Generation and Regeneration of Cells
of the Liver and Pancreas
Kenneth S. Zaret1* and Markus Grompe2
Liver and pancreas progenitors develop from endoderm cells in the embryonic foregut. Shortly
after their specification, liver and pancreas progenitors rapidly acquire markedly different cellular
functions and regenerative capacities. These changes are elicited by inductive signals and genetic
regulatory factors that are highly conserved among vertebrates. Interest in the development and
regeneration of the organs has been fueled by the intense need for hepatocytes and pancreatic b
cells in the therapeutic treatment of liver failure and type I diabetes. Studies in diverse model
organisms have revealed evolutionarily conserved inductive signals and transcription factor
networks that elicit the differentiation of liver and pancreatic cells and provide guidance for how to
promote hepatocyte and b cell differentiation from diverse stem and progenitor cell types.
in the liver and the regulation of blood glucose
levels by insulin secreted from b
cells in the pancreas. Liver hepato-
cytes are large, often polyploid cells
that secrete serum proteins, express
enzymes that neutralize toxicants,
produce bile acids to aid in diges-
tion, and control the bulk of inter-
mediary metabolism. Biliary ducts
of cholangiocytes, the other epithe-
lial cell type in the liver, serve pri-
marily as conduits of secreted bile.
In contrast, the distinct pancreatic
functions are partitioned into many
more cell types. Pancreatic cells in-
clude insulin (b), glucagon (a), so-
matostatin, ghrelin, and pancreatic
polypeptide–secreting endocrine types,
each of which produces a single
hormone. The pancreas also contains
exocrine cell types, which constitute
the bulk mass of the tissue and in-
enzymes and duct cells that provide
conduits to the gut for the enzymes.
The greater diversity of cell types in
regulatory factors and lineage deci-
sions during organogenesis.
Clinical studies have shown that
transplantation of hepatocytes can support the
functions of a failed liver and correct metabolic
he liver and pancreas coordinately control
body metabolism, including the modifica-
tion of digested nutrients by hepatocytes
liver disease in the long term (1). Similarly,
cadaveric islets can, for several years, support
glucose homeostasis in type I diabetic indi-
viduals, in whom the b cells have been de-
stroyed by an autoimmune reaction (2). In
both transplantation settings, the quality and
amount of donor cells are severely limiting, as
is the ability to expand the terminally differen-
tiated cell populations. These limitations have
led to a search for other progenitor cell sources
of hepatocytes and b cells and intense interest in
how the differentiation of such progenitors can
be directed, or “programmed,” efficiently. The
programming efforts are founded on understand-
ing how hepatocytes and b cells are normally
generated in the embryo and how they arise
during regeneration in adults, in response to
tissue damage and disease. Here we provide an
overview of the cells’ development and regen-
eration and highlight unresolved issues in the
Two Progenitor Domains for Each Tissue
The liver and pancreas in terrestrial vertebrates
each develop from two different spatial domains
of the definitive endodermal epithelium of the
embryonic foregut. Fate-mapping experiments
have shown that the liver arises from lateral
domains of endoderm in the developing ventral
foregut (3, 4) as well as from a small group of
endodermal cells tracking down the ventral mid-
line (4) (Fig. 1A). During foregut closure, the
medial and lateral domains come together (Fig.
1A, green arrows) as the hepatic endoderm is
specified. The pancreas is also induced in lateral
endoderm domains, adjacent and caudal to the
lateral liver domains, and in cells near the dorsal
midline of the foregut (5, 6) (Fig. 1A). These
events occur at 8.5 days of mouse gestation
(E8.5), corresponding to about 3 weeks of human
gestation. After the domains are specified and
initiate morphogenetic budding, the dorsal and
ventral pancreatic buds merge to create the gland.
Despite differences in how the different progen-
itor domains are specified, descendants of both
pancreatic progenitor domains make endocrine
and exocrine cells, and descendants of both liver
1Epigenetics and Progenitor Cells Program, Fox Chase Cancer
Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA.
2Oregon Stem Cell Center and Papé Family Pediatric Research
Institute, Oregon Health and Science University, 3181 South-
west Sam Jackson Park Road, Portland, OR 97239, USA.
