LIVER BIOLOGY/PATHOBIOLOGY
Locating the Stem Cell Niche and Tracing Hepatocyte
Lineages in Human Liver
Tariq G. Fellous,1 Shahriar Islam,1 Paul J. Tadrous,2 George Elia,3 Hemant M. Kocher,4 Satyajit Bhattacharya,5
Lisa Mears,6 Douglas M. Turnbull,7 Robert W. Taylor,7 Laura C. Greaves,7 Patrick F. Chinnery,7 Geoffery Taylor,7
Stuart A.C. McDonald,3,8 Nicholas A. Wright,1,3 and Malcolm R. Alison1
We have used immunohistochemical and histochemical techniques to identify patches of
hepatocytes deficient in the enzyme cytochrome c oxidase, a component of the electron
transport chain and encoded by mitochondrial DNA (mtDNA). These patches invariably
abutted the portal tracts and expanded laterally as they spread toward the hepatic veins. Here
we investigate, using mtDNA mutations as a marker of clonal expansion, the clonality of
these patches. Negative hepatocytes were laser-capture microdissected and mutations identified by polymerase chain reaction sequencing of the entire mtDNA genome. Patches of
cytochrome c oxidase– deficient hepatocytes were clonal, suggesting an origin from a longlived cell, presumably a stem cell. Immunohistochemical analysis of function and proliferation suggested that these mutations in cytochrome c oxidase-deficient hepatocytes were
nonpathogenic. Conclusion: these data show, for the first time, that clonal proliferative units
exist in the human liver, an origin from a periportal niche is most likely, and that the
trajectory of the units is compatible with a migration of cells from the periportal regions to
the hepatic veins. (HEPATOLOGY 2009;49:1655-1663.)
A
dult tissue-specific stem cells are thought to reside
within a specialized microenvironment, known as
the niche, and it is here that stem cell behavior is
regulated and maintained.1 In epithelia with ordered
structure and in a state of continual cell renewal, there is
often a hierarchical organization with stem cells at the
Abbreviations: 3D, three-dimensional; CYP-1A2, cytochrome P-1A2; mtDNA:
mitochondrial DNA; PBS, phosphate-buffered saline; SDH, succinate dehydrogenase.
From the 1Centre for Diabetes and Metabolic Medicine, Barts and the London
School of Medicine and Dentistry, Queen Mary University of London, London,
United Kingdom; the 2Department of Histopathology, Northwick Park Hospital,
Harrow, London, United Kingdom; the 3Histopathology Unit, London Research
Institute, Cancer Research UK, London, United Kingdom; the 4Tumour Biology
Laboratory, John Vane Science Centre, Barts & The London School of Medicine &
Dentistry, London, United Kingdom; 5Barts and The London HPB Centre, London, United Kingdom: the 6Department of Histopathology, Pathology and Pharmacy Building, Barts & The London School of Medicine & Dentistry, London,
United Kingdom; the 7Mitochondrial Research Group, Institute for Ageing and
Health, University of Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom; the 8Centre for Gastroenterology, Barts and The London School of Medicine
and Dentistry, United Kingdom.
Received October 6, 2008; accepted December 7, 2008.
Supported by a Grant from Barts and The London Charitable Trust.
Address reprint requests to: Malcolm R. Alison, Centre for Diabetes and Metabolic Medicine, 4 Newark Street, London E1 2AT, UK. Email:
[email protected]; fax: (44) 207 882 2186
Copyright © 2009 by the American Association for the Study of Liver Diseases.
Published online in Wiley InterScience (www.interscience.wiley.com).
DOI 10.1002/hep.22791
Potential conflict of interest: Nothing to report.
