Omentum facilitates liver regeneration.

Ashok K Singh, Krishnamurthy P Gudehithlu, Mark Kraus,
George Dunea, Jose AL Arruda, Department of Medicine,
Stroger Hospital of Cook County, Chicago IL 60612, United States
Ashok K Singh, Nishit Pancholi, Jilpa Patel, Krishnamurthy
P Gudehithlu, Perianna Sethupathi, Mark Kraus, George
Dunea, Hektoen Institute of Medicine, Chicago IL 60612,
United States
George Dunea, Jose AL Arruda, University of Illinois at Chicago
and the Chicago VAMC, Chicago IL 60612, United States
Natalia O Litbarg, Perianna Sethupathi, Loyola-Hines
Medical Center, Maywood IL 60612, United States
Author contributions: Singh AK, Arruda JAL, Gudehithlu
KP and Kraus M designed the experiments; Pancholi N, Patel
J, Gudehithlu KP, Litbarg NO and Sethupathi P performed the
experiments; Litbarg NO contributed new reagents/analytic tools;
Singh AK, Arruda JAL, Gudehithlu KP and Pancholi N analyzed
the data; Singh AK, Dunea G and Arruda JAL wrote the paper.
Supported by An Unrestricted Grant from the Hektoen
Institute of Medicine, Chicago, IL USA
Correspondence to: Ashok K Singh, PhD, Stroger Hospital
of Cook County, 637 South Wood St, Durand Bldg 2nd Floor,
Chicago IL 60612, United States. singhashok@comcast.net
Telephone: +1-312-8644613 Fax: +1-312-8649569
Received: September 6, 2008 Revised: January 29, 2009
Accepted: February 4, 2009
Published online: March 7, 2009
Abstract
AIM: To investigate the mechanism of liver regeneration
induced by fusing the omentum to a small traumatic
injury created in the liver. We studied three groups of
rats. In one group the rats were omentectomized; in
another group the omentum was left in situ and was
not activated, and in the third group the omentum was
activated by polydextran particles.
METHODS: We pre-activated the omentum by injecting
polydextran particles and then made a small wedge
wound in the rat liver to allow the omentum to fuse
to the wound. We monitored the regeneration of the
liver by determining the ratio of liver weight/body
weight, by histological evaluation (including immune
staining for cytokeratin-19, an oval cell marker), and by
testing for developmental gene activation using reverse
transcription polymerase chain reaction (RT-PCR).
RESULTS: There was no liver regeneration in the
omentectomized rats, nor was there significant
regeneration when the omentum was not activated,
even though in this instance the omentum had fused
with the liver. In contrast, the liver in the rats with the
activated omentum expanded to a size 50% greater
than the original, and there was histologically an
interlying tissue between the wounded liver and the
activated omentum in which bile ducts, containing
cytokeratin-19 positive oval cells, extended from the
wound edge. In this interlying tissue, oval cells were
abundant and appeared to proliferate to form new liver
tissue. In rats pre-treated with drugs that inhibited
hepatocyte growth, liver proliferation was ongoing,
indicating that regeneration of the liver was the result
of oval cell expansion.
CONCLUSION: Activated omentum facilitates liver
regeneration following injury by a mechanism that
depends largely on oval cell proliferation.
© 2009 The WJG Press and Baishideng. All rights reserved.
Key words: Cytokeratin; Foreign body; Growth factors;
Oval cell; Progenitor cells
Peer reviewers: James M Millis, Professor, University
of Chicago, Section of Transplantation, MC 5027, 5841 S.
Maryland Avenue, Chicago, IL 60637, United States; Isabel
Fabregat, PhD, Associate Professor, Laboratori d’Oncologia
Molecular, Institut d’Investigación Biomèdica de Bellvitge,
Gran Via, Km 2,7, L’Hospitalet, 08907 Barcelona, Spain;
Bruno Stieger, Professor, Department of Medicine, Division of
Clinical Pharmacology and Toxicology, University Hospital,
Zurich 8091, Switzerland
Singh AK, Pancholi N, Patel J, Litbarg NO, Gudehithlu KP, Sethu-
pathi P, Kraus M, Dunea G, Arruda JAL. Omentum facilitates
liver regeneration. World J Gastroenterol 2009; 15(9): 1057-1064
Available from: URL: http://www.wjgnet.com/1007-9327/15/1057.