*To whom correspondence should be addressed. E-mail:
Fated tissue domains
prior to specification
Tissue domains at time of specification
(view into foregut)
Fig. 1. Cell domains and signals for embryonic liver and pancreas specification. (A) Fate map of progenitor
cell domains before tissue induction; view is into the foregut of an idealized mouse embryo at E8.25 (three- to
four-somite stage). Green arrows indicate movement of lateral progenitor regions toward the ventral-medial
region. (B) Sagittal view of a mouse embryo several hours later than in (A) showing the positions of the newly
specified liver and pancreas tissue domains. Signals and cell sources that pattern the endoderm are shown.
Dashed blue line indicates plane of view in (A).
5 DECEMBER 2008VOL 322
progenitor domains contribute to differentiating
liver bud cells (3–6). Genetic lineage–marking
studies are needed to determine the extent to
which different descendants within each tissue
may differ with regard to functionality and
Signals Specifying Hepatic and
Embryo tissue recombination experiments and
zebrafish have revealed that the liver and pan-
creas domains are specified within the endoder-
mal epithelium under the influence of inductive
signals from nearby mesoderm cells (7, 8). Little
is known about how the signaling genes in the
vertebrate animal models. Initially, broad sup-
pression of mesodermal Wnt and fibroblast
growth factor 4 (FGF4) signaling in the foregut
enables liver and pancreas induction, whereas
active mesodermal Wnt signaling in the posterior
gut suppresses these tissue fates (9, 10) (Fig. 1B).
Retinoic acid signaling, apparently from paraxial
mesoderm cells, helps further refine the anterior-
posterior position in which the liver and pancreas
can develop from the gut endoderm (11–14).
Subsequently, in the ventral foregut, FGF from
the cardiac mesoderm and bone morphogenetic
protein (BMP) from septum transversum mesen-
chyme cells coordinately induce the liver pro-
gram and suppress the pancreas program (15–18).
Mitogen-activated protein kinase (MAPK) is ac-
tivated in response to FGF in the lateral hepatic
progenitors well before MAPK activation in the
lateral ventral endoderm cells that move caudal to
ventral pancreatic development (20). In the dorsal
foregut, signals from the notochord that include
activin and FGF suppress sonic hedgehog (shh)
signaling within the endoderm and allow the pan-
creatic program (21, 22). All of the above events
occur within hours in the vertebrate embryo.
The newly specified hepatic cells in embryos
are referred to as hepatoblasts. These cells ex-
press serum protein genes specific to hepatocytes,
such as albumin (alb1) and transthyretin (ttr), and
appear to be bipotential and later give rise to
hepatocytes and cholangiocytes (23); however,
formal genetic lineage studies remain to be
performed. The Tbx3 gene helps expand the
hepatoblast population by suppressing p19ARF
(24). The newly specified pancreatic endoderm is
initially marked by the expression of the tran-
which are crucial for pancreatic development;
Pdx1 is also expressed in adjacent progenitors of
the duodenum (27, 28).
Changes in Signal Responses as
The cellular responses to inductive signals in-
clude the activation and repression of tran-
scription factor genes that, in turn, elicit new
gene expression programs required for cell dif-
ferentiation. The new cell type programs can
change the cellular responses to exogenous
signals. Such is the case for FGFs, which are
endoderm (19, 29) and later promote the ex-
pansion of the newly specified progenitor cell
populations (16, 30). Shh signaling initially pro-
motes dorsal pancreatic development in the
zebrafish (31, 32) and later appears to suppress
it (33). Wnt signaling initially inhibits liver in-
duction (9) but shortly afterward promotes liver
bud growth and differentiation (9, 34, 35). Each
of these changes in cellular responses to induc-
changes are not known.
Organ Morphogenesis and
After the hepatoblasts and pancreatic progenitors
are specified, the respective endoderm cells tran-
sition from a cuboidal shape to a columnar one
and then become pseudo-stratified within the
epithelium (Fig. 2). This process is similar to the
morphogenetic characteristics of neural epithelial
development and is controlled in the foregut by
the homeobox transcription factor gene Hhex
(36). The pancreatic epithelium then branches
into the stroma to create the pancreatic bud;
whereas for the hepatic epithelium the basal
laminabreaks down,and the cells proliferate into
changes are controlled by the homeobox tran-
scription factor genes Prox1 (37), Hnf6/OC-1,
and OC-2 (38) (Fig. 2). Hnf6 and OC-2 regulate
E-cadherin, thrombospondin-4, and Spp1, which
control cell adhesion and migration in various
contexts. The fetal liver serves as a transient site
for hematopoiesis in amniotes. Hence, fetal via-
bility is dependent on proper liver growth.
