Current Concepts in Liver Pathology: An Update, Part I
Moderator: Dr. Paulette Bioulac-Sage
Section 1 -
Liver Stem Cells
Neil D. Theise MD
Depts of Pathology and of Medicine
(Division of Digestive Diseases)
Beth Israel Medical Center
Albert Einstein College of Medicine
New York, NY, USA
Tania Roskams, MD, PhD
Professor in Pathology
Head Liver Research Unit
Department of Pathology
University of Leuven
(Presentation by Dr. Roskams, handout prepared by Dr. Theise)
The capacity of hepatocytes and cholangiocytes to contribute to their own maintenance has long
been recognized. Studies of normal and diseased liver tissue readily demonstrate that hepatocytes and
cholangiocytes can undergo mitotic devision and, when injury is severe enough, show regenerative changes,
such as hepatocyte binucleation, cell plate thickening, cholangiocyte hyperplasia. That hepatocytes
could do this in a virtually stem cell like fashion was further shown by the now classic experiment by
Grompe et al, in which serial transplantation of mature hepatocytes from mouse to mouse with the fumaryl
acetate hydroxylase (FAH) gene knockout knockout resulted 1000 fold regeneration from the original cells,
seemingly without limit . This elegant experiment definitively established the ability of mature
hepatocytes to repopulate an entire organ and self renew. Nevertheless, despite this finding that
hepatocytes could essentially act as their own stem cell, candidates for a bipotent stem cell, which
could also regenerate cholangiocytes, were still investigated and debated.
Intraorgan Liver Stem Cells
In rodent models, the best candidate for hepatic stem cells has long been the "oval cell"
population which emerges in the context of toxic blocking of hepatocyte regeneration followed by
additional liver damage, such as partial hepatectomy or carbon tetrachloride injury
were oval in shape, with a darkly staining nucleus and scant, basophilic cytoplasm. The source of these
cells in the normal liver was felt to be a quiescent, facultative cell compartment, difficult to observe,
lying either within the biliary tree , directly adjacent to the biliary tree within portal tracts or
in the periportal parenchyma
Though highly controversial for decades, consensus eventually
formed that these were indeed facultative stem cells rather than merely a marker of severe hepatic injury
In humans, the ductular reaction that is seen in massive hepatic necrosis was identified as a
possible human correlate to the oval cell. The morphologic appearance was different from the rodent oval
cells: referred to as "ductular hepatocytes" by Gerber and Thung , these clusters of cells often had
cholangiocytic features at one end, hepatocytic features at the other, with cells of intermediate
morphology and immunophenotyping between them
These features suggested activation of a stem
cell compartment to many of the investigators, but biliary metaplasia in cholestatic, injured hepatocytes
to others .
The question of which of these interpretations pertained was fairly well settled in two ways.
In our own work we followed up on the work from Michael Gerber's laboratory. Immunostaining for
biliary-type cytokeratins had identified small oval shaped cells in undisturbed hepatic parenchyma near
to, but not within the portal tract stroma. They suggested that these might be the human equivalent to
the oval cell . Three dimensional reconstruction of these cells and nearby bile ducts determined
that they were in fact the small cholangiocytes lining the canals of Hering, thereby revising the
accepted understanding of the architecture of these lesions, showing that they extend beyond the limiting
plate, into the proximal third of the lobule. Similar three dimensional reconstruction of the ductular
reactions in massive hepatic necrosis indicated that they were budding off of the pre-existent canal of
which had become highly proliferative . Thus, we concluded: "the canal of Hering is
comprised of, or at least harbors, facultative stem cells of the liver." Following similar procedures,
we have now also shown that small intraseptal hepatocyte nodules in cirrhosis derive from the biliary
tree as well, though not just from the canal of Hering, but also from the interlobular bile ducts, thus
the same statement may be said of more distal biliary structures .
The other convincing approach was that of Roskams et al, using serial sampling of livers with
acute injury to extend their examination into the fourth dimension, i.e. over time . This added
dimension again convincingly demonstrated that the ductular reactions are proliferative and regenerative,
not merely a non-functional sign of damage.
