Received: 2 September 2021 | Revised: 16 December 2021 | Accepted: 12 January 2022

DOI: 10.1111/liv.15174

REVIEW

Liver regeneration: Cellular origin and molecular mechanisms

Ru Huang1 | Xin Zhang2 | Jordi Gracia-­Sancho3 | Wei-­Fen Xie2

1
Department of Gastroenterology,
Shanghai East Hospital, Tongji University Abstract
School of Medicine, Shanghai, China
The liver is known as an organ with high proliferation potential. Clarifying the cel-
2
Department of Gastroenterology,
Changzheng Hospital, Naval Medical
lular origin and deepening the understanding of liver regeneration mechanisms will
University, Shanghai, China help provide new directions for the treatment of liver disease. With the development
3
Liver Vascular Biology Research Group, and application of lineage tracing technology, the specific distribution and dynamic
Barcelona Hepatic Hemodynamic Unit,
IDIBAPS, CIBEREHD, Barcelona, Spain changes of hepatocyte subpopulations in homeostasis and liver injury have been il-
lustrated. Self-­replication of hepatocytes is responsible for the maintenance of liver
Correspondence
Wei-­Fen Xie, Department of function and mass under homeostasis. The compensatory proliferation of remaining
Gastroenterology, Changzheng Hospital, hepatocytes is the main mechanism of liver regeneration following acute and chronic
415 Fengyang Road, Shanghai, 200003,
China. liver injury. Transdifferentiation between hepatocytes and cholangiocytes has been
Email: weifenxie@medmail.com.cn recognized upon severe chronic liver injury. Wnt/β-­catenin, Hippo/YAP and Notch sig-
Jordi Gracia-­Sancho, IDIBAPS Biomedical nalling play essential roles in the maintenance of homeostatic liver and hepatocyte-­to-­
Research Institute. Rosselló 149, 08036, cholangiocyte conversion under liver injury. In this review, we summarized the recent
Barcelona, Spain.
Email: jordi.gracia@idibaps.org studies on cell origin of newly generated hepatocytes and the underlying mechanisms
of liver regeneration in homeostasis and liver injury.
Funding information
This study is supported by National
Natural Science Foundation of China KEYWORDS
(82030021, 82072641). cholangiocytes, compensatory proliferation, hepatocyte regeneration, hepatocyte
subpopulation, lineage tracing, liver progenitor cells, metabolic zonation, ScRNA-­seq,
Handling Editor: Luca Valenti. transdifferentiation

The liver is the largest organ in the body and plays critical roles in
nutrient metabolism, detoxification, regulation of blood clotting fac-
tors and bile synthesis. The liver lobule, as the basic functional and
anatomical unit of the liver, is composed of hepatic cords or hepatic
plates, with central veins and portal veins at each end. Hepatocytes
and cholangiocytes are the two epithelial cell types constituting the
hepatic parenchyma. For centuries, the liver has been known as an
organ with high regenerative potential. Hepatocyte regeneration
is pivotal to the maintenance of liver function and size during ho-
meostasis and after liver injury. The cell origin of newly generated
hepatocytes and the underlying mechanisms have been extensively
explored in recent decades. This review focuses on the cellular ori-
gin and the underlying molecular mechanisms in liver regeneration
under distinct circumstances.
1 | G E N E TI C LI N E AG E TR AC I N G : S TATE-­
O F-­T H E-­A RT TEC H N I Q U E FO R TH E S T U DY
O F LI V E R R EG E N E R ATI O N
Most studies in the field of liver regeneration have been performed
in mice during the last decade. Lineage tracing technology has been
widely employed to closely monitor the migration, expansion and
transdifferentiation of specific cell populations and their progeny
during homeostasis and liver injury.1 The application of genetic la-
belling technology provides a solid basis for the cellular source of
newly-­produced hepatocytes in liver regeneration.
Currently, the Cre-­loxP system is the standard lineage tracing
strategy, which works by placing Cre recombinase under the control
of the transcriptional regulatory sequence of certain marker genes

Ru Huang and Xin Zhang contributed equally.

© 2022 John Wiley & Sons A/S . Published by John Wiley & Sons Ltd.