Additional Supporting Information may be found in the online version of this
article.
beginning of the flux, and terminally differentiated, reproductively sterile cells at the end of the flux, imminently
to be lost from the population. Many studies have attempted to identify stem cells and the location of the
niche using histological methods based on the premise
that stem cells have inherent properties such as DNA label
retention, high integrin expression, and abundant detoxifying enzyme activity.2 However, many uncertainties remain even in comparatively well-defined instances such as
the hematopoietic system.3
It has been proposed that the gold standard of stem cell
identification involves marking putative stem cells to
identify the niche, and then performing lineage tracing to
demonstrate that the proposed “stem cell” has multipotentiality.4 This approach commonly uses mice genetically engineered to have a steroid-activated version of Crerecombinase knocked into the putative stem cell marker
gene, such that Cre activation mediates excision of a roadblock sequence in the Rosa26-lacZ reporter, thus resulting
in an irreversible marker in all the descendants of the
putative stem cell. Using this technology, it has been
shown, for example, that mouse hair follicle bulge cells
expressing K15 generate all the epithelial cells in the lower
hair follicle,5,6 whereas in the murine small intestine,
long-lived cell clones containing all the intestinal cell lineages can be generated from both leucine-rich repeat-containing G protein– coupled receptor 5 [Lgr5]– expressing7
and polycomb ring finger oncogene [Bmi1]– expressing
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FELLOUS ET AL.
cells,8 possibly indicating the presence of distinct subpopulations of stem cells.
In human epithelial tissues, and in this case the liver,
progress in identifying the stem cell niche, the stem cells
themselves, and lineage tracing from these stem cells has
been practically impossible. Indeed, although the liver has
a remarkable ability to regenerate from its usually proliferatively quiescent hepatocyte population,9 the fact that
in young rats the response to two-thirds partial hepatectomy involves all hepatocytes traversing the cell cycle at
least once10 has called into question the need for a distinct
stem cell compartment. By definition, stem cells should
be self-renewing and clonogenic, and models of hepatocyte transplantation have shown that transplanted hepatocytes are capable of significant clonal expansion in
diseased liver,11 and it was suggested that perhaps a subpopulation of hepatocytes was responsible for the colonization, cells that were termed “regenerative transplantable
hepatocytes.” Maybe these regenerative transplantable
hepatocytes are analogous to the small hepatocyte-like
progenitors described by Gordon et al.,12 observed by
us,13 and associated with compromised hepatocyte replicative efficacy in the face of a regenerative demand. From
retroviral lineage tracing studies in rats, the origin of these
cells is thought to be hepatocytes,14 but their location is
unclear, seeming to be located at random in the parenchyma rather than confined to a particular niche.
In the mouse, the location of label-retaining cells has
suggested a parenchymal stem cell niche close to the portal area,15 whereas in human liver, rare putative stem cells
strongly expressing signal transducer and activator of
transcription protein 3 and the embryonic stem cell–associated pluripotency-associated factors Oct4 and Nanog
are also located near portal tracts.16 A niche close to portal
tracts would be also compatible with the “streaming liver”
hypothesis.17,18
We describe a new technique for lineage tracing in
human liver. It has recently been shown that human gastrointestinal stem cells and their progeny contain nonpathogenic mutations in their mitochondrial DNA,
including mutations in the cytochrome c oxidase gene, a
component of complex IV of the respiratory chain, that
are relatively common.19-21 Each mitochondrion contains
several hundred molecules of its own circular genome,
and in most cells several thousand of these organelles exist.
The mitochondrial genome is prone to mutation because
of a lack of protective histones and poor DNA repair
mechanisms. Mutations can expand stochastically within
a cell and over time cells will become either homoplasmic—all the mitochondria in the cell are mutated, or
heteroplasmic—the cell contains a mixture of mutated and
wild-type mitochondria. This stochastic expansion is a
HEPATOLOGY, May 2009
lengthy process, often taking many years,22 and for a mutated cellular phenotype to be observed, homoplasmy or a
high degree of heteroplasmy must be present. Thus, stem
cells are the only cells that have a sufficient lifespan to
accumulate these mitochondrial mutations to a level that
results in a detectable biochemical deficiency. We have
found many patches of cytochrome c oxidase–negative
hepatocytes in human liver, invariably connected to the
portal areas, and mtDNA sequencing of laser-captured
individual hepatocytes from these patches provided unequivocal proof that each patch was monoclonally derived. Thus, for the first time, we have identified clonal
proliferative units in the human liver, whose location suggests an origin from an area close to the limiting plate—
the stem cell niche.
Patients and Methods
Patients. Fifteen fresh frozen blocks of human liver,
of normal histological appearance, were obtained from
hepatic resections for metastatic colorectal carcinoma.