Extraorgan Liver Stem Cells
Two different lines of investigation led to the discovery that there were circulating cells, at
least partially bone marrow-derived, which can also contribute to liver turn over and repair following
injury. Bryon Petersen took note that he and several other investigators had identified markers on oval
cell populations in rodents and in humans that were commonly thought of as hematopoietic markers,
including Thy-1, CD34, and c-kit
Speculating that these marker profiles might be indicative of
marrow origin he performed bone marrow transplantations and liver transplantations in rodents utilizing
various markers to indicate cells of donor vs. recipient origin. Using the well established protocol of
AAF/CCL4 for liver injury he showed both oval cells and hepatocytes deriving from the marrow
On the other hand, we recognized that our hypothesis that the canal of Hering was the source of
ductular/oval cell reactions could not explain the findings of Sell et al that in response to periportal
necrosis from allyl alcohol toxicity in rats the oval cells were devoid of all usual markers of oval
cells in the first day of proliferation, including the biliary cytokeratins . This suggested an
alternate source of cells to the canal of Hering. We performed bone marrow transplants in mice using
both whole bone marrow and CD34+lin- marrow cells. In the absence of overt injury
(i.e. without necrosis, regenerative changes, or an oval cell reaction) isolated hepatocytes were shown
to be bone marrow derived .
Thus, liver regeneration appeared, surprisingly, to be a three-tier process with mature cells,
intraorgan stem cells, and extraorgan stem cells all contributing to liver turn over and regeneration
after injury. It seemed that it was the proportional contribution of these compartments that shifted
depending on the type and extent of injury. But the fact of these multi-tier processes led to more
questions than ever.
Confirmation and Elaboration
First, there was confirmation that circulating cells were not merely a rodent phenomenon. We
examined human tissues that were correlates of both our own and Petersen's marrow-derivation studies,
looking at tissue specimens from women with male bone marrow transplants with minimal injury and from men
who received liver allografts from female donors, with subsequent injury severe enough to cause a
ductular reaction . In all of these specimens there was significant engraftment from outside the
liver, and at much higher intervals than that seen in the rodents. In fact, in one patient with the most
severe form of recurrent hepatitis C, the fibrosing cholestatic variant, up to nearly 40% of hepatocytes
and cholangiocytes were derived from circulating cells. Moreover, as Petersen had shown in the rodents,
the ductular reaction had significant numbers of cells also extrahepatically derived, suggesting a flow
through of cells through the ductular intermediates. Virtually simultaneously, Alison et al similarly
confirmed human hepatocyte derivation from marrow derived cells, using similar techniques of in situ
hybridization for Y chromosomes in gender mismatched donor/recipient chimeric organs . Subsequent
work from Korbling et al revealed similar findings 
The next confirmation of the marrow to liver pathway was a paper by Lagasse et al using the FAH
knockout mouse described above . Using different subpopulations of marrow-derived cells instead of
hepatocytes, the investigators once again achieved metabolic rescue. However, the rescue was slower and
less efficient than that seen in the original hepatocyte transplantation work, suggesting that for some
types of therapeutic interventions hepatocytes might prove a better option.
Questions and controversies
Now that these various phenomena have been well established in experimental animals and in
humans, research efforts are now turning to more finely detailed elucidation of the mechanisms whereby
cells enter the liver from the circulation.
Which circulating cells can regenerate hepatocytes? The initial
animal studies primarily used whole, unfractionated bone marrow to accomplish the experiments. However,
in our own study, two archival mice initially transplanted with 200 CD34+lin- cells
also demonstrated the same engraftment potential . Thus, CD34 positive cells are capable of hepatic
engratment. In the FAH knockout model, the authors' emphasized the complete restitution of function only
by their favored hematopoietic subpopulation: c-kit+sca1+lin- .
While it is true that this population is the most efficient and successful at metabolic rescue, it is not
the only population – as the authors claim – that can regenerate hepatocyes. Indeed they cite data that
shows hepatocyte regeneration from all fractionated subpopulations: positive and negative for c-kit,
sca1, and mature lineage markers. Thus, while these other subpopulations are less efficient, they still
are capable of plasticity in the direction of hepatocytes, detected under the extreme pressure of the
injury model. The data thus solidly demonstrates the stochastic nature of these cell lineage phenomena
and points up the likely folly of definitive statements as to which cells are capable of hepatocyte
differentiation. The answer is that all cells are, it just depends on how they are conditioned and what
environment encourages the process
Other mesoderm-derived cells have experimentally been demonstrated to give rise to hepatocytes,
even if they are less likely to contribute to physiologic processes. Experimentally, human cord blood
stem cells have engrafted as albumin and alpha-1-antitrypsin producing hepatocytes in SCID mice .