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wileyonlinelibrary.com/journal/liv Liver International. 2022;42:1486–1495.

HUANG et al.
and inserting a two-­loxP-­flanked stop cassette in an open gene locus
with a downstream reporter gene in mice. 2,3 Once the expression
of the Cre recombinase is activated, the stop cassette between the
loxP sites is excised, and the reporter genes (GFP, tdTomato, etc.)
start expressing to label certain cell populations. Since Cre-­mediated
site-­specific recombination is passed on to their progeny, the cell
lineage could be labelled permanently from the timepoint of Cre ex-
pression. While constitutive Cre restricts reporter gene expression
spatially, an inducible Cre strategy further controls gene expression
temporally by fusing Cre with a T2 mutant of the oestrogen recep-
tor, which is activated through tamoxifen induction.4 However, com-
pared to constitutive Cre, inducible Cre activity is difficult to fully
activate. In addition, high dose of tamoxifen administration has been
reported to lead to liver injury with ectopic stem cell marker ex-
pression.5,6 Thus, it is important to determine tamoxifen quantities
and efficacy before induction of liver injury. Due to the difference
in Cre reporter gene recombination sensitivities, the efficacy of cell
labelling differs, with divergent results obtained from different lab-
oratories. Therefore, interpretation of lineage tracing results should
be conducted carefully, with immunostaining of liver cell markers
incorporated.
2 | CO NTROV E R S Y OV E R TH E E X I S TE N C E
O F LP C s I N LI V E R R EG E N E R ATI O N
Unlike skin tissue and intestinal epithelial tissue where stem cells
or progenitor cells exist, there is no consensus about the exist-
ence of liver progenitor cells (LPCs) during liver regeneration.7 Oval
cells emerging from canals of Hering, which located at the termi-
8
nal branches of bile ducts, were identified by Farber and Popper
9
et al. in 2-­acetylaminofluorene (2-­A AF, a hepatotoxic carcinogen)
treated rats in mid-­1950s. Following studies characterized oval cells
as hyperplastic epithelial cells with small cellular size, a high nu-
clear/plasma ratio and expression of the hepatoblast marker alpha-­
fetoprotein (AFP).8-­11 Oval cells were considered to be bipotential
progenitor cells as they showed the capacity to differentiate into
hepatocytes and bile duct cells in vitro.12 Oval cells were also re-
ported as LPCs in human chronic liver disease, and their numbers
are associated with disease severity.13 However, due to the lack of
specific markers for oval cells and the inapplicability of 2-­A AF/PHx
model in mice, there is still lack of direct in vivo evidence showing
that oval cells give rise to mature hepatocytes and cholangiocytes in
any of the classical mouse injury models to date.
Through the years, adult LPCs have been investigated
through in vitro cultivation, liver repopulation assay and lin-
eage tracing.14-­19 Robust activation of LPCs was detected in
AhCre+Mdm2flox/flox mice model with extensive hepatocytes dam-
age and transplantation of EpCAM+CD24 +CD133+ LPCs restored
liver mass in AhCre+Mdm2flox/flox mice. 20 Moreover, through em-
ploying scRNA-­seq and organoid cultivation, Aizarani et al. identi-
fied EpCAM+TROP2intCK19low progenitor cell in human liver, with
21
strong organoid forming capability. Although such hepatocyte
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1487
Key points
• Lineage tracing technology provides a solid basis for
identifying the role of hepatocyte subpopulations and
cholangiocytes during liver regeneration.
• Hepatocytes self-­replication is the major contributor to
liver regeneration.
• The conversion between hepatocytes and cholangio-
cytes during liver injury demonstrates the high plasticity
of liver cells.
• Further characterization of cellular and molecular mech-
anisms underlying liver regeneration is in great demand
to help provide reliable approaches towards the man-
agement of liver diseases.
subpopulations have been defined functionally through in vitro cul-
tivation and cell transplantation, it remains to be determined how
they function in vivo under homeostasis and liver injury.7 Further
characterization of those cell subpopulations through combination
of spatial transcriptomes and scRNA-­seq or by lineage-­tracing stud-
ies are in demand.
Multiple groups have performed fate tracing using cholangio-
cytes markers (including SOX9, HNF1β, CK19 or OPN) to label LPCs
(Table 1). Intriguingly, Furuyama et al. found Sox9-­expressing cells
contributed significantly to the hepatocytes pool under homeostasis
and liver injury induced by CCl4, BDL or MCDE in SOX9IRES-­CreERT
knockin mouse strain, 22 whereas Tarlow et al showed hepatocytes
rarely came from Sox9+ cells in various liver injury using BAC trans-
genic SOX9CreERT2 mice strain. 23 The discrepancy between these
studies could be due to the different promotor specificity and sen-
sitivity in the SOX9 genetic labelling mouse tools. HNF1β, CK19 or
OPN labelled cholangiocytes also show limited contributions to liver
regeneration under physiological conditions and during liver inju-
ry. 24-­27 One explanation underlying the limited role of those cells is
the incomplete blockade of hepatocyte proliferation in mouse liver
injury model, which could not faithfully reflect the impaired prolifer-
ation of hepatocytes in human chronic liver disease.16,28 This notion
is supported by the study that inhibition of hepatocyte proliferation
through overexpressing p21 leads to significant regenerated hepato-
cytes from CK19+ cholangiocytes. 29
3 | D I S TR I B U TI O N O F H E PATO C Y TE
S U B P O PU L ATI O N S U N D E R LI V E R
H O M EOS TA S I S
Hepatocytes, accounting for approximately 70% of total liver
cells, renew at a slow rate under physiological conditions. The life
cycle of hepatocytes is approximately 200 to 300 days in mice.30
Hepatocytes are highly heterogeneous and have different functions
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1488 HUANG et al.