asp DOI: http://dx.doi.org/10.3748/wjg.15.1057
INTRODUCTION
The omentum has been called the “policeman of the
abdomen” because after traumatic injury it migrates to
the injured site, adheres to the wound, and promotes
healing[1,2]. These properties have found clinical
application where the omentum is surgically brought
into contact with injured tissues such as ischemic heart,
fractured bones, or injured spinal cord[3-6]. We have
recently shown that introducing a foreign body into the
peritoneal cavity further enhanced the healing power
Omentum facilitates liver regeneration
Ashok K Singh, Nishit Pancholi, Jilpa Patel, Natalia O Litbarg, Krishnamurthy P Gudehithlu,
Perianna Sethupathi, Mark Kraus, George Dunea, Jose AL Arruda
ORIGINAL ARTICLES
www.wjgnet.com
Online Submissions: wjg.wjgnet.com World J Gastroenterol 2009 March 7; 15(9): 1057-1064
wjg@wjgnet.com World Journal of Gastroenterology ISSN 1007-9327
doi:10.3748/wjg.15.1057 © 2009 The WJG Press and Baishideng. All rights reserved.

of the omentum by causing it to expand, surround the
foreign body, and transform itself from mostly fatty
tissue to tissue abundant in progenitor cells and rich in
growth and angiogenic factors (activated omentum)[7,8].
Because liver regeneration can be brought about by
resident stem cells (oval cells) even in the absence of
hepatocyte multiplication[9,10], we attempted to use the
activated omentum to facilitate liver regeneration. The
procedure involved removing a small wedge of tissue
(traumatic injury) in rats and allowing the omentum
to adhere to the wound in order to supply the liver
with stem cells. We also studied two other groups
of rats (controls); one in which the omentum was
left in its native state (inactivated omentum), and the
other in which the omentum was surgically removed
(omentectomized), and focused on the cellular and
developmental gene activation at the site of injury and
omental adhesion.
MATERIALS AND METHODS
Traumatic injury of the liver
Animal experimentation was conducted according to
the approval of the Institutional Animal Care and Use
Committee (IACUC).
Under general anesthesia, male Sprague-Dawley rats
(200-250 g) were laparotomized and the most anterior
and prominent of the liver lobes lying in the middle
of the abdominal cavity was exposed. Using a pair of
fine scissors a small V-shaped cut was made in the lobe
(3-4 mm on each side) and the wedge of liver was removed
and later used as normal tissue for immunostaining and
quantitative reverse transcription polymerase chain
reaction (RT-PCR) (Figure 1). The rats were divided into
three groups. In the activated omentum group, before
the incision was sutured, 5 mL of polydextran particle
slurry (Biogel P-60, 120 μmol/L; Biorad Laboratories,
Richmond, CA, USA) (1:1 in normal saline) was
introduced into the abdominal cavity to activate the
omentum. The inactivated omentum group underwent
similar hepatic wedge injuries. However, polydextran
slurry was not placed in the abdomen and, thus the
omentum was not activated (inactivated omentum).
The omentectomized group underwent similar hepatic
wedge resections, in addition to an omentectomy. An
omentectomy was performed by surgically excising
the entire omentum from the lower curvature of the
stomach.
The animals were maintained on normal rat chow
and water ad libitum from three to twenty eight days. At
the time of sacrifice, the livers were examined, wholly
removed, and weighed. Liver mass was expressed
conventionally as a percent ratio: liver weight/body
weight. Pieces of the re-grown liver from the point of
omental fusion, and at 0.5 cm and 1.0 cm away from the
wound as well as portions from an uninjured lobe were
collected for immunostaining and quantitative RT-PCR.
To test whether liver regeneration by omental
intervention depended upon hepatocyte proliferation,
rats were injected intraperitoneally daily for four days
with 2-acetyl-amino-fluorene, which inhibits hepatocyte
proliferation (2-AAF; 30 mg/kg dissolved in M400
polyethylene glycol (Avg. MW = 400); both chemicals
were obtained from the Sigma Chemical Company,
St Louis, MO, USA), followed by liver wounding and
omental activation (at day 5 by intraperitoneal injection
of polydextran), and further daily injections of 2-AAF
for four days to inhibit expansion of hepatocytes. The
rats were sacrificed 14 d after liver wounding and the
livers were examined, wholly removed, and weighed.
