Hindawi Publishing Corporation
Journal of Transplantation
Volume 2011, Article ID 892453, 15 pages
β-CellGeneration:CanRodent StudiesBeTranslated to Humans?
Franc ¸oise Carlotti,1ArnaudZaldumbide,2Johanne H.Ellenbroek,1H.SiebeSpijker,1
Rob C.Hoeben,2and Eelco J. de Koning1,3
1Department of Nephrology, Leiden University Medical Center, Postal Zone C3-P, P.O. Box 9600, 2300 RC Leiden, The Netherlands
2Department of Molecular Cell Biology, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands
3Hubrecht Institute, 3584 CX Utrecht, The Netherlands
Correspondence should be addressed to Franc ¸oise Carlotti, email@example.com
Received 31 May 2011; Revised 31 July 2011; Accepted 31 July 2011
Academic Editor: Thierry Berney
Copyright © 2011 Franc ¸oise Carlotti et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
β-cell replacement by allogeneic islet transplantation is a promising approach for patients with type 1 diabetes, but the shortage of
organ donors requires new sources of β cells. Islet regeneration in vivo and generation of β-cells ex vivo followed by transplantation
represent attractive therapeutic alternatives to restore the β-cell mass. In this paper, we discuss different postnatal cell types that
have been envisaged as potential sources for future β-cell replacement therapy. The ultimate goal being translation to the clinic,
a particular attention is given to the discrepancies between findings from studies performed in rodents (both ex vivo on primary
cells and in vivo on animal models), when compared with clinical data and studies performed on human cells.
Type 1 diabetes results from the specific destruction of
β-cells by the immune system. The insulin-producing β-
cells are the most abundant cell type residing in the islets
of Langerhans, which are microorgans that are scattered
throughout the exocrine tissue and represent only 1 to 2%
of the total organ mass. β-cell replacement is considered the
best therapeutic option, given the capacity of this particular
cell type to accurately respond to highly variable changes
in blood glucose level and as such to maintain glucose
homeostasis. Islet transplantation via the portal vein of the
liver is a promising approach and considerably less invasive
than total organ transplantation. However, among other
difficulties, the scarcity of donor material will always remain
a major hurdle [1, 2].
Adult β-cell mass is known to be dynamic and to be
able to respond to physiological changes in insulin demand
such as obesity, pregnancy, and starvation. In theory, β-
cell mass adaptation can occur via modification of cell size
(hypertrophy versus atrophy) or modification of cell number
(proliferation of existing mature β-cells or formation of new
β-cells from progenitor cells versus apoptosis).
The origin of newly formed β-cells has long been
debated [3–9], part of the observations reported remaining
controversial and partial. In addition, many statements rely
on studies performed in animal models or animal cell(s)
(lines). As the ultimate goal is translation to the clinic, the
aim of this paper will be to compare rodent and human data
regarding postnatal cell types envisaged as potential sources
to generate β-cells during the past decade.
We will focus on the replication capacity of the β-
cell itself then address the unexpected recent plasticity of
differentiated cell types developmentally close to β-cells.
For many years the dogma was upheld that terminally
differentiated cells were committed to a specific function
and could no longer change their identity. In contrast, pro-
genitor/stem cells are expected to retain some multipotency
capacities and therefore be more suitable for tissue replace-
ment strategies. Nowadays more and more examples of
efficient “transdifferentiation” have been reported, without
an apparent need for a progenitor cell intermediate stage.
Finally, given their various capacities, adult mesenchymal
stem cells (MSC) are increasingly considered for clinical use
in many different applications. We will review the recent
reports about both pancreatic and extrapancreatic sources of
2 Journal of Transplantation
MSC and the possible application of these cells for type 1
Several groups reported on a successful derivation of
both murine and human embryonic stem cells (ESs) to
β-cell-like cells able to revert hyperglycemia in diabetic
mouse models (for detailed review see ). Mimicking
normal pancreatogenesis appears to be the best strategy
to differentiate ES cells toward the endocrine lineage,
although large differences in differentiation capacity exist
between different ES cell lines using similar protocols .
to ES cells when they remain undifferentiated, as well as
ethical principles about any therapeutical use of human ES
cells currently hold back extensive clinical applications of
these cells. Recent developments on technology to generate
induced pluripotent stem cells (iPS) hold great promise [12–
16]. However, safety issues due to genetic and epigenetic
abnormalities during reprogramming or in subsequent cell
culture are a major hurdle for clinical transplantation. A
more immediate application of ES and iPS cells is more likely
cell sources The data of this part are summarized in Table 1.