As the progenitors of both tissues bud into
the stroma, they are adjacent to and receive
stimulatory signals from nearby endothelial cells
(39–41). Endothelial cells also promote liver
regeneration after tissue damage, apparently by
hepatocyte growth factor (HGF) signaling (42).
The specific molecular signals produced from
endothelial cells in the embryonic context have
not been described, but sphingosine-1-phosphate
in the circulation, delivered by the endothelium,
promotes dorsal pancreatic budding (43). The
emerging vascular systems in the liver and
E 8.5 10 somite pairs (10S)
Fig. 2. Stages of liver bud organogenesis. Hepatoblasts are stained blue
(HexLacZ+), cells with orange nuclei are gut endoderm (FoxA2+), and all nuclei
were stained green by 4´,6´-diamidino-2-phenylindole. White arrows point to
E 9.0 (21S)
E 10.0 (27S)
Emergence into stroma
the hepatic cells. Genes and signals that promote each transition are indicated.
Similar morphogenetic stages occur during pancreas bud organogenesis.
Images are adapted from (36). S, pair somite stage; E, embryonic day.
VOL 3225 DECEMBER 2008
pancreatic buds also provide oxygen and nutri-
Neural crest cells migrate into the develop-
ing pancreas and, as they develop into neurons,
affect the numbers of b cells (45). The stimu-
latory roles of endothelial cells and neural crest
cells illustrate how crucial is the codifferentia-
tion of the stromal environment with that of the
hepatic and pancreatic progenitors.
Within the liver and pancreas buds, Notch
signaling components are important for creating
the proper balance in the numbers of hepatocytes
and cholangiocytes from hepatoblasts (46, 47)
and of endocrine and exocrine cells from pan-
creatic progenitor cells (48–50). Loss of Notch
signaling allows the endocrine lineage, which is
marked by and requires the transcription factor
gene Ngn3 (25, 48, 51).
the pancreatic bud develops into an organ, Cpa1-
positive cells in the distal tips of the branching
epithelium are multipotent progenitors that give
rise to duct and endocrine descendants along
the trunk of the branches, until about E14 in the
mouse (52). Afterward, the Cpa1-positive cells
give rise to acinar cells; this corresponds to the
time of the “secondary transition,” when defini-
tive b cells are generated under the influence of
the Mafa transcription factor (53, 54). Further
genetic lineage studies, the transcription factor
genes that elicit pancreatic and hepatic cell dif-
ferentiation, and the parameters that affect cell
growth are shown in Fig. 3 and have been re-
viewed extensively elsewhere (23, 55–57). There
tral pancreatic progenitors are specified (Fig. 3),
which suggests flexibility in the ways by which
pancreas cells could be specified from stem cells.
Liver regeneration after most forms of injury
does not rely on stem or progenitor cells but
instead involves the mitosis of mature cells (58).
