rou-
tinely observed. With fetal liver stem/progenitor cells,
we have observed additionally that cells engrafting into
the hepatic cords differentiate into hepatocytes, where-
as cells engrafting into the bile ducts differentiate into
bile duct epithelial cells (Sandhu et al., 2001). As indi-
cated previously, some cells exhibit bipotency and
generate clusters containing both hepatocytes and bile
ducts. These are the first studies demonstrating in vivo
the bipotency of isolated liver epithelial cells, a property
required for a cell to be considered a stem cell. When
transformed, undifferentiated hepatoma cells are trans-
planted into the liver, they also assume a mature
hepatocytic phenotype and become arrested in cell
growth (Coleman et al., 1993). Thus, the local liver envi-
ronment plays an important role in determining the
phenotype of transplanted cells.
ENRICHED POPULATIONS OF LIVER
STEM/PROGENITOR CELLS
Studies are now progressing to enrich for or purify liver
stem/progenitor cells by selection with specific antibod-
ies and FACS or immuno-magnetic bead separation of
antibody tagged cells. However, specific selection mark-
ers for fetal liver stem/progenitor cells need to be
identified. Fetal liver cells have been sorted for those
that are positive for expression of α6 (CD49f) integrin
and β1 (CD29) integrin, which are known to be present
on hepatocytic cells, and negative for CD45 and Ter119,
which are specific markers for hematopoietic cells
(Suzuki et al., 2000). Interestingly, cells showing the
greatest enrichment for liver progenitor cells in a colony-
forming assay were negative for c-kit (Suzuki et al., 2000),
which is a positive sorting marker for hematopoietic
stem cells and is also detected in liver oval cells (Fujiio et
al., 1994). In contrast, c-kit positive selection has been
used to enrich for human liver stem-like progenitor cells
that can differentiate in culture into biliary epithelial
cells (Crosby et al., 2000). In other studies, CD34 has
been used for selection of hematopoietic stem cells that
can differentiate into hepatocytes (Theise et al., 2000a).
Kubota and Reid (2000) have used entirely different
genes (MHC class I, OX18, and ICAM-1) to select for ED13
fetal rat cells that express phenotypic markers for hepa-
tocytes (AFP and albumin) and bile duct epithelial cells
(CK-19) in culture. Interestingly, in several of these stud-
ies (Suzuki et al., 2000; Crosby et al., 2000; Kubota and
Reid, 2000), cells with similar marker expression patterns
 4SCIENTIFIC REVIEW
Liver Stem/Progenitor Cells and their Transplantation: From Fantasy to Reality as We Enter the New Millennium

FIGURE 9 Differentiation of tissue derived stem cells into different organ-specific phenotypes after
their transplantation.

FIGURE 10 Developmental origin of liver stem cells

and intermediate steps in their maturation pathways
leading to differentiation into hepatocytes and bile
duct epithelial cells.

The Einstein Quarterly Journal of Biology and Medicine 29


Liver Stem/Progenitor Cells and their Transplantation: From Fantasy to Reality as We Enter the New Millennium
could be isolated from the normal adult liver, albeit at
reduced numbers, suggesting that tissue-specific
stem/progenitor cells may be present in the mature
organ. These findings are consistent with our original
observation of undifferentiated AFP positive epithelial
cells in the periportal region of the adult rat liver (Alpini
et al., 1992).
In most spectacular recent studies, extensive liver repop-
ulation has been observed with highly purified
hematopoietic stem cells in the FAH null mouse (Lagasse
et al., 2000). In this model, donor hematopoietic stem
cells differentiated into hepatocytes, but not into bile
duct epithelial cells. Therefore, either there is no defi-
ciency in bile ductular cells in the FAH null liver, so that
transplanted bone marrow cells or hematopoietic stem
cells do not need to differentiate along this lineage, or
hematopoietic stem cells in mice are not bipotent for
liver epithelium. As noted previously, stem cells have
been found in other tissues, such as the brain, and when
transplanted, these cells differentiate into different
mature phenotypes depending on the organ environ-
ment in which they are engrafted. Thus, it is clear that
liver stem/progenitor cells, their hematopoietic cousins,
and perhaps other stem cell relatives, have a bright
future in the treatment of liver, as well as other diseases.
SUMMARY
During the past decade, it has become clear that the
mammalian liver contains stem/progenitor cells. These
cells are derived embryologically from the foregut endo-
derm (see Figure 10) and seed the bone marrow, giving
rise to hematopoietic stem cells. A role for stem/progen-
itor cells in maintaining the liver parenchyma during
normal liver cell turnover or in restoring liver mass
during liver regeneration has not been established.
However, these cells can be induced to proliferate under
circumstances in which the proliferative capacity of
mature hepatocytes is blocked. Both hepatocytes and
stem/progenitor cells can repopulate the liver under
highly selective conditions of severe continuous liver
injury or disruption of endogenous hepatocytes prolifer-
ation, but only stem/progenitor cells can do this in a
normal liver environment. Recently, it has been discov-
ered that hematopoietic stem cells can migrate to the
liver and differentiate into hepatocytes in both rodent
models and in humans. However, a role for these cells in
liver regeneration or repopulation remains to be estab-
lished. All of these findings provide hope that under
selected circumstances cell transplantation will become
a therapeutic reality in the treatment of inherited or
acquired chronic liver diseases.
Acknowledgments
The authors would like to give thanks to present and
30 EINSTEIN QUARTERLY, Copyright © 2002
4SCIENTIFIC REVIEW
past students, postdoctoral fellows, and the technical
staff of our laboratory for their contributions to this
research; to other faculty members in the Marion Bessin
Liver Research Center for their contributions to this
work and other studies cited; and to Anna Caponigro
for secretarial assistance.
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