TA B L E 1 Genetic labelling of
Targeting
cholangiocytes during liver regeneration
Mouse line strategy Circumstances References

Sox9-­CreERT2 Knockin (3’-­UTR) Homeostasis 22

Chronic liver injury
Transgenic Homeostasis 23
Chronic liver injury
Ck19-­CreERT Knockin (ATG) Homeostasis, Acute and Chronic liver 103
injury
Hnf1β-­CreERT2 Transgenic Homeostasis, 26,104
Chronic liver injury
OPN-­CreERT2 Transgenic Homeostasis, Acute and Chronic liver 27
injury

TA B L E 2 Characterization of
Hepatocytes Zone 1 Zone 3
hepatocytes in zone 1 and zone 3
Location Periportal area Pericentral area
Zonation marker Gls2, Pck1, Arg1 GS, Oat, Cyp1a2, Cyp2e1, Glul
Metabolic function Gluconeogenesis Glycolysis
Glycogen and Protein synthesis Lipid Glutamine synthesis
metabolism Xenobiotic metabolism
Ureagenesis

Abbreviations: Arg1, arginase 1; Cyp1a2, cytochrome P450 family 1 subfamily A member 2; Cyp2e1,
cytochrome P450 family 2 subfamily E member 1; Gls2, glutaminase 2; Glul, glutamate-­ammonia
ligase; GS, glutamine synthetase; Oat, ornithine aminotransferase; Pck1, phosphoenolpyruvate.

F I G U R E 1 Hepatocyte subpopulation
distribution under homeostasis and liver
injury. (A) The liver lobule is divided into
three zones, with periportal area as zone
1, pericentral area as zone 3, and the
region between them as zone 2. Mfsd2a+
hepatocytes and hybrid hepatocytes
(HybHPs) are sited in zone 1, while Axin2+
hepatocytes are mainly located in zone 3.
Terthigh hepatocytes are scattered in the
liver lobule. Hepatocytes self-­replication
contribute to the maintenance of liver
function and mass under homeostasis. (B)
In chronic periportal liver injury, HybHPs
and Mfsd2a+ hepatocytes are mostly
impaired. Midlobular and pericentral
hepatocytes, especially TERThigh
hepatocytes, contribute to hepatocytes
regeneration. (C) During chronic
pericentral injury, TERThigh, HybHPs and
Mfsd2a+ hepatocytes proliferate and
expand significantly to repopulate the
liver. Abbreviations: PV, portal vein; BD,
bile duct; CV, central vein; HA, hepatic
artery