The regenerative capacity of hepatocytes can be
assessed in animal models of liver repopulation,
in which transplanted cells have a selective ad-
vantage over the host (59). By use of such
models, it has been shown that mature polyploid
hepatocytes have a stem cell–like regenerative
capacity rivaling that of hematopoietic stem cells
and are able to divide more than 100 times with-
out loss of function (60). Human hepatocytes are
also highly regenerative (61), and this capacity
for regeneration is established at the earliest em-
bryonic stages (36). Unfortunately, it has not yet
proved possible to grow and expand populations
of hepatocytes in cell culture or maintain their
differentiation. Even with the most sophisticated
growth media, hepatocytes dedifferentiate exten-
the functions of adult hepatocytes, including cell
division, appear to depend on complex inter-
actions with other cells in a three-dimensional
matrix. Coculture systems attempting to mimic
The adult liver also harbors facultative pro-
genitors that can be activated in response to spe-
cific injuries (Fig. 4), usually under conditions of
rise to an intermediary cell type, often termed
“oval cells,” which are thought to differentiate
However, oval cells are not a homogeneous
population (64), and their apparent multipoten-
tiality has not been demonstrated by definitive
lineage tracing. In the rat, oval cells resemble
embryonic hepatoblasts in that they express both
bile duct and hepatocyte markers as well as a-
fetoprotein (58). Thus, progenitor cell activation
in the adult employs some of the same genetic
programs used during development (65). Details
about the precise origin of adult liver progenitors
Whereas hepatocytes are capable of extensive
regeneration, the ability of b cells to expand is
more limited, especially in the adult. Some de-
gree of regeneration can occur in young animals
after physiologic stimuli such as pregnancy (66)
or injury (partial pancreatectomy) (67). How-
ever, this partial growth ability is insufficient to
permit recovery from cell loss in type I diabetes;
yet it might, with suppression of autoimmunity
(68). The restricted regenerative ability of the
endocrine pancreas may be related to the de-
fined number of pancreatic progenitors, which is
not capable of compensatory growth in response
to cell loss (69). In contrast, hepatoblasts can
increase their proliferative rate in response to
dysfunctional cells in their midst (36). The lack
of regeneration in b cells has raised considerable
interest in the potential of tissue repair by resi-
dent stem cells. It remains controversial whether
progenitors exist in the adult pancreas. It is clear
that the majority of new b cells derive from pre-
existing insulin-expressing cells after surgical in-
jury (67, 70), but recent work has shown that
duct ligation can activate Ngn3-positive b cell
(bile duct cell)
Mafa, Pdx1, Hlxb9
Pax4, Pax6, Isl1
Fig. 3. Regulatoryfactorscontrollingcelltypelineageswithintheliverandpancreas.Transcriptionfactor
genes are shown in bold; their functions have been reviewed in the text and elsewhere (23, 55–57),
except for vHnf1 in hepatic development (81). Pdx1 initially marks duodenum and caudal stomach
progenitors (not shown) as well as the pancreatic domains (28).
5 DECEMBER 2008VOL 322
precursors in the ductal epithelium (71). Thus,
adult pancreatic progenitors exist and their acti-
vation depends on the specific injury, as does
oval-cell initiation in the liver (Fig. 4).
Creating Hepatocytes and b Cells de Novo
The current inability to expand human hepato-
cytes in vitro is an obstacle not only for cell
therapy but also for pharmaceutical drug devel-
opment because of the cells’ importance in as-
sessing the metabolism of xenobiotics. Thus, the
generation of hepatocytes from expandable pre-
cursors is of considerable interest. Cells with
properties virtually identical to those of hepatic
oval cells can also emerge in the pancreas, es-
pecially after the ablation of acinar cells (72).
cells can differentiate into functional hepatocytes
and bile ducts (73). Several reports have sug-
gested that the reciprocal transdifferentiation is
also possible; that is, the conversion of liver cells
toward the pancreatic endocrine fate (74). Forced
insulin expression in the liver and corrects ex-
perimental diabetes (75). Together, these findings
suggest that both the adult liver and pancreas
b cell precursors in the adult liver is of obvious
medical interest. Because pancreatic exocrine
cells greatly outnumber b cells, it is also exciting
that they can be reprogrammed to make func-
tional b cells in vivo by viral delivery of the de-
velopmental transcription factors Pdx1, Ngn3,
and Mafa (76).
Pluripotent stem cells, including embryonic
stem cells (ESCs) and induced pluripotent stem
cells (iPSCs), are a potentially abundant source
of hepatocytes and b cells. Numerous groups
have been developing ESC differentiation pro-
tocols that attempt to mimic normal embryonic
development. The first step of both pancreatic
and hepatic development is the induction of a
definitive endoderm by using activin A (77).
Further treatment with BMP-4 and bFGF can
then direct cells toward the hepatic lineage (78).
endocrine pancreatic cells, definitive endoderm
was treated insequential stages with keratinocyte
growth factor (KGF), retinoic acid, Noggin, and
cyclopamine (79, 80). Despite remarkable prog-
ress, the resulting cells often fail to achieve com-
plete function sufficient for regenerative therapy,
remaining only, “hepatocyte- or b cell–like.” It is
not yet clear how precisely the known develop-
mental signals must be orchestrated to properly
program hepatic and pancreatic cells at will, but
detailed studies of the activated signaling path-
ways and their cross-regulatory interactions dur-
ing embryogenesis will be informative.