2.1. β-cells. During adult life, β-cell replication appears to
be a predominant mechanism of β-cell mass expansion
in healthy mice (Figure 1). Dor et al. demonstrated that
major source of new β-cells during adult life and after partial
pancreatectomy . The study relies on a transgenic mouse
model carrying a rat insulin gene promoter-controlled
expression cassette encoding a tamoxifen-dependent CRE
recombinase. A pulse treatment of tamoxifen irreversibly
induced the expression of a reporter gene (human placen-
tal alkaline phosphatase (HPAP)) through CRE-mediated
excision of a STOP sequence specifically in β-cells. Intrigu-
ingly, the percentage of HPAP-positive β-cells immediately
after tamoxifen treatment and 4, 6, 9, and 12 months
later remained very similar, indicating that during normal
turnover in murine β-cells originate exclusively from preex-
or by β-cell ablation using β-cell specific expression of
Diphtheria toxin A resulting in 70% and 80% reduction
in β-cell mass, respectively [18, 19], new β-cells appeared
to be formed largely from genetically labeled preexisting β-
cells and not from neogenesis or from expansion of non-
β-cell precursors. Nevertheless, murine β-cell replication
capacity appears to decline with age [20–22]. Furthermore,
the question was addressed whether all β-cells contribute
equally to growth and maintenance of β-cell mass or if
distinct subpopulations exist. Label-retaining techniques
were applied. Brennand et al. performed an in vivo pulse-
chase labeling experiment using Histone 2B-GFP as reporter
was observed. Next, a clonal analysis of dividing β-cells
Table 1: Potential sources of de novo β-cells among differentiated
adult cell types.
in vitro in vivo in vitroin vivo
Figure 1: Section of murine pancreas stained with anti-insulin
(red) and anti-BrdU (brown) antibodies. C57BL/6 mice were on
a high-fat diet for 6 weeks. BrdU was injected for one week by
s.c. injections, thereby labeling all proliferating cells. A number of
replicating β-cells (arrows) and acinar cells are detected.
revealed that all clones were of similar sizes. Altogether this
suggests that, in mice, the pool of β-cells is homogenous
and all cells are able to replicate at the same rate. Therefore
all β-cells can be candidate for in vitro expansion. Similar
conclusions were obtained from a parallel study, in which
a DNA analog-based lineage tracing method was developed
to detect sequential cell division in vivo . Recently Dor
and colleagues further investigated the replication dynamics
of adult murine β-cells and showed that replicated β-cells are
able to reenter the cell division cycle shortly after mitosis.
This short quiescence period of several days was found to
be lengthened with advanced age  and shortened during
injury-driven β-cell regeneration and following treatment
data of this part are summarized in Table 1.
In humans, Meier et al. investigated the β-cell mass from
infancy to adulthood by performing immunohistochemistry
and morphometric analyses on pancreas obtained at autopsy
from 46 donors (from 2 weeks to 21 years of age) and
determined pancreas volume by CT scan in 135 individ-
uals of similar age . The authors concluded that the
predominant expansion of β-cell mass in humans occurs in
early childhood (2.6% of β-cells positive for Ki67), without
secondary growth phase of β-cell mass during adolescence,
in contrast to rodents. Interestingly, the islets appear to
increase in size rather than in number. Moreover, although
the authors identified some β-cells (both individual cells
Journal of Transplantation3
and in small clusters) near the ducts, the number of these
insulin-positive cells increased to a similar extent as the
overall expansion of β-cell numbers in the islets, consistent
with the hypothesis of β-cell replication. It is important to
note that an efficient β-cell mass regeneration approach in a
therapeutic perspective would require a similar rate of β-cell
replication. Other studies confirmed these results. Kassem
et al. reported that β-cell replication decreased progressively
from 3.2% at 17–32 weeks of gestation to 1.1% perinatally
. After birth, levels of β-cell replication would drop
further to reach less than 0.1% in young adults . These
data also correlate with the findings that β-cell mass is
established by the first two or three decades of human
life as determined by measuring accumulation of lipofuscin
bodies as a marker to estimate β-cell longevity  or by
in vivo thymidine analog incorporation and radiocarbon
dating . Remarkably, the adaptive increase of β-cell
mass in adult humans appears to be modest in response to
obesity (2- versus 10-fold) , as well as during pregnancy
(1.4 fold versus 2 to 5-fold) compared to rodents [33, 34].