and metabolic expression profiles along the portal-­

central axis (Figure 1A).31 Hepatocytes adjacent to the portal area belong to
31-­33
(Table 2). Based on their metabolic features, hepatocytes are zone 1, with access to blood containing high concentrations of oxy-
roughly divided into three regions, known as metabolic zonation gen and nutrients delivered by the hepatic artery and portal vein,
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HUANG et al. 1489

respectively. Periportal hepatocytes are mainly involved in liver met-
abolic functions, including gluconeogenesis, glycogen and protein
synthesis, lipid metabolism and ureagenesis. Hepatocytes near the
central vein belong to zone 3, where glycolysis, glutamine synthesis
and xenobiotic metabolism take place. Zone 2 is located between
34
the above two regions. Single-­cell RNA sequencing (scRNA-­seq)
combined with spatial reconstruction provides in-­depth characteri-
zation of liver gene expression patterns and cell population zonation
21,35,36
along the portal-­
central axis. Lineage tracing studies per-
formed by labelling hepatocyte subpopulations have revealed their
specific distribution in the liver lobule during homeostasis and liver
regeneration (Table 3).
Lgr5+/Axin2+ cells, which utilize WNT/β-­c atenin signalling
to sustain their high proliferative capacity, are considered tissue
stem cells in many tissues. 37 Hepatocytes present a centro-­portal
WNT/β-­c atenin signalling gradient, in which Lgr5 and Axin2 are co-­
expressed in pericentral hepatocytes. 38,39 The controversial roles
+ +
of Lgr5 /Axin2 cells in homeostasis have been reported. Using
Axin2-­CreERT2 mice expressing CreERT2 under control of the en-
dogenous Axin2 promoter, Wang et al. demonstrated that Axin2+
hepatocytes are a self-­renewing cell population and expand to 30%
of the area of the entire liver for replacing other hepatocyte sub-
40
populations over 1 year during homeostasis. However, another
study using bacterial artificial chromosome (BAC)-­transgenic mice
to trace Axin2+ cells showed that pericentral Axin2+ hepatocytes
do not have superior proliferative capacity compared with other
41
hepatocyte subpopulations. Consistently, Ang et al. showed
that the number of pericentral Lgr5+ hepatocytes remained stable
under homeostasis.42
The contribution of periportal hepatocytes has also been investi-
gated. Font-­Burgada et al. identified a subpopulation of hybrid peri-
portal hepatocytes (HybHPs) coexpressing the mature hepatocyte
marker HNF4α and low levels of the bile duct cell-­specific gene SOX9.
Although HybHPs contribute to hepatocyte regeneration upon in-
jury, the population of HybHPs remains at approximately 4.5% under
physiological circumstances.43 In addition, the proportion of another
periportal subpopulation—­Mfsd2a+ hepatocytes—­
was decreased
from 40% at 6 weeks to 20% at 20 weeks and then remained stable
under homeostasis.44 Furthermore, a type of hepatocyte subpopu-
lation with high expression of telomerase (TERTHigh), which is distrib-
uted randomly in the liver lobules, expanded 10-­fold (from 2.8% to
nearly 30%) in 1 year during homeostasis.45 These findings suggest
that periportal hepatocytes play a limited role during homeostasis.
More recently, the proliferative capacity of hepatocytes in three
zones has been thoroughly explored by two independent groups
employing multiple genetic lineage tracing approaches. He et al.
developed a dual recombinase-­mediated genetic system involving
DreER, Ki67-­CrexER and R26-­GFP reporter (named ‘ProTracer’) to
record cell proliferation continuously during a given time window,
and found that hepatocytes in zone 2 showed a higher proliferation
rate than hepatocytes in zones 3 and 1.46 Wei et al. performed lin-
eage tracing in 14 CreER knock-­in mouse models, labelling distinct
zonal hepatocyte subpopulations, and found that hepatocytes in
zone 2 were major contributors to homeostasis repopulation and
regeneration.47 These studies further demonstrated that the self-­
replication of hepatocytes is responsible for the maintenance of
liver function and mass under homeostasis. In particular, midlobular
hepatocytes contribute the most to the homeostasis repopulation.

