Download Pancreatic stellate cells can form new β-like cells

Biochem. J. (2009) 421, e1–e4 (Printed in Great Britain)
e1
doi:10.1042/BJ20090779
COMMENTARY
Pancreatic stellate cells can form new β-like cells
Kevin DOCHERTY1
School of Medical Sciences, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, U.K.
Regenerative medicine, including cell-replacement strategies,
may have an important role in the treatment of Type 1 and Type 2
diabetes, both of which are associated with decreased islet cell
mass. To date, significant progress has been made in deriving
insulin-secreting β-like cells from human ES (embryonic stem)
cells. However, the cells are not fully differentiated, and there is
a long way to go before they could be used as a replenishable
supply of insulin-secreting β-cells for transplantation. For this
reason, adult pancreatic stem cells are seen as an alternative source
that could be expanded and differentiated ex vivo, or induced to
form new islets in situ. In this issue of the Biochemical Journal,
Mato et al. used drug selection to purify a population of stellate
cells from explant cultures of pancreas from lactating rats. The
selected cells express some stem-cell markers and can be grown
for over 2 years as a fibroblast-like monolayer. When plated on
extracellular matrix, along with a cocktail of growth factors that
included insulin, transferrin, selenium and the GLP-1 (glucagonlike peptide-1) analogue exendin-4, the cells differentiated into
cells that expressed many of the phenotypic markers characteristic
of a β-cell, and exhibited an insulin-secretory response, albeit
weak, to glucose. The ability to purify this cell population opens
up the possibility of unravelling the mechanisms that control selfrenewal and differentiation of pancreatic cells that share some of
the properties of stem cells.
The hunt has been on for many years to track down and
characterize the elusive adult pancreatic stem cell. The idea is
that such a cell, with the ability to self-renew and differentiate
into functional insulin-secreting β-cells, would prove invaluable
in providing new therapeutic options for the treatment of Type 1
and Type 2 diabetes, since both forms of the disease are associated
with a decline in β-cell mass [1]. Type 1 diabetes is caused by the
autoimmune destruction of the insulin-secreting β-cells of
the islets of Langerhans, whereas Type 2 diabetes is marked
by both resistance of target tissue to the effects of insulin and
a marked impaired function of the β-cell, leading to a gradual
decline in β-cell mass. Restoration of β-cell mass either from ex
vivo expansion and differentiation of stem cells as a replenishable
source of tissue for transplantation or in situ activation of a β-cell
progenitor pool are currently viewed as viable cell therapeutics. Of
course, for Type 1 diabetes, any cell-replacement therapy would
need to overcome the problem of autoimmunity by encapsulating the cells in a matrix that protects the transplanted cells
from the host immune system, whereas for Type 2 diabetes, cell
therapy would best be used in conjunction with drugs directed
at ameliorating insulin resistance. With the worldwide number
of people affected by diabetes running at over 200 million and
increasing [2], the stakes are high. Nonetheless, despite many
years of effort, we know very little about adult pancreatic stem
cells. This is in contrast with the huge advances that have been
made in characterizing their embryonic counterparts.
In the developing mouse, the pancreas first appears at around
embryonic day 9.5 (E9.5) as dorsal and ventral evaginations of a
subregion of the foregut in which Sonic Hedgehog is repressed
(Figure 1) [3]. This allows expression of the homeodomain transcription factor Pdx1 (pancreatic and duodenal homeobox 1),
which, along with the bHLH (basic helix–loop–helix) transcription factor Ptf1a, plays important roles in the formation of
the pancreatic buds. The pancreas then undergoes a period of
expansion, during which a branching epithelial network forms.
Genetic-lineage-tracing studies provide evidence for a pool
of MPCs (multipotent progenitor cells) within the expanding
pancreatic bud [4]. These MPCs give rise to all pancreatic cell
types, including exocrine (acinar and ducts) and endocrine cells.