Two basic opportunities for medical application
of the knowledge of the developmental biology
of the liver and pancreas have emerged. The first
is the application of the precise conditions that
exist within the embryo to differentiate pluri-
potent stem cells. The sequential and exactly
timed use of extracellular factors and accessory
cell types (such as endothelium and mesen-
chyme) is predicted to mimic embryogenesis
and thus yield highly functional derivatives for
transplantation and other applications. In this
setting, competent cells respond to extrinsic
signals that act on their epigenome. The second
approach is to use genetic reprogramming to
directly change cell fates by taking advantage of
transcriptional activators, repressors, and chro-
matin modifiers. This method can work in vivo
as well as in culture and can be applied to adult
it may be possible to enhance tissue regeneration
in situ without the complications of cell engraft-
ment and immunological rejection. However, it
will be important to overcome the potential
problems of insertional mutagenesis, in which
stable gene integration is involved, as well as
undesired transgene expression changes and
phasis is required on both approaches to use the
signals and regulatory factors from developmen-
tal biology to sculpt the differentiation of pro-
genitor and stem cells to liver and pancreas cell
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not cite because of space constraints. We thank D.
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preparing the manuscript. K.S.Z. is supported by NIH
grants R37 GM36477, U01 DK072503, and
P30CA06927, and M.G. by grants U01 DK072477 and
ROI DK05192 and Juvenile Diabetes Research Foundation
grant 18508680-36749. The authors have patents
pending related to the work in this article. M.G. is a
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advisory board for Johnson and Johnson.
Cardiogenesis and the Complex
Biology of Regenerative
Kenneth R. Chien,1,2* Ibrahim J. Domian,1Kevin Kit Parker3
The heart is a complex organ system composed of a highly diverse set of muscle and nonmuscle
cells. Understanding the pathways that drive the formation, migration, and assembly of these cells
into the heart muscle tissue, the pacemaker and conduction system, and the coronary vasculature is
a central problem in developmental biology. Efforts to unravel the biological complexity of in vivo
cardiogenesis have identified a family of closely related multipotent cardiac progenitor cells. These
progenitors must respond to non–cell-autonomous signaling cues to expand, differentiate, and
ultimately integrate into the three-dimensional heart structures. Coupling tissue-engineering
technologies with patient-specific cardiac progenitor biology holds great promise for the
development of human cell models of human disease and may lay the foundation for
novel approaches in regenerative cardiovascular medicine.
“There is always an easy solution to every
human problem—neat, plausible and wrong.”
is clearly more than muscle, with a panoply of
diverse cardiac and smooth muscle, valvular,
—H. L. Mencken (1)
contractile, electrical, and vascular roles (Figs. 1
and 2). To form a fully functional heart organ sys-
tem, a set of embryonic precursor cells must give
rise to these distinct cell types, which must ulti-
mately assemble and align within specific heart
compartments to form ventricular chambers, cor-
onary arteries, and the conduction system. In this
regard, recent studies have identified a novel set
of multipotentheartprogenitorsthatcan giverise
same time, a host of clinical studies in regenera-
tive cardiovascular medicine have attempted to
reverse heart muscle failure by augmenting the
amount of functional human cardiac muscle via
transplantation of a diverse group of adult pro-
genitor cell types (2–8). The concept itself is rel-
atively simple: Progenitor cells isolated from
outside the heart are transplanted into the adult
heart, with the hope that they will eventually
expand and integrate into the intact myocardial
tissue and thereby improve cardiac function.
Unfortunately, to date, the results have largely
been ambiguous, marginal, or negative, suggest-
ing that simply transplanting adult nonheart pro-
will not necessarily lead to substantial, long-term
clinical improvement (9). The lessons from these
(10–18) point to the need to account for the bio-
logical complexity of in vivo embryonic car-
diogenesis. How is the diversity of heart cells
generated? Is there a “master” heart progenitor
that can give rise to the major muscle and non-
1MGH Cardiovascular Research Center, Massachusetts
General Hospital, Boston, MA 02114, USA.2Department
of Stem Cell and Regenerative Biology, Harvard University
and the Harvard Stem Cell Institute, Cambridge, MA
Engineering and Applied Sciences, Harvard University,
Cambridge, MA 02138, USA.
*To whom correspondence should be addressed. E-mail:
3Disease Biophysics Group, School of
5 DECEMBER 2008VOL 322