TA B L E 3 Lineage tracing of hepatocyte subpopulation during homeostasis and liver injury

Hepatocyte Percentage of
Zonation subpopulation Mouse line Targeting strategy Total hepatocytes Circumstances Refer-­ences

Zone 1 HybHP Sox9-­CreERT2 Transgenic 4.53 ± 0.24% Homeostasis 43

Acute and chronic liver
injury
SOX9+ Sox9-­CreER; Knockin Around 2.55% Homeostasis 74
Hnf4a-­DreER Acute and chronic liver
injury
Mfsd2a+ Mfsd2a-­CreER, Knockin (ATG) Around 40% Homeostasis 44
Acute and chronic liver
injury
Zone 3 Axin2+ Axin2-­CreERT2 Knockin (ATG) Around 5% Homeostasis 40,105
Axin2-­CreERT2 Transgenic ND Homeostasis 41
Acute liver injury
GS+ GS-­CreER Knockin (3’-­UTR) 9.4 ± 0.4% Homeostasis 47
Chronic liver injury
Lgr5+ Lgr5-­r tTA-­IRES Transgenic 2 ~ 5% Homeostasis 42
Acute liver injury
Distributed Terthigh Tert-­CreERT2 Knockin (ATG) 2.8 ± 0.4% Homeostasis 45
throughout Chronic liver injury
the liver Hamp2+ Hamp2-­CreER Knockin (3’-­UTR) 7.4 ± 0.7% Homeostasis 47
lobules Chronic liver injury
 |
1490
4 | CO M PE N S ATO RY PRO LI FE R ATI O N
O F R E M A I N I N G H E PATO C Y TE S I N ACU TE
LI V E R I N J U RY
In contrast to the slow turnover of hepatocytes during homeostasis,
hepatocytes show remarkable regeneration capability upon injury.
Partial hepatectomy (PHx) is the classic acute liver injury model that
removes certain lobes of the liver.48 The most common PHx model, in
which 70% of the liver is removed, induces hypertrophy and compen-
satory proliferation of the remaining hepatocytes to restore liver mass
49
within 3 weeks. Lineage tracing studies showed that hepatocyte
subpopulation gives rise to its own lineage during the recovery from
PHx.41,42 Shortly after PHx, the activity of urokinase-­type plasminogen
activator (uPA) and metalloproteinases increases, leading to remodel-
ling of the extracellular matrix and formation of active heterodimeric
50,51
hepatocyte growth factor (HGF). The peak of liver regeneration
following 70% PHx occurs within 48 hours, measured by the number
of hepatocytes incorporating 3H-­
thymidine in S phase.52 A recent
study showed that periportal hepatocytes are the first to proliferate
after PHx, followed by midlobular and pericentral hepatocytes, as
demonstrated by the hepatocyte-­specific ProTracer system.46
Hepatocyte nuclear factor 4α (HNF4α) plays an indispensable role
in hepatocyte differentiation and maintenance of hepatocyte function
53,54
during embryonic development and homeostasis. A recent study
showed that HNF4α is down-­regulated at the beginning of regener-
ation following PHx while re-­expression of HNF4α is pivotal for the
termination of regeneration and recovery of hepatocyte function,55
which indicates that hepatocyte might undergo de-­differentiation with
partial loss of its mature hepatocyte markers following acute liver in-
jury and the restoration of its profiles is essential for its full recovery.
Both carbon tetrachloride (CCl4) and acetaminophen (APAP)
have been employed to induce acute liver injury with pericentral
hepatocyte necrosis. In CCl4 model, proliferation of remaining he-
patocytes peaked at 2–­3 days after CCl4 injection and liver archi-
25,56
tecture was completely restored at 10 days after CCl4 injection.
Pericentral Lgr5+ hepatocytes depleted in acute CCl4 injury were
gradually replaced by Lgr5− hepatocytes, which regained Lgr5 ex-
pression to adapt their identity along the portal-­central axis during
42
recovery. Functionally, remaining hepatocytes upregulated the ex-
pression level of pericentral hepatocytes genes, such as Cyp2e1, to
compensate for the function of lost pericentral hepatocytes at 24 h
57