The samples were split into two, with one half being snap
frozen in liquid nitrogen and the other fixed in 4% formalin for 24 hours and then stored in 70% alcohol ready
for paraffin block processing. Another 45 formalin-fixed
paraffin-embedded tissue sections were also available.
Multicenter ethical approval was sought and obtained as
per the requirements of the United Kingdom Human
Tissue Act (2006). Ethical approval was granted from the
Redbridge and Waltham Forest Local Research Ethical
Committee (REC) on the May 9, 2006, to use human
tissue collected from the Royal London Hospital REC
(REC reference number 2006/Q0601/33).
Antibodies. The mouse monoclonal anti-OxPhos
Complex IV subunit I antibody was purchased from Molecular Probes Invitrogen (Paisley, UK). Anti-albumin
rabbit polyclonal was purchased from Dako (Cambs UK
A-0001). Anti-CYP 1A2 rabbit polyclonal was a gift from
Dr. Robert Edwards of Imperial College (Hammersmith
Campus, London, UK). Alexa Fluor 488 goat anti-rabbit
immunoglobulin G A-11008 and Alexa Fluor 555 goat
anti-mouse immunoglobulin G1 A-21127 were purchased from Invitrogen (Paisley, UK).
Cytochrome c Oxidase Immunohistochemistry. Immunohistochemical staining was performed on 4-␮m sections cut from formalin-fixed paraffin tissue blocks and
allowed to air-dry overnight. Paraffin sections were dewaxed in xylene and rehydrated through decreasing alcohol
concentrations and then blocked for endogenous peroxidases in methanol/0.18% H2O2. Antigen retrieval pretreatment was performed, boiling slides for 15 minutes in
10 mM sodium citrate pH 6.0. Sections were treated with
HEPATOLOGY, Vol. 49, No. 5, 2009
an avidin-biotin blocking kit (DakoCytomation) to block
endogenous biotin and nonspecific binding. Sections
were incubated for 1 hour at room temperature with the
primary anti-OxPhos Complex IV subunit 1 antibody
diluted 1:400. Sections were then incubated for 35 minutes with a rabbit anti-mouse biotinylated secondary antibody diluted 1:300 (DakoCytomation), followed by a
final tertiary layer of streptavidin-horseradish peroxidase
diluted 1:500 (Dako) for 30 minutes. Peroxidase activity
was revealed using 4 mmol/L 3, 3-diaminobenzidine as a
chromogen in phosphate-buffered saline (PBS) containing 0.2% H2O2. All sections were washed 3 ⫻ 5 minutes
in PBS between each step. Sections were counterstained
with Mayer’s hematoxylin and then dehydrated through
alcohol, cleared with xylene, and mounted in DePeX resinous mounting medium.
Dual Immunofluorescence for Hepatocyte Function. Paraffin sections (4 ␮m) were dewaxed in xylene,
and endogenous peroxidases were blocked by incubation
in 0.18% hydrogen peroxide in methanol, followed by
rehydration through graded alcohols to PBS. Sections
were microwaved for 10 minutes in boiling 10 mM sodium citrate buffer (pH 6.0), cooled in water, and rinsed
in PBS. Slides were pre-incubated in a protein block (DakoCytomation) for 15 minutes. For double-labeling, sections were incubated with the diluted primary antibody
against either albumin (1:800) or cytochrome P-1A2
(CYP-1A2) (1:200), followed by the appropriate fluorescently labeled secondary antibody (1:200). Slides were
then incubated with the anti- cytochrome c oxidase primary antibody (1:500), followed by the appropriate fluorescently labeled secondary antibody (1:200). For all
stages, antibodies were diluted in PBS, incubated for 40 to
45 minutes at room temperature, and sections were
washed in PBS between each step. Finally the doublestained sections were mounted in Vectorshield with 4⬘,6diamidino-2-phenylindole (Vector Lab Inc, Burlingame,
CA, H-1200) and coverslipped.