Histological processing and immunostaining of the
regenerated liver
Pieces of normal and regenerated liver (including
the omental attachment) were fixed for histology and
immunostaining by immersion in Histochoice® (Amersco
Inc., Solon, OH, USA). Following dehydration and
paraffin embedment, tissues were sectioned (5 μm thick)
and stained with hematoxylin-eosin (HE) or Trichrome
stain. Immunostaining was carried out by first pressure-
cooking the sections for 10 min in a solution of
BorgDecloaker® (Biocare Medical; Walnut Creek, CA,
USA) for antigen enhancement. For immunofluorescent
staining the sections were incubated with monoclonal
(mouse) anti-rat cytokeratin-19 (Sigma Chem. Co,
St Louis, MO, USA) followed by washing and re-
incubating with fluorescein (FITC) labeled anti-mouse
IgG antibody (Sigma Chem. Co., St Louis, MO, USA).
The slides were washed and wet-mounted in glycerol-
PBS. For immunoperoxidase staining, sections were
sequentially incubated with monoclonal (mouse) anti-
rat cytokeratin-19, anti-mouse IgG-biotin conjugate,
avidin-horse radish peroxidase and finally developed
with diaminobenzidine-H2O2 (brown color) (Vector
Laboratories, Inc. , Burl ingame, CA, USA). The
slides were examined either by epifluorescent or light
microscopy and digitally photographed (Nikon Inc.,
New York, NY, USA).
www.wjgnet.com
Figure 1 The traumatic liver injury model used to induce regeneration.
The wound was created in one of the lobes of rat liver by removing a wedge of
tissue (3-4 mm on each side) with a pair of fine scissors. In rats with activated
or unactivated omentum, the wound was filled with new liver tissue by day 7.
On the other hand, in omentectomized rats the original wound edges as seen
in the picture remained visible for up to 28 d. The horizontal black bar in the
picture represents 3 mm.
1058 ISSN 1007-9327 CN 14-1219/R World J Gastroenterol March 7, 2009 Volume 15 Number 9

Quantification of mRNA for selected developmental
genes in regenerated liver by RT-PCR
Liver tissues at the point of omental attachment or wound
edge (in omentectomized rats), at 0.5 and 1.0 cm away
from the omental attachment, and from a remote uninjured
lobe were tested for expression of developmental genes.
The specific genes and their respective forward and reverse
primer sequences are listed in Table 1. The liver tissue was
cleared of the attached omental tissue and processed for
total RNA extraction by Trizol using a RNA purification
kit (Invitrogen, CA, USA). The RT-PCR procedure
was carried out in one step using 3 μg of total tissue
RNA and primers using the Invitrogen RT-PCR system
(Invitrogen, CA, USA). The system uses Superscript Ⅱ
reverse transcriptase for first strand synthesis and Taq
DNA polymerase for second strand cDNA synthesis
and amplification (30 cycles). β-actin amplification was
performed from the total RNA preparations (60 ng) as
a control. The RT-PCR products were quantitated as the
ratio of gene band density/β-actin band density by image
analysis using MIPAV software (JAVA imaging software
inspired by the National Institutes of Health).
Statistical method
Quantitative data presented in Figures 2 to 6, which
compare the differences between different groups,
were analyzed by student’s t test. The differences were
considered significant when P < 0.05.
RESULTS
Fusion of the omentum to the wounded liver resulted in
new liver growth
In all rats in which the liver was traumatically wounded
and the omentum was intact, whether activated (n = 24)
or inactivated (n = 12), there was fusion between the
omentum and the wound edge of the liver, and the
omentum remained attached to the injury site for up to
28 d. On gross inspection, by day 14 new tissue filled
the resected wedge, and the location of the resection
site was only identifiable by the omental attachment. In
omentectomized rats (n = 12), there was an absence of
omental attachment and of liver growth at the wound,
making the wound edges noticeable until at least day 28
(Figure 1).
In rats with activated omentum, there was additional
liver growth, especially in the wounded lobe at the point
of omental attachment. There was also growth in other
lobes which was suggested by alterations in the natural
contours of the edges of the uninjured liver lobes.