post-­APAP injury. Taken together, hepatocytes not only proliferate
to repopulate the liver mass but also transcriptionally regulate its
expression to maintain liver function and re-­establish liver metabolic
zonation at the same time after acute liver injury.
5 | TH E RO LE S O F H E PATO C Y TE S A N D
TH E I R S U B P O PU L ATI O N S I N C H RO N I C
LI V E R I N J U RY
Chronic liver injury models encompass repetitive administration
of chemicals and genetic modulation in transgenic mice through
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HUANG et al.
related genes. 20,29,58,59 CCl4 and thio-
interfering with cell cycle-­
acetamide (TAA) are employed to induce pericentral liver injury with
fibrosis.60,61 A 3,5-­diethoxycarbonyl-­1,4-­dihydrocollidine (DDC)-­
enriched diet is usually used to mimic chronic cholestatic liver injury
by damaging the biliary compartment.62,63
Hepatocyte self-­replication remains to be the main contributor
to liver mass restoration during chronic liver injury, while certain
hepatocyte subpopulations exhibit superior proliferative capabil-
ity. 24,64,65 Consistent with the finding of homeostasis, TERTHigh he-
patocytes have higher proliferative ability than TERTLow hepatocytes
in CCl4-­and DDC-­induced liver injury models.45 On the other hand,
HybHP expands significantly in chronic liver injury models without
causing hepatocellular carcinoma.43 During the recovery, the newly
generated hepatocytes reprogram to adapt their metabolic function
with their zonation in the liver lobule.43,44 Meantime, the biliary tract
is also remodelled with structural transformation in response to liver
injuries.66
The cell origin of newly regenerated hepatocytes differs slightly
in various liver injury models. Due to the differences in areas of
injury inflicted through the liver injury model, hepatocyte subpop-
ulations located outside targeted areas are more capable of prolif-
erating to restore liver mass and maintain physiological function
(Figure 1B-­C).46,57,67 The periportal subpopulation HybHP and
+
Mfsd2a hepatocytes expand significantly to replace pericentral
hepatocytes in CCl4-­induced chronic liver injury, but they show
limited proliferative ability under DDC-­induced periportal liver
injury.43,44
6 | TR A N S D I FFE R E NTI ATI O N B E T W E E N
H E PATO C Y TE S A N D C H O L A N G I O C Y TE S
Both hepatocytes and cholangiocytes originate from hepatoblasts
during embryonic development.5,68 The conversion between hepat-
ocytes and cholangiocytes during adulthood has also been observed
(Figure 2). Loss of hepatocyte cellular identity was found in non-­
alcoholic steatohepatitis (NASH), DDC mouse model and alcoholic
hepatitis (AH) mouse and human samples.69-­71 In both DDC and AH
liver, hepatocytes transcriptional reprogramming was also accompa-
nied by enrichment of cholangiocyte markers. Several studies have
shown that hepatocytes spontaneously reprogram and convert into
cholangiocytes in vivo during liver regeneration.72-­74 Tarlow et al.
demonstrated that mature hepatocytes exhibit cholangiocyte phe-
notypes following DDC injury and revert to their original identity
when the injury subsides.75 In vitro studies showed that mature
hepatocytes can be converted into bipotent cells by the addition
of small molecules, and these bipotent cells could repopulate liver
when transplanted in Fah−/− and uPA/SCID liver injury models.76-­78
Furthermore, Johanna et al. found de novo formation of mature
cholangiocytes with bile-­
draining function from hepatocytes in
Alb-­cre+/-­Rbpjf/fHnf6f/f transgenic mice that lack peripheral bile
ducts at birth, confirming the transdifferentiation of hepatocytes to
cholangiocytes.79