Enzyme Histochemistry for Cytochrome c Oxidase/
Succinate Dehydrogenase. Frozen sections were cut at a
thickness of 15 ␮m. Sequential cytochrome c oxidase and
succinate dehydrogenase (SDH) histochemistry was performed as previously published.19 SDH staining was used
to highlight cytochrome c oxidase– deficient cells. The
frozen sections were briefly air-dried before being incubated in cytochrome c oxidase medium containing 100
mmol/L cytochrome c, 20 ␮g/mL catalase, and 4 mmol/L
diaminobenzidine tetrahydrochloride in 0.2 mol/L phosphate buffer, pH 7.0, all sourced from Sigma Aldrich
(Poole, UK) for approximately 50 minutes at 37°C. Sections were then washed in PBS, pH 7.4, for 3 ⫻ 5 minutes
and then incubated in SDH medium (130 mmol/L so-
FELLOUS ET AL.
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dium succinate, 200 mmol/L phenazine methosulfate, 1
mmol/L sodium azide, and 1.5 mmol/L nitroblue tetrazolium in 0.2 mol/L phosphate buffer, pH 7.0) for approximately 45 minutes at 37°C, or until a strong blue
stain had developed. Sections again were washed in PBS
for 3 ⫻ 5 minutes and dehydrated in a graded ethanol
series (70%, 90%, 100%, and 100%), and cleared in Histoclear (Lamb Laboratory Supplies, Eastbourne, UK) and
mounted in Permount.
Isolation of DNA from Individual Cells. Frozen
15-␮m sections were fixed onto P.A.L.M. membrane
slides (P.A.L.M. Microlaser Biotechnologies, Bernried,
Germany) that had been sterilized under ultraviolet light
for 30 minutes. Cytochrome c oxidase– deficient hepatocytes or cytochrome c oxidase–positive hepatocytes were
collected in sterile 0.5-mL AdhesiveCap (Zeiss, Bernried)
tubes using a P.A.L.M. microdissection system. The tubes
were then centrifuged (7000g for 10 minutes) and the cell
was lysed in 20 ␮L lysis buffer (50 mmol/L tris(hydroxymethyl)aminomethane HCl pH 8.5, 1 mmol/L ethylenediaminetetra-acetic acid, 0.5% Tween-20, 200
ng/mL proteinase K, all sourced from Sigma) at an optimum temperature of 65°C for 3 hours, and then the
reaction was stopped by raising the temperature to 95°C
for 10 minutes to denature the proteinase K.
mtDNA Sequencing of Individual Hepatocytes.
Once the entire DNA was extracted from a hepatocyte, its
mtDNA was then selectively amplified and sequenced to
identify the entire mitochondrial genome sequence. The
mtDNA was amplified by two successive rounds of nested
polymerase chain reaction designed to generate overlapping
fragments, each tagged with an M13-tail to facilitate the
direct sequencing of polymerase chain reaction–amplified
products.20 Polymerase chain reaction products were sequenced using BigDye Ver3.1 terminator cycle sequencing
chemistries on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA) and compared directly
with the revised Cambridge reference sequence, using SEQUENCE ANALYSIS and SEQSCAPE software (Applied
Biosystems), as described.20
Three-Dimensional Modeling. Using custom-written software, we constructed fully interactive three-dimensional (3D) models as previously described,13
allowing visualization of cytochrome c oxidase– deficient
patches identified on serial sections throughout a paraffin
block of liver. One hundred serial sections of human liver
tissue were cut and placed on slides. Immunohistochemistry (as described above) was used to detect cytochrome c
oxidase– deficient patches. Each cytochrome c oxidase–
deficient patch was serially photographed through 100
sections at a resolution of 300 pixels/inch. The serial digital images were registered (aligned relative to each other)
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FELLOUS ET AL.
HEPATOLOGY, May 2009
Fig. 1. Immunohistochemistry on sections of morphologically normal liver tissue illustrating cytochrome c oxidase– deficient patches closely
apposed to portal tracts (PT). Note three patches are composed of heteroplasmic hepatocytes (A, D, F), whereas the three others are almost
homoplasmic (B, C, E). Patient was a 60-year-old woman.
using freely available automated histological tissue section
image registration software (Autoreg). (Tadrous PJ. Autoreg version 2.61, 2004, and FATCAM image processing software, Version 1.03 alpha, 2003.) The 3D models
were generated as fully manipulatable semitransparent
virtual reality modeling language models using the same
freely available 3D rendering sofware (FATCAM). Using
these models, one can make out the spatial relationship of
these patches in relation to structures, that is, portal tracts;
they also allow us to trace these patches over large areas.