Figure 2 shows the liver mass (as a percent ratio to body
www.wjgnet.com
Table 1 Primer sequences of the selected developmental genes that were tested in the regenerating liver tissue by
RT-PCR technique
Gene1 Primer Predicted size (bp) Accession number2 Entrez gene ID2
β-actin F (926) 5'-TCATGAAGTGTGACGTTGACATCCGT-3'3
R (1210) 5'-CCTAGAAGCATTTGCGGTGCACGATG-3' 285 NM_031144 81822
Wnt-4 F (127) 5'-GAAACGTGCGAGAAGCTCAAAG-3'
R (513) 5'-AAAGGACTGTGAGAAGGCTACG-3' 387 NM_053402 84426
WT-1 F (1059) 5'-TGAGAAACCATACCAGTGTGAC-3'
R (1458) 5'-GTAGGTGAGAGGGAGGAATTTC-3' 400 NM_031534 24883
Nanog F (541) 5'-ATCCATTGCAGCTATTCTCAGG-3'
R (850) 5'-CTTCCAAATTCGCCTCCAAATC-3' 310 XM_575662 414065
AFP F (1421) 5'-CAGTGAGGAGAAACGGTCCG-3'
R (1672) 5'-ATGGTCTGTAGGGCTCGGCC-3' 252 NM_012493 24177
Oct-4 F (633) 5'-GGAGATATGCAAATCGGAGACC-3'
R (984) 5'-CGAGTAGAGTGTGGTGAAATGG-3' 352 NM_001009178 294562
HNF-6 F (1698) 5'-AAGACCAGGACCTCAAGATAGC-3'
R (2001) 5'-GCAGTGTGGTGGAACAGATAAG-3' 304 NM_022671 25231
1Wnt-4: Wingless-type mouse mammary tumor virus integration site family, member 4[21,26,27]; WT-1: Wilm’s tumor suppressor gene[16,22];
Nanog: One of the gene markers of pluripotency[24]; AFP: α-fetoprotein[15,23]; Oct-4: Octomer-4[25]; HNF-6: Hepatic nuclear factor-6[16,28];
2Accessed from http://www.ncbi.nlm.nih.gov; 3Numbers in parentheses after forward (F) and reverse primers (R) denote the nucleotide
number in the cDNA sequence.
7
6
5
4
3
2
1
0
Ra
tio
:
liv
er
w
t/
bo
dy
w
t
(%
)
Normal 3 d 7 d 14 d Omentx Inact oment
Activated omentum
a a
a,b
Figure 2 Liver mass (as a ratio of body weight) at different times after
injury and fusing of the activated omentum to the wound. The ratio of liver
wt/body wt in normal rats was established to be 3.85% ± 0.07%. ‘Omentx’ are
rats in which the omentum was removed before liver injury (n = 12) and ‘inact
oment’ are rats in which the liver was injured, but the omentum was inactivated
(n = 12). Liver regeneration following wounding and fusion of activated
omentum was rapid, and by day 3 the liver grew to 110% of the original mass.
The liver continued to grow, reaching a maximum size of 150% of the original
mass by day 14, after which growth stopped (day 28, data not shown). Normal
= 15 rats and there were 6 in each of the 3, 7, 14 and 28 d groups. aDenotes
statistical difference from normal or ‘omentx’ or ‘inact oment’ groups at P < 0.05.
bDenotes statistical difference from day 3 and day 7 groups at P < 0.05. With
regard to ‘omentx’ and ‘inact oment’ groups, no differences were seen at days 3,
7, 14 and 28 compared to Normal (only day 14 data is shown in the figure; n = 3).
Singh AK et al . Omentum facilitates liver regeneration 1059

weight) at different times after wounding and fusing of
the omentum to the wound. The percent ratio in normal
rats was established to be 3.85 ± 0.07 [Normal (n = 15);
Figure 2]. In the activated omentum group, the liver grew
to 110% of its original mass by day 3 (percent ratio: 4.4 ±
0.24) and to a maximum size of 150% by day 14 (percent
ratio: 6.0 ± 0.16) (n = 6 at days 3, 7, 14 and 28; Figure 2).
From day 14 to day 28, the liver did not grow any further,
but remained enlarged (data not shown).
In rats with inactivated omentum, growth was
observed at the site of omental fusion and filled the
resected site with new tissue, however, the overall
liver mass did not increase at any of the time points
compared with the established normal liver mass [n = 3
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Figure 3 Histology of the boundary between the growing edge of the liver and the activated omentum. A: Normal rat liver; B: 7 d after liver injury the liver and
the omentum were separated by a wide and compact interlying tissue (400-600 μm). On one side of the interlying tissue (T) lay the omental tissue (OM) with the
embedded polydextran gel particles and on the other side was the liver tissue (L). Occasionally, islands of liver tissue were observed in the interlying tissue (Figure
4G). The compactness and the width of the interlying tissue was maximal between 3 and 7 d after liver injury (B) which became thinner (100-150 μm) and looser by
day 14 (C). By 28 d the interlying tissue was barely appreciable and looked like a tissue septum (picture not shown). Trichrome staining. The horizontal white bar in
the pictures represents 100 μm.