The establishment of the various pancreatic lineages proceeds in
a stepwise manner in response to signalling molecules generated
from various surrounding cell types, including those present in the
mesenchyme and vasculature. At around E14.5, the commitment
to expand further or differentiate is governed by Delta/Notch
signalling on adjacent cells. Notch-mediated signalling ensures
maintenance of the progenitor cell population through activation
of the bHLH protein Hes1. In a population of Pdx1-positive
cells that evades the Notch ligand, repression of Hes1 leads to
activation of the bHLH transcription factor Ngn3 (neurogenin
3) that specifies the endocrine lineage. Within this lineage, the
transcription factors Nkx2.2, Nkx6.1, Pax4, MafB/A and Pdx1
drive formation of β-cells, while Pax6, Arx and Brn4 drive
formation of glucagon-secreting α-cells. This scenario, which
appears also to operate in Xenopus, chicks and zebrafish as well as
in humans, emphasizes the importance of these key transcription
factors in the development of the different cell lineages. The
crucial role of Sonic Hedgehog and Notch in the maintenance
of the progenitor state is supported further by the presence of
Sonic Hedgehog and Notch signalling in pancreatic cancer cells
that appear to be trapped in the process of self-renewal, i.e. a
stem-cell phenotype [5].
With regard to generating a replenishable supply of islets
for transplantation in the treatment of Type 1 (and presumably
Type 2) diabetes, this understanding of the developing pancreas
has provided the basis for strategies to induce differentiation of
human ES (embryonic stem) and iPS (induced pluripotent stem)
Key words: cell therapy, diabetes mellitus, islet of Langerhans,
pancreatic stem cell.
Abbreviations used: ABC, ATP-binding cassette; bHLH, basic helix–loop–helix; E, embryonic day; ES, embryonic stem; iPS, induced pluripotent stem;
MPC, multipotent progenitor cell; Ngn3, neurogenin 3; Pdx1, pancreatic and duodenal homeobox 1.
1
email [email protected]
c The Authors Journal compilation c 2009 Biochemical Society
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Figure 1
K. Docherty
Simplified schematic diagram showing stages in the developing pancreas of the mouse
The pancreas is formed from ventral and dorsal evaginations of the foregut, whereas the liver is formed from the ventral region. Transcription factors involved in the specification of the various
lineages are shown in bold type. The approximate embryonic day (E) is shown at the bottom. For reasons of clarity, the other endocrine cell types, i.e. the somatostatin-secreting γ -cell, the pancreatic
polypeptide-secreting PP cell and the ghrelin cell, have been omitted. The exocrine pancreas comprises acinar and duct cells. Further information can be found in [21].
cells into functional islets [6]. The problem is that, although
a high proportion of the differentiated cells express insulin at
levels close to those seen in human islets, the cells are not
fully differentiated, lacking a secretory response to glucose, and
many of the cells co-express more than one hormone. The ESderived MPCs (generated at an intermediate stage) can, however,
be induced to differentiate into functional β-cells following
prolonged culture (up to 72 days) under the kidney capsule or fat
pads of mice. This suggests that ES- (or iPS-) derived functional
β-cells may one day become available for therapeutic trials.
So, where does this leave the elusive adult pancreatic stem
cell? There is a perception that such a cell would have important
therapeutic implications. If it were present in biopsied material
and could be induced to proliferate and differentiate ex vivo, then
it would provide a patient-specific supply of transplantable cells
that would circumvent the requirement for immunosuppression
to prevent foreign tissue allograft rejection. In addition, it may
be more practical to obtain fully differentiated functional islet
cells from such dedicated ‘pancreatic progenitors’ than from
ES/iPS cells, and possibly also reduce the chances of teratoma
being formed. Finally, a better understanding of the properties
of such a cell and how it becomes activated would represent
an important breakthrough in the development of strategies to
regenerate islets, particularly in Type 2 diabetes. The problem
in this case is that, like most solid tissues, the pancreas is
terminally differentiated with a very low turnover rate under
normal physiological conditions. Recent elegant genetic lineagetracing studies in mice have shown that β-cell replication is
the principal mechanism involved in the maintenance of βcell mass [7]. This was subsequently confirmed using a DNAanalogue-based lineage-tracing technique [8], whereas autopsy
studies in humans provide strong supportive evidence that β-cell
replication is the primary mechanism underlying β-cell expansion
in childhood [9] and potentially also in obesity/pregnancy.