Results
Immunohistochemical Analysis of Cytochrome c
Oxidase Activity. Immunostaining of normal human
liver identified patches of cytochrome c oxidase–negative
hepatocytes located in close proximity to or in direct contact with the portal tract region (Fig. 1). The patches
varied in size but had clearly defined boundaries, seem-
ingly always extending to the perivenous regions, though
not always appreciated in two-dimensional sections.
Some of the patches were composed of heteroplasmic
hepatocytes with residual cytochrome c oxidase activity
(Fig. 1A,D,F), whereas others were more homoplasmic
(Fig. 1B,C,E). Close inspection of a homoplasmic patch
(Fig. 2) revealed the sinusoid-lining cells to be cytochrome c oxidase positive, indicative of a separate lineage.
Other areas of liver had sharply defined patches of cytochrome c oxidase– deficient hepatocytes extending from
portal tract to the hepatic vein (Fig. 3A,B), whereas in
other sections we picked up extraordinarily linear patches
of hepatocytes seemingly also migrating toward the hepatic veins (Fig. 3C,D). These cytochrome c oxidase–
deficient patches were not noticeably deficient in either
albumin or cytochrome P450 1A2 (Fig. 4), and neither
were they more proliferative (Ki-67 staining) nor expressive of alpha-fetoprotein (data not shown). In total, 60
HEPATOLOGY, Vol. 49, No. 5, 2009
FELLOUS ET AL.
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Fig. 2. (A) A single-cytochrome c oxidase– deficient patch, appearing to emanate from the portal tract. (B) High-power magnification illustrates
that within the patch there are cytochrome c oxidase–positive sinusoid-lining cells (*), clearly pointing to the fact that, as expected, such cells are
of different cell lineages from hepatocytes. Patient was a 61-year-old man.
patient samples (average age, 61 years) were analyzed, and
of these 60% had no detectable cytochrome c oxidase–
deficient patches, but in many cases we were restricted to
the analysis of a single section. A 33-year-old man was the
youngest patient from whom we detected a cytochrome c
oxidase– deficient patch, albeit a very small one.
Clonal mtDNA mutations were present within each
patch (Figs. 5 and 6 and Table 1), thus demonstrating the
clonal expansion of a population of cells within morphologically normal liver tissue. Because of the long time
required for mtDNA mutations to become established in
the population, it is likely that these negative patches represent the progeny of a long-lived cell type, such as a stem
cell or committed progenitor cell.
One hundred serial sections of paraffin-embedded normal human liver were immunostained for cytochrome c
oxidase complex IV subunit 1 and reconstructed to generate a 3D model (Fig. 7 and Supporting Fig. 1). This
showed that these patches can be large, stretching over
600 ␮m, underlining the clonogenicity of the mutated
cell. In these 3D models, a clear association of the patches
with portal areas can be appreciated.
Discussion
Fig. 3. (A) Well-demarcated patch of cytochrome c oxidase– deficient
hepatocytes stretching from the portal tract to the hepatic vein. (B)
High-power view of the perivenous area illustrated in (A); note the
pyknotic hepatocyte nuclei (arrow) consistent with the “streaming liver”
hypothesis that predicted cell death at the perivenous region. (C, D)
Remarkably linear patches of cytochrome c oxidase–negative hepatocytes seemingly feeding into the hepatic veins (HV). (A, B, D) From a
71-year-old man and (C) from a 51-year-old woman.
The liver in its usual state is largely proliferatively quiescent, with only a minority of hepatocytes replicating at
any one time. We have highlighted the presence of defined populations of clonal cells in the nonregenerative
liver, apparently originating from the periportal area. We
would therefore propose that this constitutes the liver
stem cell niche, which houses stem cells capable of giving
rise to very large populations of hepatocytes. The fact that
we cannot always trace, in a single section, a patch of
cytochrome c oxidase–negative hepatocytes extending
across the entire proposed trajectory from portal rim to
hepatic vein is consistent with the complex 3D architecture of the liver. Nevertheless, 3D reconstruction of multiple serial sections (Supporting Fig. 1) did confirm their
extensive nature.