L
OM
OM
L
T T
A B C
T T
Figure 4 Immunostaining of normal and regenerated rat liver (from activated omentum) for cytokeratin-19, a marker of oval cells. A: Normal or uninjured liver
lobe showing widespread presence of oval cells in the lining of bile ducts lying around a central vein; B, D, E, G: Different areas of injured liver showing extensions of
cytokeratin-19 positive bile ducts in the interlying tissue between the liver and the activated omentum; C: Tissue section shown in B stained with Trichrome to show
the bile ducts lying in the interlying omental tissue; F: Occasionally, the growing edge of the liver lying in the interlying tissue was seen to be entirely covered with
cytokeratin-19 positive cells; G: Islands of liver tissue, probably newly formed, were seen in the interlying tissue (white arrows; also seen in D); A, B, D-F were stained
by immunofluorescence (green); G was stained by immunoperoxidase (brown). The horizontal white bar in F represents 100 μm for all pictures.
←
A B C D
E F G
←
1060 ISSN 1007-9327 CN 14-1219/R World J Gastroenterol March 7, 2009 Volume 15 Number 9
A B
Figure 5 Histology and cytokeratin-19 immune
staining of the liver at the boundary between
the growing edge of the liver and the inactivated
omentum at day 3 or 7 after injury. A: Trichrome
stained section showing the adherent omental
tissue with a thinner interlying tissue (blue stained)
than that seen in the activated omentum group
(Figure 3 for comparison); B: Cytokeratin-19
positive bile ducts were seen in the interlying tissue
(same section as A) although these were much less
frequent than those seen in the activated omentum
group (Figure 4 for comparison). The horizontal
white bar in pictures represents 100 μm.

at days 3, 7, 14 and 28 (day 14 data shown in Figure 2)].
In omentectomized rats, the liver did not grow and
the resection site remained visible up to day 28. The
percent ratio of liver wt/body wt in this group was
similar to that of normal rats at all time points [n = 3 at
days 3, 7, 14 and 28 (day 14 data shown in Figure 2)].
Histology of the regenerated liver
In the activated omentum group, in which liver tissue
grew to more than normal size, histological examination
at the site of injury revealed normal hepatic architecture
up to the point of omental fusion (Figure 3B). At the
site of fusion there was a wide and compact band of
interlying tissue between the omentum and the growing
edge of the liver (Figure 3B). On one side of the
interlying tissue lay the liver tissue and on the other side
was the omental tissue with the embedded polydextran
gel particles. The compactness and the width of the
interlying tissue was maximal between 3 and 7 d after
liver injury (400-600 μmol/L) (Figure 3B) and became
thinner (100-150 μmol/L) and less compact by day 14
(Figure 3C). In the interlying tissue, small islands of liver
tissue, probably newly formed, were seen (Figure 4G).
By day 28 the interlying tissue was barely visible and
appeared like a tissue septum (not shown).
In the inactivated omentum group in which liver
growth was minimal, although the adherent omental
tissue was clearly visible, a thinner interlying tissue than
that seen in the activated omentum group was observed
(Figure 5A). In the omentectomized group, the void left
by the injury was visible at all time points. The injury site
lacked omental attachment, but showed a layer of dead
tissue (approx. five cells thick) which sloughed off from
the wound edge by day 14 (not shown).
Immunostaining of the regenerated tissue for
cytokeratin-19
Immunosta in ing of nor mal adu l t ra t l iver for
cytokeratin-19, a well-known marker for bile ducts as
well as oval cells (oval cells are presumed to be liver
stem cells), showed the expected widespread presence
of oval cells in the lining of bile ducts lying in the liver
triads (Figure 4A). In the liver with or without omental
attachment, cytokeratin-19 positive bile duct staining
in the triads of the uninjured lobes was similar in
intensity to that seen in the normal liver (not shown).
In the liver tissue with activated omentum, remarkably,
cytokeratin-19 positive bile ducts in the regenerated liver
(day 3 or 7) extended into the interlying area between
the liver and the omentum (Figure 4B-E). Occasionally,
the growing edge of the liver at the interlying tissue was
seen to be entirely covered with cytokeratin-19 positive
cells (Figure 4F). Islands of liver tissue, probably
newly formed, were present in the interlying tissue
(Figure 4G).