"Multipotent adult progenitor cells", obtained from long term culture and conditioning of marrow-derived
stromal cells, as per Verfaillie et al, can give rise to hepatocytes in vitro and in vivo
findings further emphasize the far greater possibilities for plasticity than imagined until recently, and
indicate that sources of cells for therapeutic options are not limited to those which play important
What signaling regulates hepatic engraftment from marrow cells?
Recent work by Petersen demonstrates that in severe toxic injury to the liver, hepatocytes produce
SDF-1. This chemokine binds to CXCR4, present on some circulating cells, and acts as a chemoattractant.
Originally defined as a signaling pathway whereby hematopoietic cells are summoned to the stem
cell:stromal cell niche in the marrow, this work suggests that it is part of a more ubiquitous
engraftment modulating pathway. Interestingly, in Petersen's experiments, partial hepatectomy did not
result in the same production of SDF-1 and in our own experiments (author's unpublished data), partial
hepatectomy did not upregulate engraftment from the marrow.
A second signaling pathway that may be important is that of c-kit and its ligand, SCF (stem
cell factor). Direct experimental evidence for this is, as yet, lacking. However, the canals of Hering
are c-kit positive as are the proliferating ductular reactions in massive hepatic necrosis  and c-kit
positive cells isolated from human livers have been demonstrated in vitro to behave like an hepatic
Moreover, recent work implicates MMP-9-stimulated SCF production in
activation and recruitment of hematopoietic stem cells in the marrow . These isolated findings are
suggestive that c-kit/SCF is another important pathway for multiorgan engraftment processes.
The work of Orlic et al, regarding mouse models of myocardial infarction and marrow derived
cardiac myocytes implicates a third pathway . By administering G-CSF to their experimental mice,
thereby promoting mobilization of "hematopoietic stem cells" (classically speaking) from the bone marrow,
they achieved higher levels of cardiac engraftment from the marrow. Whether this was a purely artificial
effect, or reflects a physiological pathway of control of mobilization of cells for healing of injury in
distant organs remains unclear.
Fusion vs. Direct Differentiation The most vociferous and, perhaps, most interesting controversy turns out to be no controversy
at all as data has accumulated. The original marrow-to-liver findings were based on chromosome tracking,
mostly using male cells into female recipients. If one stained for only the Y chromosome, this would not
preclude the possibility that rather than a direct differentiation from marrow cell to liver,
investigators might have actually been observing fusion of the engrafting cell with a target hepatocyte
(or other cell). Indeed, when full cytogenetics was performed in the FAH-null mouse, indeed fusion
(perhaps sometimes followed by reduction division)
was indeed identified . Subsequent studies using
more sophisticated transgenic approaches to sorting out fusion from direct differentiation have indicated
that both may occur.
While some will strive to maintain the controversy, usually for reasons
independent of the actual investigative findings , consensus of most people actively working in the
field suggests that both these pathways of plasticity exist and their presence depends on whether there
is injury present and, if so, the nature and the extent of that injury
We are at the beginning of a new era in understanding of liver regeneration. One main avenue
of investigation will explore the physiological pathways by which liver tissue is maintained or repaired
after injury. The other avenue will involve elucidation of the technical aspects of therapeutic
interventions, from which cells can be employed, to methods of cell application (transplantation,
construction of artificial organs, growth of new organs, targets for gene therapy). Undoubtedly, the
latter avenue will exploit details elucidated in the former; however, there will certainly also be
creative use of non-physiological possibilities. In this latter category we may consider hepatic
progenitor sources such as cord blood or embryonic stem cells.
Ultimately, therapeutic success will be achieved when the combinations of techniques yield
efficient and cost-effective production and expansion of cells in vitro, or of the cytokine/chemokine
elements that could stimulate our own bodies to accomplish therapeutic outcomes. On the other hand,
despite the occasional ineffectiveness of the system – such as in fulminant hepatic failure – the
majority of us have livers that function steadily, without significant defect, over a lifetime and which
recover from even severe insults. Therapeutics will focus on rescuing the comparatively rare failures
of this system, but the rest of us will rely on the rather wondrous ability of the body to keep our liver
and us alive in that long period stretching from birth to death (from non-hepatic causes).
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