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HEPATOLOGY, May 2009
Fig. 4. Dual immunofluorescence for cytochrome c oxidase/albumin (A-C) and cytochrome c oxidase/CYP 1A2 (D-F). (A) Cytochrome c
oxidase–positive hepatocytes stain red and the cytochrome c oxidase-negative cells are highlighted by a blue nuclear counterstain (4⬘,6-diamidino2-phenylindole). (B) Albumin staining showing uniform expression throughout the cytochrome c oxidase–negative patch and surrounding tissue. (C)
Merged image of (A) and (B) demonstrating albumin expression in the cytochrome c oxidase–negative patch. (D) Staining for cytochrome c
oxidase–negative patch as in (A). (E) CYP1A2 staining showing uniform expression throughout the cytochrome c oxidase–negative patch and
surrounding tissue. (F) Merged image of (D) and (E) demonstrating CYP1A2 expression in the cytochrome c oxidase–negative patch. Therefore,
cytochrome c oxidase–negative cells are neither synthetically nor metabolically impaired and exhibit identical staining to that of the surrounding
cytochrome c oxidase–positive hepatocytes. Patient was a 56-year-old woman.
Our observations are consistent with a recent report of
DNA label-retaining cells in mouse liver being found
within or close to the biliary ducts15 and the observation
of rare periportally located hepatocytes expressing embryonic stem cell–associated transcription factors in the human liver.16 In the mouse, Theise and colleagues15 labeled
cells with bromodeoxyuridine after a necrogenic dose of
acetaminophen, and then administered another dose 2
weeks later to induce several divisions of previously labeled cells. Label-retaining cells, considered to be slowly
dividing stem cells, were found, both as cholangiocytes of
interlobular ducts and peribiliary hepatocytes and socalled null cells. In human liver, rare putative stem cells
that strongly express signal transducer and activator of
transcription protein 3 and the embryonic stem cell–associated pluripotency-associated factors octamer 4 and
nanog were also located near portal tracts.16 Interestingly,
in a conditional cytochrome c oxidase knockout mouse, in
which Cre recombinase was under the control of an albumin promoter, repopulation occurred through the occurrence of cytochrome c oxidase–positive hepatocytes in the
periportal regions, suggesting that progenitor cells could
be undifferentiated albumin-negative cells.23
A niche close to portal tracts would also be compatible
with the “streaming liver” hypothesis. Gershom Zajicek
and colleagues17,18 proposed that the liver, and other very
slowly turning-over glandular cell populations such as
kidney and salivary glands, are in fact organized like the
intestine, with a unidirectional flux of cells.17,18 In the
case of liver, cells would be born at one end of the flux, the
portal area, and migrate down a path leading to the hepatic (central) vein. This hypothesis was put forward
HEPATOLOGY, Vol. 49, No. 5, 2009
FELLOUS ET AL.
1661
Fig. 5. (A) Two-color enzyme histochemistry can be used to simultaneously detect activity of the mtDNA-encoded cytochrome c oxidase and
nuclear DNA-encoded SDH; cells lacking in cytochrome c oxidase activity appear blue; cytochrome c oxidase–active cells appear brown. (B) Post-laser
capture. (C, D) Sequencing revealed an A⬎G point mutation at position 7570 in the cytochrome c oxidase– deficient cells but (E) not in the
cytochrome c oxidase–positive cells. Patient was a 68-year-old woman.
based on some very simple observations; intact adult rats
were injected with tritiated thymidine and from those
killed 1 hour later, labeled hepatocytes were on average
some 70 ␮m away from the portal rim. The remaining
rats were killed at intervals up to 4 weeks later, and the
average distance of labeled cells from the portal rim gradually increased to 140 ␮m, leading to the conclusion that
hepatocytes migrated at a velocity of over 2 ␮m/day from
the portal rim to the central vein. Not all agree with this
model; Bralet and colleagues24 labeled proliferating hepatocytes at 24 hours after a two-thirds partial hepatectomy
through retroviral mediated gene transfer of the Escherichia coli beta-galactosidase gene, and then studied the
fate of the labeled cells over the next 15 months.24 Over
Fig. 6. (A) Histochemistry of another patch (as Fig. 5). (B) Post-laser capture. (C, D) Sequencing revealed a C⬎T point mutation at position 6301;
note degree of heteroplasmy in (D) either because the hepatocyte is heteroplasmic or because of contamination with a sinusoid-lining cell. (E) No
such mutation in the cytochrome c oxidase–positive hepatocytes. Patient was a 71-year-old man.