In contrast to rodents again, in both obese subjects and
pregnant women, the adaptive increase in β-cell number
was accompanied by an increased number of small new
islets, indicative of neogenesis (cf. paragraph 2.3), rather
than an increase in islet size or number of β-cells per islet,
on human pancreatic tissue collected from 13 patients who
underwent a partial pancreatectomy showed that, unlike in
rodents, a 50% pancreatectomy does not trigger any β-cell
regeneration in adult humans. This corroborates with the
high incidence of diabetes after partial pancreatectomy .
In summary, in vivo β-cell replication capacity in humans
appears to be mostly limited to the very early postnatal
period, and the triggers of such a process are still unknown.
Data available on β-cell proliferation in vitro are very
limited when restricted to primary β-cells. An early report
claimed that human β-cells were able to replicate efficiently
(69% of BrdU/insulin double-positive cells) when exposed
to a specific matrix (matrix produced by the rat bladder
carcinoma cell line 804G) in the presence of hepatocyte
growth factor/scatter factor (HGF/SF) . However HGF
induced a rapid decrease in insulin content . This work
was rapidly challenged by another report showing that the
defined culture conditions were favorable for replication of
ductal cells and not for β-cells . In a comparative study
between in vitro proliferation of purified human and rat β-
cells, Parnaud et al. also failed to detect any β-cells that were
of exposure . However a clear proliferation of purified
rat β-cells was observed and could be further enhanced by
cells in (intact) isolated human islets was also assessed in the
same study and remained undetectable. Therefore it appears
that culture conditions for efficient human β-cell replication
ex vivo have not been clearly identified.
2.2. α Cells. Differentiation of endocrine non-β-cells to β-
cells is an interesting alternative mechanism for increasing
the β-cell mass. A limited number of studies supported this
new concept. Collombat et al. showed in various transgenic
mouse models that forced expression of Pax4 at different
stages (in pancreatic progenitor cells, endocrine precursor
cells, or in mature α cells) resulted in a shift of all endocrine
lineages toward a β-cell fate . A bicistronic vector (Pax4-
IRES-β-galactosidase) was used in order to follow cells of
interest by staining for β-galactosidase activity. Importantly,
the authors observed an age-dependent increase in islet size
and in number of insulin/β-galactosidase double-positive
cells and a concomitant decrease in α-cell content. These
observations indicate that ectopic Pax4 expression can also
force conversion of adult glucagon-expressing cells into β-
cells. Remarkably, the subsequent decrease in glucagon was
found to activate the differentiation of ducts-associated
progenitor cells α cells (via an intermediate stage of Ngn3
positive cells). However the newly formed α cells failed
to correct the hypoglucagonemia since they were shown
to be rapidly converted into β-cells upon Pax4 ectopic
expression. Notably, the expression of Pax4 in glucagon-
positive cells has been reported to be sufficient to restore
a functional β-cell mass in diabetic mice, although only
in the young animals. Thorel et al. developed a transgenic
mouse model of near total β-cell ablation that relies on
the specific expression of Diphtheria Toxin Receptor (DTR)
in pancreatic β-cells (expression driven by the rat insulin
promoter) . After administration of Diphtheria Toxin, a
rapid and extreme (>99%) β-cell destruction by apoptosis
was observed resulting in a characteristic diabetes within
a couple of weeks. If given insulin, the mice survived
and showed a slow β-cell mass regeneration. Only after 5
months the regenerated β-cell mass was able to maintain
glucose homeostasis without exogenous insulin administra-
tion. Using a doxycycline inducible α-cell lineage tracing
system, the authors established that one month after β-
cell ablation, a large but variable (32 to 81%) fraction of
newly formed β-cells resulted from the transdifferentiation
of about 5 to 10% of α cells. Importantly, almost all (∼90%)
were bihormonal (still expressing glucagon even as far as
10 months after ablation). Of note, no β-cells were found
in extrainsular locations. Therefore, in contrast to previous
studies , the β-cell mass regeneration was attributed to
neogenesis through α-cell transdifferentiation, rather than to
the slow self-replication mechanism of preexisting mature
β-cells. The authors suggested that the amount of β-cell
loss and the type of injury would determine the mechanism
of regeneration. This theory of unexpected pancreatic cell
plasticity triggered by extreme β-cell loss was supported by a
used was a combination of pancreatic duct ligation (PDL)
with specific elimination of preexisting β-cells by alloxan
. No lineage tracing method was used in this work,
and results are based on immunohistochemistry stainings
performed at 7 and 14 days. The authors reported a rapid
regeneration of β-cell mass within weeks and suggested that
the newly formed β-cells resulted mainly from conversion
of adult α cells. Interestingly the authors also observed
that under injury conditions, α cells were able to replicate.