In the inactivated omentum group, the interlying
t issue attached to the wound edge also showed
extensions of the cytokeratin-19 positive bile ducts (at
days 3 and 7) (Figure 5B), although these were much
less frequent than those seen in the activated omentum
group. In omentectomized rats, the wound edge was
devoid of omental attachment, and as expected no
cytokeratin-19 positive bile ducts were seen outside the
liver tissue (not shown).
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Figure 6 Activation of developmental genes in the regenerated liver at the wound site at days 3 and 7 after injury and fusion of the activated omentum.
The regenerating part of the liver (part of liver attached to the omentum) showed high expression levels (7 to 20-fold) of WT-1, Wnt-4, Nanog, AFP by RT-PCR (A)
compared with normal rat adult liver tissue (control). Wnt-4 (C), Nanog (D), and AFP (E) were maximally activated at day 3, while WT-1 (B) showed maximal activation
at day 7. Tissue from regenerated liver, from sites further away from the wound area (0.5 cm, 1.0 cm further away in the same lobe and from an uninjured lobe),
showed reduced activation of WT-1, Wnt-4 and AFP genes (although higher in all cases compared with normal adult liver), suggesting that the regeneration stimulus
‘rippled’ throughout the liver from the wound area (data not shown). n = 3 in each bar and the differences amongst the bars within each of the figures B, C, D, and E
are statistically significant (P < 0.05).
30
20
10
0
Fo
ld
in
cr
ea
se
Control 3 d 7 d
10
5
0
Fo
ld
in
cr
ea
se
Control 3 d 7 d
WT1
Wnt4
Nanog
AFP
Control 3 d 7 d
A B C
30
20
10
0
Fo
ld
in
cr
ea
se
Control 3 d 7 d
10
5
0
Fo
ld
in
cr
ea
se
Control 3 d 7 d
D E
Singh AK et al . Omentum facilitates liver regeneration 1061

Expression of developmental genes in the liver tissue
attached to the activated omentum
To further investigate if activated omentum fused to
the injured liver triggered developmental events in the
adult liver, we determined the expression levels of
several important genes associated with (1) pluripotent
embryonic stem cell activity (Nanog, Oct-4), (2) liver
differentiation (WT1, Wnt-4, HNF-6) and (3) fetal liver
synthetic activity (α-fetoprotein; AFP) at days 3 and 7
after wounding using RT-PCR. Comparisons between
normal rat adult liver and the regenerated liver attached
to the activated omentum showed high expression levels
(7-20 fold) of four of these genes (WT-1, Wnt-4, Nanog,
AFP) in the regenerated liver tissue (Figure 6) (Oct-4
and HNF-6 levels were negative; not shown). Wnt-4,
Nanog, and AFP were maximally activated at day 3,
while WT-1 showed maximal activation at day 7.
Regenerated liver tissue from sites further away
from the injured area (0.5 cm and 1.0 cm away from the
activated omentum in the same lobe, and tissue from an
uninjured lobe), showed reduced expression of WT-1,
Wnt-4, and AFP genes (although higher in all cases
compared to normal adult liver (Nanog, Oct-4, AFP did
not change), suggesting that the regeneration stimulus
‘rippled’ from the injured area further into the liver
tissue (data not shown).
In contrast, in the inactivated omentum group
(compared to normal adult liver) the expression level of
WT-1, Wnt-4 and Nanog increased to a much smaller
degree than that observed in the activated omentum
group at days 3 and 7 (WT-1 by 1.5-fold, Wnt-4 by
1.9-fold and Nanog by 1.2-fold), while AFP decreased
by 0.8-fold (P < 0.05 in all cases; no detectable changes
were seen in Oct-4 and HNF-6).
In the omentectomized group, while the expression
levels of WT-1 and Nanog did not change, the levels of
Wnt-4, Oct-4, AFP and HNF-6 decreased by 0.78-fold-
undetectable levels compared with normal adult liver
(P < 0.05 in all cases).