HUANG et al.
F I G U R E 2 Conversion between
cholangiocytes and hepatocytes. In
chronic injury, hepatocytes spontaneously
transdifferentiate to cholangiocytes.
In liver injury with bile duct impaired,
the biliary system is formed through
hepatocytes transdifferentiation.
As the liver injury becomes severe
and prolonged or with hepatocytes
proliferation inhibited, cholangiocytes
transdifferentiate into hepatocytes to
replenish the liver parenchyma
The role of cholangiocytes in liver regeneration has been elu-
cidated through multiple genetic labelling mice (Table 1). Lineage
tracing through labelling cholangiocytes with HNF1β, CK19, OPN
or SOX9 promoters showed their limited contributions to liver
regeneration in classic liver injury model. 24-­27 However, when
the extent of liver injury becomes severe, the outcome differs.
In a metronidazole-­induced zebrafish liver injury model in which
nearly all hepatocytes were lost, the newly regenerated hepato-
cytes mainly arose from transdifferentiation of biliary cells. 80
During this process, cholangiocytes first dedifferentiate into
hepatoblast-­like cells as they proliferate, and then differentiate
into proliferative hepatocytes to promote restoration of the liver
parenchyma. 81 Raven et al. found significant regenerated hepato-
cytes from cholangiocytes in CK19CreERTtdTomato LSL mice with
impaired hepatocyte proliferation by overexpressing p21 or ab-
lating Itgb1. 29 We also demonstrated that nearly 10% of newly
regenerated hepatocytes were derived from biliary epithelial cells
82
(BECs) in mice with prolonged chronic liver injury. Moreover,
biphenotypic cells co-­e xpressing hepatocyte marker HNF4α and
cholangiocyte marker CK19 or SOX9 were detected in human
samples with several types of liver diseases as well as in injured
mouse liver, suggesting high cellular plasticity in both human and
rodents liver.72,82 Another study showed that CDE diet-­induced
severe liver injury stimulates the formation of cholangiocytes-­
derived hepatocytes in hepatocyte-­s pecific β-­c atenin knockout
83
mice. These studies support the notion of cholangiocytes-­to-­
hepatocytes conversion in severe liver injury. Thus, both hepato-
cytes and cholangiocytes have the capability to convert into each
other, though the severity of liver injuries for induction of such
conversion is quite not the same.
7 | TH E S I G N A LLI N G PATH WAYS
I N VO LV E D I N LI V E R R EG E N E R ATI O N
During homeostasis and liver injury, fine-­tuning of Wnt/β-­
catenin, Hippo/YAP and Notch signalling in hepatocytes and/or
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1491
cholangiocytes is required for the maintenance of liver metabolic
function, proliferation and transdifferentiation ability.
The Wnt/β-­
catenin signalling gradient along the centropor-
tal axis controls homeostatic liver metabolic zonation.38,84,85
Overexpression of active β-­catenin promoted hepatocyte-­to-­
cholangiocyte transdifferentiation in DDC liver injury model.86 A
recent study showed that upregulation of Wnt/β-­catenin activity in
hepatocytes by deletion of ZNRF3 and RNF43 increased hepatocyte
proliferation, impaired hepatocyte metabolic zonation and promoted
liver tumours.87 In addition, the Wnt-­producing niche provided by
the endothelium and macrophages plays an indispensable role in
the maintenance of functional compensation and the proliferative
response of hepatocytes.40,57
Highly conserved Hippo/YAP signalling plays a prominent role
in regulating liver size and regeneration. 88 The activation of YAP
in hepatocytes led to loss of hepatocyte identity, conversion of
hepatocytes into cholangiocytes and weakened liver regenera-
tion.70,89 Furthermore, clinical samples from alcoholic hepatitis
patients showed profound YAP activation in hepatocytes ac-
companied with increased expression of biliary marker (SOX9,
HNF1β).70 Studies have shown transcriptional heterogeneity of
YAP signalling in homeostatic cholangiocytes and upregulation
of YAP signalling in cholangiocytes and hepatocytes upon CCl 4
and DDC injury.90,91 Verboven et al. showed that the conditional
knockout of the Hippo pathway downstream effectors YAP/TAZ
in mature hepatocytes did not affect hepatocyte proliferation
after acute liver injury, while the deletion of YAP/TAZ in chol-
angiocytes impaired liver regeneration with bile duct deteriora-
tion.90,92 These findings highlighted the pivotal role of Hippo/YAP
signalling in periportal hepatocytes and cholangiocytes during
liver regeneration.
Notch signalling is required for the formation and repair of the