However no α-cell division was required for conversion
into β-cells. Importantly the regenerated β-cell mass was
4 Journal of Transplantation
not sufficient to revert hyperglycemia, possibly due to the
persistent inflammation caused by the PDL. In summary,
three independent studies performed in transgenic mouse
models showed that α- to β-cell conversion can occur.
One could wonder how this relates with the developmental
process. In rodents, the glucagon gene is expressed in the
earliest endocrine cells that can be detected . However,
Herrera et al. demonstrated that, during murine embryo-
genesis, mature glucagon- and insulin-producing cells share
a common precursor but belong to separate developmental
In human cells, the embryologic situation appears to
be different, since insulin-positive cells are the first to
be detected during development . Therefore it is not
known whether this extraordinary islet cell plasticity exists
in adult human α cells in vitro or in vivo. If so, α cells,
especially if their capacity to replicate under injury condition
is confirmed, could be an ideal intraislet source for in vivo
regeneration of β-cells.
2.3. Ductal Cells. Pancreatic ductal cells have long been
thought to be the main source for progenitor cells in the
pancreas (cf. reviews [3, 8]).
Several rodent studies based on immunohistochemical
observations in animal models, suggested a possible mecha-
nism of islet neogenesis via recapitulation ofthe embryologi-
cal development after activation and differentiation of ductal
progenitors [46–50]. Recently a number of lineage tracing
studies in transgenic mouse models have been performed in
order to identify putative endocrine progenitors in adults,
but provided contradictory conclusions. Bonner-Weir and
collaborators made use of the human carbonic anhydrase II
bined to a tamoxifen inducible CRE-ER/Lox recombination
system . CAII-expressing cells within the pancreas acting
as precursor cells gave rise to both pancreatic endocrine and
β-cells were labeled. The authors proposed that ductal cells
represent a pool of homogenous cells from which a fraction
can dedifferentiate into progenitor cells that are able to
regenerate both endocrine and exocrine tissues. In a parallel
study, Heimberg and colleagues suggested a slightly different
model: a rare subpopulation of endocrine progenitor cells,
located in the ductal lining, could be activated upon PDL in
adult mouse pancreas and subsequently started to express an
embryonic key endocrine developmental factor, Ngn3 .
Differentiation of an Ngn3-positive subpopulation gave rise
to all islet cell types, including glucose responsive β-cells
both in situ and when cultured in embryonic pancreatic
explants. However, in a subsequent study a lineage tracing
of HNF1β-positive ductal cells was performed. 65% of
pancreatic ductal cells were labeled. The authors show
that the ductal epithelium does not make a significant
contribution to acinar or endocrine cells during neonatal
growth (6-month observation period) or upon regeneration
condition (PDL or Alloxan followed by EGF/gastrin treat-
ment) . Of interest, earlier lineage studies by Melton’s
group pointed out on the heterogeneity in developmental
potential among “duct-like structures” in early embryo .
Figure 2: Section of human pancreatic exocrine tissue stained
with an anti-insulin antibody (red). Nuclei are stained by DAPI
(blue). An insulin-positive cell (arrow) is detected in the duct lining
suggesting a process of β-cell neogenesis.