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HEPATOLOGY, May 2009
Table 1. Mutations Highlighting Monoclonal Nature of
Cytochrome c OxidaseⴚDeficient Patches of Hepatocytes
Mutation
Cytochrome c
oxidase⫺deficient cells with
mutant mtDNA
Cells within cytochrome c
oxidase⫺deficient patch with
wild-type mtDNA
Cytochrome c oxidase⫺positive
cells with wild-type mtDNA
6301 C>T
Age 69, Male
7570 A>G
67, Female
2571
G>A
70, Male
8/10
10/10
9/10
2/10
0/10
1/10
5/5
5/5
5/5
In each case, at least 20 cells were collected evenly over a number of serial
sections to find at least 10 cells with good quality mtDNA from which the entire
genome could be sequenced.
6301C⬎T ⫽ Cytochrome c oxidase subunit I coding region.
7570A⬎G ⫽ tRNA aspartic acid coding region.
2571G⬎A ⫽ 16S ribosomal RNA coding region.
time, not only did each labeled cell develop into a small
cluster of labeled cells but their pattern of distribution
over the three zones did not alter in 15 months: if the
livers “streamed,” one might have expected a significant
shift of the labeled clusters out of the periportal zone
toward the hepatic veins.
Ianacconne and colleagues25 found large patches of
hepatocytes composed of two genetically distinguishable
strains in chimeric rats. However, no particular relationship to vascular structures was noted, but serial sectioning
and 3D-modeling, as performed in our study, was not
attempted. Thus, a relationship to portal areas or hepatic
veins could not be ruled out. In a younger group of human immunodeficiency virus–positive patients (median
age, 43 years), a very heterogeneous distribution of cytochrome c oxidase– deficient hepatocytes was observed, but
this was associated with overt mitochondrial cytopathy
related to treatment with a nucleoside reverse transcriptase inhibitor.26 In the current study, we found no evidence that the patches of cytochrome c oxidase– deficient
hepatocytes increased in size with age, though they increased in frequency with age. We would not expect an
increase in size if measuring proliferative units in the normal liver, though such entities might be larger in pathological situations in which an increase in lobular size had
occurred. These observations are entirely consistent with
a previous study that found an age-related increase in
frequency of cytochrome c oxidase– deficient patches in
human liver, but no increase in overall size; moreover,
there was no consistent genetic abnormality between
patches, suggesting a stochastic process, such as free radical damage.27 Overall, these observations are consistent
with cytochrome c oxidase deficiency accruing over time
in long-lived cells (stem cells), that eventually become
recognized histologically, thus giving rise to similarly deficient progeny (recognized as patches of cytochrome c
oxidase– deficient hepatocytes). In the human gut, crypts
with cytochrome c oxidase deficiency are not seen before
approximately 40 years of age, again consistent with a
lengthy time for a stem cell to acquire a histologically
detectable deficiency in cytochrome c oxidase.20
It is possible that the biliary epithelium can give rise
to hepatocytes in the normal liver, as in the damaged
liver, and proof of this concept would lie in the demonstration of the same clonal mutation in the biliary
Fig. 7. Serial sections of human liver immunostained for cytochrome c oxidase, illustrating every 20th section. There are two patches, invariably
closely apposed to portal tracts, one is almost homoplasmic with almost no cytochrome c oxidase activity (a), whereas the other exhibits some
cytochrome c oxidase activity presumably indicative of an origin from a more heteroplasmic stem/progenitor cell (b). Patient was a 70-year-old man.
HEPATOLOGY, Vol. 49, No. 5, 2009
epithelium and associated mutated hepatocytes. Regardless of whether this is the case, it appears that, in
the human liver, there are at least three options
whereby liver tissue can regenerate: (1) via replication
of preexisting hepatocytes, (2) through differentiation
of progenitor cells feeding into the liver cell population
from a stem cell niche located in the periportal region,
and (3) from a facultative stem cell population located
within the intrahepatic biliary tree.
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