There is a view that β-cell replication alone may be sufficient
to account for maintaining the mass of the pancreas, which
c The Authors Journal compilation c 2009 Biochemical Society
appears, at least in rodents, to be set by the size of the
progenitor pool in the early embryo [10]. However, there is
also strong evidence that new β-cells can be generated by a
process of neogenesis from a stem-cell population residing in
the pancreatic duct [1]. Thus it was shown over 10 years ago
that long-term culture of islet-producing stem cells could be
established from ductal epithelial cells or digested pancreatic
tissue freshly explanted from human organ donors or prediabetic
NOD (non-obese diabetic) mice. There now exists a significant
body of data supporting a role for differentiated adult ductal
cells as a source of pancreatic progenitors. Increased budding
of endocrine cells from the ducts has been observed in rodents
undergoing partial pancreatectomy, and in response to treatment
with the GLP-1 (glucagon-like peptide-1) analogue exendin4, and Betacellulin or overexpression of IFN-γ (interferon-γ )
or TNFα (tumour necrosis factor α). Lineage tracing of
genetically marked ductal cells shows that, in mice, they can
give rise to both new islets and acinar tissue after birth and
injury [11]. An elegant study in adult mice has shown that
new β-cells can be formed from non-β-cells located in the
lining of the duct during regeneration of the pancreas in response to duct ligation. Shortly after duct ligation, there was an
increased number of cells expressing Ngn3, which is not normally
expressed in the adult pancreas [12]. These Ngn3-positive cells
were sorted by flow cytometry and implanted into pancreatic buds
from Ngn3−/− mice. Under these conditions, the Ngn3-positive
cells from the regenerating adult pancreas differentiated into
β-cells and other endocrine cell types. As noted above, there
is still some controversy over the relative contribution of β-cell
replication and neogenesis to the maintenance of β-cell mass
under normal physiological conditions, but clearly neogenesis
appears to be important in compensatory responses to increased
metabolic demands, as seen with increased age, obesity and
pregnancy.
New islets can also be formed by a process of transdifferentiation. Acinar cells, for example, when placed in culture
Commentary
will spontaneously dedifferentiate and redifferentiate [13]. During
the redifferentiation process, the cells acquire characteristics
of ductal cells through a process that mimics early stages of
pancreatogenesis. The progenitor cells derived from adult acinar
cell cultures can be directed towards an hepatocyte lineage
by treatment with the glucocorticoid dexamethasone, while
treatment with EGF (epidermal growth factor) and LIF (leukaemia
inhibitory factor) can induce formation of β-cells, albeit at
low efficiency. Interestingly, liver cells, which incidentally share
a common ancestry with the pancreas (both arise from the
same region of the ventral foregut; Figure 1), can also undergo
transdifferentiation to β-like cells following overexpression
of pancreatic transcription factors such as Pdx1 or NeuroD1
[14]. The presence of pancreatic endocrine-hormone-producing
cells in the gall bladder and biliary duct emphasizes further
the liver as a potential source of new cells for treating
diabetes.
It is against this background that, in this issue of the Biochemical
Journal, Mato et al. [15] report that stellate cells present within
explants of pancreas from lactating rats can be induced to
differentiate into insulin-expressing cells. It is not altogether clear
why they used lactating rats, although presumably in these animals
β-cell mass would be increasing to compensate for the increased
metabolic demands during lactation. Their strategy was to select
for cells that expressed ABCG2, a member of the ABC (ATPbinding cassette) superfamily of membrane proteins. Expression
and activity of these transporters is elevated in haemopoietic and
non-haemopoietic stem cells. Their ability to facilitate efflux of
lipophilic, fluorescent DNA-intercalating agents such as Hoechst
33342 has led to their identification in fluorescent cell sorting as
SP (side population) cells that do not retain the fluorescent dye.