Omentum-assisted liver regeneration in rats treated with
2-AAF, a drug that blocks proliferation of hepatocytes
Because liver mass can increase due to either progenitor
cell activation or hepatocyte multiplication, we performed
the wedge wound experiment in a small group of rats
in which hepatocyte multiplication was blocked by
treatment with 2-AAF. This was performed to confirm
that liver regeneration was via progenitor cells and not by
hepatocyte expansion in our model. Fourteen days after
wounding, 2-AAF treated rats showed complete healing
of the wedge wound and increased the liver mass to 135%
of the original mass [liver weight/body weight ratios:
5.1 ± 0.2 in 2-AAF treated (n = 4) versus 3.85 ± 0.07
in normal controls (n = 15); P < 0.05], confirming that
omentum-assisted liver regeneration was not mediated by
hepatocyte expansion, but by progenitor cell activation.
DISCUSSION
For many years the omentum was believed to have
no specific function. During the course of the 19th
century, however, several investigators recognized that it
possessed healing properties. These were later exploited
in a variety of surgical procedures designed to facilitate
the healing of bone fractures, spinal cord injuries, and
heart ischemia[1-6]. In previous studies we investigated this
process and found that these properties can be enhanced
by physically expanding the omentum with foreign
particles. Under such conditions, the expanded omentum
becomes rich in growth and angiogenic factors, and has
abundant progenitor cells[7,8]. In a separate study we used
this approach to generate new β-cells from the diabetic
pancreas[11].
Here we applied these findings in an attempt to re-
generate liver tissue by creating a surgical wound and al-
lowing the omentum to fuse with the wound. We studied
groups of rats with (1) no omentum (omentectomized),
(2) inactivated omentum, and (3) omentum pre-activated
by foreign polydextran particles. We found no liver
growth in omentectomized rats. In rats with inactivated
omentum, the omentum fused with the injured site and
although new growth was noted, this did not result in a
significant increase in liver mass. However, in rats with
activated omentum, following omental fusion, the liver
grew to fill the wound and continued to grow, both at
the wound site and globally, to a level 50% greater than
the original mass. These findings suggest that the omen-
tum plays an important role in bringing about growth
and regeneration of the injured liver. The amount of
liver growth induced by the omentum was proportional
to the degree of omental activation, consistent with our
previous observations that the concentration of growth
factors and the number of progenitor cells in the omen-
tum increase with increased activation[7,8].
Clinicians have long known that the liver has the abil-
ity to regenerate. Experimentally, a 70% hepatectomy
(either surgically or chemically) induces a form of liver
regeneration in which growth is largely due to hepato-
cyte proliferation[9-13]. When hepatectomy is carried out
following the administration of drugs which inhibit he-
patocyte proliferation, the regeneration is mainly due to
the expansion of oval cells[14-17]. In these various models,
there is a massive loss of functional liver tissue, which
then systemically triggers a cascade of cytokines (such
as tumor necrosis factors-α, IL-6 and growth factors).
In our model, the injury was so slight that regeneration
would not occur unless the omentum was activated, as
shown in our omentectomized control rats.
In further studies we attempted to understand the
mechanism by which activation of the omentum causes
liver regeneration. Histologically, at the fusion site be-
tween the activated omentum and the liver, we found a
wide and compact interlying band of tissue into which
tubular structures resembling bile ducts extended and
proliferated. On staining, these structures were strongly
positive for cytokeratin-19, a known marker for oval
cells, believed to be liver progenitor cells[18-20]. At an early
stage of regeneration the oval cells in the interlying tis-
sue were seen near small islands of liver tissue; later
these islands became integrated into the native liver, so
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that the border between the native and the new liver
could no longer be discerned in stained sections. Because
proliferation of the progenitor oval cells took place in
the omentum rather than in the liver, they may have had
more room to proliferate and expand, accounting for the
robust, supra normal liver growth.
Because liver tissue can also regenerate by hepato-
cyte proliferation we repeated our experiment in rats
treated with 2-AAF, a drug commonly used to inhibit
hepatocyte multiplication. Our finding that the activated
omentum continued to exert its regenerative property in
2-AAF-treated rats by increasing the liver mass to 135%
of the native mass, further suggested that the prolifera-
tion of progenitor oval cells rather than proliferation of
hepatocytes was responsible for liver regeneration in our
model.