Lineage tracing experiments suggested that Ngn3-positive
cells are indeed islet progenitors but distinct from duct
progenitors. On the other hand, the authors could not rule
out the possibility that a minor population of mature ductal
cells is able to transiently activate Ngn3 gene expression
and subsequently contributes to islets neogenesis. Lineage
tracing of Muc1 ductal/acinar cells confirmed that Muc1-
positive cells give rise to endocrine cells in utero, in line
with embryological development . However, after birth,
Muc1 lineage-labeled cells were found to be restricted to
the exocrine compartment, with no detectable contribution
to islet cells. No injury or regeneration models have been
tested in that particular model. Along the same line, another
in early pancreatic progenitors) and resulting in 70% of
labeled pancreatic ductal cells established that very few
nonendocrine cells continue to arise from Sox9-positive
precursors in early postnatal life, but no endocrine or
acinar cell neogenesis from Sox9-positive cells occurs during
adulthood . Intriguingly, in contrast with an earlier
study , the authors found that after PDL, Sox9-positive
cells give rise to Ngn3-positive cells but these cells do not
contribute to islets. Thus, although the function of ductal
cells as endocrine progenitors in embryonic development
is commonly accepted, more insight is needed into the
potential of cells from the ductal compartment as an
alternative source for the formation of new β-cells in adult
Immunohistochemistry stainings performed on tissues
obtained at autopsy or after pancreatectomy are obviously
the only possible technique to evaluate the β-cell mass
in humans. The occurrence of neogenesis is suggested by
the presence of insulin-positive cells in the ductal area
(ruling out the expansion of a preexisting insulin-positive
repeated observations in different contexts can result in
strong indications. Meier et al. analyzed pancreatic tissues
obtained at autopsy from 46 children aged from 2 weeks to
21 years and observed about 0.5% of insulin-positive ductal
cells . A similar percentage has been reported by Reers
Journal of Transplantation5
et al. that studied pancreatic tissues from 20 donors aged
from 7 to 66 years . The rate of islet neogenesis does
not seem to be affected by aging. Importantly, Butler et al.
observed that ductal cells positive for insulin were increased
by 2- and 3-fold during obesity and pregnancy, respectively
[32, 33]. Therefore it appears that in humans islet neogenesis
from ductal cells is stimulated during metabolic situations
that require an increase in insulin level. It should be noted
of β-cell mass is much more limited in humans compared
to rodents. In pathophysiological condition such as chronic
pancreatitis, Phillipset al.alsoobserved a significant increase
of insulin-positive ducts in adult human pancreas of 11
patients compared to control group . In another study,
the question was addressed whether β-cell neogenesis occurs
in the transplanted pancreas of type 1 diabetic patients
(SPK) . Pancreatic tissues from 9 SPK patients and 16
nondiabetic organ donors were examined by immunohisto-
chemistry. Remarkably, high numbers 33 to 90% of ductal
cells were found to be insulin positive, and 17% to 95% of
the ducts harboured insulin-positive cells in SPK patients
with recurrent autoimmunity and diabetes. A high degree of
neogenesis was observed in patients with the most severe β-
cell loss, whereas a low degree of neogenesis was observed
in normoglycaemic patients, suggesting that in the particular
inflammation may strongly stimulate β-cell neogenesis from
ductal cells. Intriguingly, a study by Bouwens et al. analyzing
pancreatic tissues from nine adult donors revealed that 15%
of all β-cells were located in units with a diameter less than
20 mm and without associated glucagon-, somatostatin-, or
pancreatic polypeptide cells . These single β-cell units
were situated in or along ductules, from which they appear
to bud as previously noticed in fetal and neonatal pancreas.
Furthermore, simultaneous presence of Ki67-positive ductal
cells (0.05%) and absence of Ki67-immunoreactive budding
β-cells suggested that β-cell neogenesis depends on ductal
cell proliferation and differentiation in humans.
About ten years ago murine ductal cells cultured in
vitro were reported to form 3D clusters that differentiate
to functional islet cells, which are able to respond to a
glucose challenge and to reverse diabetes in mice . An
interesting strategy for the prospective isolation of putative
progenitors from an enriched ductal cell population is
also being pursued by Taniguchi and colleagues [61, 62].
The approach combines immunohistochemical analysis of
mouse pancreas to define new phenotypic markers and flow
cytometry cell sorting to isolate clonal cell populations that
are able to differentiate toward the endocrine lineage in vitro
or in vivo. The data suggest that a population of progenitor
cells was present among CD133-positive ductal cells.