bile duct during development and liver injury.93-­96 Activation of
Notch signalling in hepatocytes by enhancing NOTCH1 intracel-
lular fragment expression induced the reprogramming of hepato-
cytes into biliary cells during homeostasis and the formation of
intrahepatic cholangiocarcinoma.72,97 Inhibition of Notch signalling
 |
1492
in hepatocytes through deletion of the Notch signalling effector
RBP-­Jkappa reduced hepatocyte-­to-­biliary reprogramming in DDC
injury.72 Since Notch signalling acts downstream of Hippo/YAP, the
effect of Hippo/YAP signalling in liver injury could be attributed
to the regulation of Notch signalling.89 Furthermore, activation of
TGF-­β signalling through constitutively active TGFBR1 expression
in hepatocytes promoted hepatocyte-­to-­cholangicyte transdiffer-
entiation and the formation of bile ducts in an Alagille syndrome
mouse model (Alb-­cre+/-­Rbpjf/fHnf6f/f ), demonstrating TGF-­β signal-
ling as a key driver during transdifferentiation independent of Notch
signalling.79
The crosstalk between parenchymal cells and non-­parenchymal
cells (NPCs), which include liver sinusoidal endothelial cells (LSECs),
hepatic stellate cells (HSCs) and immune cells, play important role
in liver regeneration (reviewed previously).98-­100 Moreover, a recent
study showed that the dynamic ratio between mesenchymal cells
and ductal cells regulate the proliferation of epithelial cell through
101
organoid co-­culture, which shed light on the effect of cell–­cell
contact during liver regeneration.
8 | CO N C LU D I N G R E M A R K S A N D FU T U R E
D I R EC TI O N S
Recent work has significantly advanced our understanding of
the cellular origins and underlying mechanisms of liver regenera-
tion. Multiple lines of evidences suggest that liver regeneration is
a process of hepatocyte self-­duplication in all regions of the liver
lobule during homeostasis and following injury, with midlobular
hepatocytes as the main contributor to the homeostatic turnover of
hepatocytes. In the meantime, newly-­generated hepatocytes tran-
scriptionally regulate their zonally-­expressed genes to adapt their
identities along the portal-­central axis. The conversion between
hepatocytes and cholangiocytes in several severe injury models
demonstrated the high plasticity of liver cells. But the precise mech-
anisms underlying their proliferation and high plasticity remain to be
further elucidated. Future studies should further characterize the
signalling pathways driving hepatocyte self-­replication and cell fate
plasticity, particularly in situations of ageing. It also remains to be
clarified how non-­parenchymal cells contribute to the local niche,
which helps re-­establish the metabolic zonation of newly gener-
ated hepatocytes. Single-­cell sequencing technology has overcome
the shortcomings of bulk sequencing in the heterogeneous tissues
with mixed transcriptomic characterization and will help to further
identify dynamic signalling pathways in liver cells. 36 With emerging
technology, the field of liver generation is on the verge of making
great progress in addressing those questions, which will ultimately
deepen our understanding of liver regeneration. Clinically, the gen-
eration of functional and transplantable hepatocytes presents as a
promising strategy for patients with acute liver failure or metabolic
liver diseases.102 With further mechanisms underlying liver regener-
ation uncovered, it is conceivable that reliable approaches towards
14783231, 2022, 7, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/liv.15174 by Egyptian National Sti. Network (Enstinet), Wiley Online Library on [27/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
HUANG et al.
the management of liver diseases would be provided through direct
reprogramming of differentiation pathways and indirect modifica-
tion of surrounding niche in the liver.
C O N FL I C T S O F I N T E R E S T
The author(s) declare that they have no conflicts of interest.
E T H I C A L S TA N DA R D S
This article does not contain any experiments with human or animal
subjects conducted by any of the authors.
DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were
generated or analysed in this study.
ORCID
Ru Huang https://orcid.org/0000-0003-0236-3940
Wei-­Fen Xie https://orcid.org/0000-0002-7137-112X
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How to cite this article: Huang R, Zhang X, Gracia-­Sancho J &
Xie W-F. Liver regeneration: Cellular origin and molecular
mechanisms. Liver Int. 2022;42:1486–1495. doi: 10.1111/
liv.15174