As genes and proteins involved in liver develop-
ment (Wnt-4, WT-1, HNF-6, AFP) may become re-
activated during liver regeneration[15-17,21-23], we tested
these developmental genes and also Nanog and Oct-4,
markers of early progenitor cells[24,25], to see if they were
altered in regenerating liver tissue. We found that many
of these genes, silent in the adult liver, were highly up-
regulated (7 to 20-fold) after fusion of the activated
omentum. Nanog was up-regulated by 20-fold three
days after omental fusion and returned to undetectable
levels by day seven, suggesting the transient presence
of early progenitor cells. Gene expression of Wnt-4
and AFP was highest at day 3 and decreased by day 7,
in contrast to WT-1 which was unchanged at day 3 and
highest at day 7. These findings are consistent with the
transient activation of these transcription factors (WT-1
and Wnt-4) known to occur in liver re-modeling and dif-
ferentiation[16,26,27]. HNF-6, a marker strongly associated
with hepatocyte proliferation[28], was unchanged, as also
noted in a previous study of non-hepatocyte mediated
(but progenitor cell mediated) liver regeneration[16]. This
was not surprising because liver growth by omental fu-
sion was via oval cells and not dependent on hepatocyte
proliferation. Interestingly, we also found a few selected
genes (WT-1, Wnt-4 and AFP) to be activated in regions
of the native liver 0.5 cm and 1.0 cm from the wound
edge. The level of activation decreased as the distance
from the wound edge increased, suggesting that a para-
crine effect was exerted by the omentum. Importantly,
we found lower activation levels of genes in the inac-
tivated omentum group (1.2-1.9-fold), consistent with
reduced liver growth seen in these rats. Furthermore, in
omentectomized rats where there was no liver growth,
a decrease in gene expression levels was observed com-
pared with normal liver.
The present study is the first to demonstrate the
unique role of the omentum in traumatic wound
healing of the liver. We have shown previously that
omental derived factors stimulate wound healing and
can be upregulated by pre-activating the omentum. By
bringing the omentum into close contact with injured
liver we observed a vigorous regeneration of liver
tissue. Although the liver is known to regenerate to
the original size following a significant loss of hepatic
tissue, there are no reports of liver regeneration up
to 150% of the original size as noted in our study. As
both cytokeratin-19 positive cells and expression of
developmental genes were increased, we postulate that
both growth factors and stem cells are conveyed to the
site of injury by omental fusion. It may be argued that
bringing the omentum into contact with damaged organs
after controlled deliberate wounding may have immediate
clinical applicability. Also, the use of progenitor cells
isolated from the activated omentum or of the growth
factors secreted by these cells holds further promise of
other exciting therapeutic possibilities.
ACKNOWLEDGMENTS
The authors wish to thank Lev Rappoport, MD for
tissue processing and histology.
COMMENTS
Background
Although the liver is a unique tissue that can regenerate after an acute injury, it
has been a challenge to induce such regeneration after chronic liver disease.
It is, therefore, important to study mechanisms of liver regeneration in order to
devise new approaches for regeneration following damage by chronic disease.
Although embryonic stem cells have the power to regenerate liver tissue, their
use is hampered by ethical, political and safety concerns. In that regard, the
use of adult stem cells derived from the patient’s own tissue to regenerate the
liver is free of such concerns and, therefore an alternative approach.
Research frontiers
Stem cells have been derived from several adult organs such as bone marrow,
skin, hair, kidney and dental pulp. Although these cells express stem cell
markers and differentiate to other cell phenotypes in culture they seem to
lack the potency to regenerate an organ in vivo. Identifying a source of adult
stem cells that could regenerate liver or other organs would be an immense
advantage.
Innovations and breakthroughs
Singh and his colleagues devised a methodology to harness adult stem cells
to regenerate the liver by first activating the omentum using a foreign body
to increase its content of stem cells and growth factors. They then cut and
removed a small piece of the liver tissue and let the activated omentum adhere
to the wound in order to supply stem cells to the injured liver. They found that
the liver of these rats with an activated omentum expanded to a size 50%
greater than the original, an outcome never reported before. This approach
represents an application of adult stem cells to regenerate an organ in vivo.
Applications
This method of liver regeneration is novel and could be attempted in patients
with liver failure in order to regenerate new liver tissue.
Terminology
Activated omentum, which is central to this methodology of liver regeneration,
was created by injecting polydextran particles (foreign body) into the abdominal
cavity. As the omentum naturally grows to encapsulate the particles individually
it expands 20-30 times its original size and has abundant stem cells and
growth factors, which appear to be the basis of the regenerating power of the
omentum.
Peer review
Reviewers considered the use of the omentum to regenerate the liver as
meritorious and interesting. Further they thought the paper was well written,
results were clear, and the data supported the conclusions reached by the
authors.
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S- Editor Tian L L- Editor Webster JR E- Editor Ma WH
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