Mato et al. [15] generated a pancreatic ABCG2-positive cell line
by selecting for cells that were resistance to the anti-cancer drug
mitoxantrone. The selected cells could be grown for over 2 years
as fibroblast-like cultures that could spontaneously form clusters.
The cells were identified as stellate cells on the basis that they
expressed vimentin, desmin, α-actin, GFAP (glial fibrillary acidic
protein) and exhibited Oil Red O staining of liposoluble material
in the cytoplasm, which was identified as vitamin A on the basis
of its characteristic fading fluorescence.
This is not the first time that stellate cells have been detected
in the pancreas [16]. They have been identified as myofibroblastlike cells that share many features of their hepatocyte counterparts.
They can be activated to proliferate and migrate to sites of tissue
damage, where they synthesize extracellular matrix to promote
tissue repair. Their sustained activation has been associated
with the fibrosis that accompanies chronic pancreatitis and with
pancreatic cancer. The importance of the study by Mato et al.
[15] is that the mitoxantrone-resistant cells could be induced to
differentiate into pancreatic endocrine cells. In basal medium the
cells expressed Pdx1, as well as the stem cells markers nestin and
Thy1.1. The presence of a differentiation cocktail of high glucose,
HGF (hepatocyte growth factor), Betacellulin and nicotinamide
increased the levels of Ngn3. However, the most dramatic effect
was observed with ITS (insulin, transferrin and selenium) and
exendin-4 when the cells were cultured on MatrigelTM for 2 weeks.
Under these conditions the cells expressed Ngn3, NeuroD1,
Pax6, Pax4, Pdx-1, GLUT-2, insulin, IAPP PC1/3, PC2, CK19
and glucagon, with reduced levels of the stem cell markers.
There was no expression of the exocrine marker amylase or of
somatostatin. The differentiated cells exhibited a weak (1.4-fold)
insulin-secretory response to glucose (2.8 cf. 20 mM), although
it is difficult to assess these data, which were expressed as pg per
100 cellular clusters rather than per μg of DNA, and clearly more
detailed studies are required to determine whether this is a robust
e3
effect. Differentiation could also be achieved by overexpression
of exogenous Ngn3, but the effects were weak, suggesting that
Ngn3 alone was not sufficient.
In summary, the study by Mato et al. [15] increases the
number of pancreatic and liver cell types that have been shown to
(trans)differentiate into β-like cells. One of the strengths of the
system is the ability to select a population of cells that can be
characterized in detail. It will be of interest to determine whether
similar mitoxantrone-sensitive cells can be isolated from mouse
pancreas, and thus allow genetic lineage tracing of the ABCG2positive cells using transgenic technology. Also, given the role
of Sonic Hedgehog and Notch [and Wnt (Wingless)] signalling
in pancreatic cancer-initiating cells that exhibit some properties
of stem cells, this may be an ideal model to investigate the
role of these pathways in the maintenance and differentiation of
pancreatic stem cells [17]. It will also be important to determine
how these cells compare with ABCG2-expressing cells that have
previously been identified in cultured islet preparations or with the
CD133+ (a stem-cell marker) cells present in the ductal network
[18–20]. Finally, it would be of interest to determine whether
these cells could be expanded to produce the sufficient quantities
(1 billion) that would be required for therapeutic purposes. They
might even provide some incisive insights that will finally nail the
adult pancreatic stem cell.
FUNDING
The author’s work is supported by the Juvenile Diabetes Research Foundation (JDRF) [grant
number 99-1009-410]; Diabetes U.K. [grant number RD05/003106]; and the Wellcome
Trust [grant number 080241].
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Received 22 May 2009/2 June 2009; accepted 2 June 2009
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c The Authors Journal compilation c 2009 Biochemical Society
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