Koblas et al. confirmed that a subpopulation of CD133-
positive cells in human islet-depleted tissue was able to
differentiate into functional insulin-producing cells in vitro
and to secrete insulin in a glucose-dependent manner
. Furthermore, Bonner-Weir and collaborators showed
that human primary ductal cells could be isolated from
islet-depleted pancreatic tissue, expanded in culture, and
triggered to differentiate towards glucose responsive islet-
like clusters . These results were confirmed by Gao et
al. who further characterized the nature of these pancreatic
progenitor cells . During monolayer expansion, two
subpopulations of proliferating cells were observed, CK19-
positive ductal cells at an early time point (day 3) and
nestin-positive cells at a later time point (day 7). Under
serum-free conditions and Matrigel covering of the cells,
the CK19-positive cells, but not the Nestin-positive cells,
were able to form islet-like clusters that contain insulin- and
glucagon-positive cells. When transplanted under the kidney
capsule of nude mice, one out of five grafts demonstrated
further growth with foci of both endocrine and exocrine
cells. Next, Bonner-Weir and colleagues used magnetic cell
sorting and antibodies raised against the ductal surface
marker CA19-9 to isolate ductal cells from islet-depleted
tissue . Transplantation experiments of purified ductal
cells versus unpurified preparations (56% CK19-positive
cells only) into normoglycemic NOD/SCID mice revealed
that differentiation of ductal cells to insulin-producing cells
was dependent on the presence of nonductal cells, probably
pancreatic stromal cells as suggested by the authors. Of
for human cells [67, 68].
Although some lineage tracing studies in rodents have
provided contradictory results, most in vivo and in vitro data
from both human studies indicate that cells from the ductal
compartment are an attractive putative cell source for β-cell
2.4. Acinar Cells. Besides the ductal origin hypothesis, a
number of studies show that acinar cells might also display
a certain degree of plasticity.
Melton and colleagues suggested that acinar cells and
endocrine cells share a common progenitor after the ductal
cell lineage has already separated . On the other hand,
Bouwens and colleagues proposed a variant of the ductal
origin hypothesis, in which acinar cells would be able to
transdifferentiate towards β-cells, through an intermediate
dedifferentiated ductal-like stage [49, 69]. This model was
supported by more reports. β-cell mass regeneration has
been studied after streptozotocin treatment in a transgenic
mouse model expressing interferon gamma under the con-
trol of the insulin promoter . New β-cells appeared
to result primarily from the formation of new islets from
small pancreatic ducts. However, interestingly, some putative
transitional cells were identified harboring both exocrine
and endocrine granules, indicative of acinar cells as possible
precursor cells. Similar observations were made in a parallel
study after PDL . Furthermore, in order to visualize
better possible intermediate stages during acinar-to-ductal
transdifferentiation in a PDL injury model, Lardon et al.
took advantage of the fact that dexamethasone treatment
inhibits the loss of amylase from acinar cells . Puta-
tive transitional cells coexpressing acinus-specific (amylase)
and duct-specific (CK20) markers were identified in vivo.
Furthermore, acinus-to-islet conversion was confirmed in
vitro after isolation of acini and identification of putative
transitional cells coexpressing acinus-specific (amylase) and
6 Journal of Transplantation
β-cell-specific (insulin) markers. Several groups recently
performed some in vivo lineage tracing analyses using
of preexisting acinar cells is seen as the major mechanism
for regeneration of the acinar tissue. Moreover, acinus-
to-duct transdifferentiation has been reported to occur in
vivo, although at a very low frequency, in mouse models
for pancreatitis , and in a mouse model that devel-
ops insulin-positive cell-containing hyperplastic ducts in
response to the growth factor TGFα . However, the same
authors also showed that the insulin positive cells adjacent
to acinus-derived ductal cells arose from preexisting insulin-
positive cells and not from acinar cells. Along the same
line, Stoffers and collaborator failed to observe any acinus-
to-β-cell transdifferentiation in adult mice under normal
induced pancreatitis . On the other hand, Melton et al.
reported that adenovirus-mediated coexpression of Pdx1,
Ngn3, and MafA in vivo in pancreas from adult mice
was sufficient to induce the transdifferentiation of mature
exocrine cells into β-cells that are indistinguishable from
endogenous islet β-cells in size, shape, and ultrastructure
. However the question remains open whether acinar
cells or other possible precursor cells were reprogrammed in
In vitro, rat exocrine cells treated with dexamethasone
can convert to hepatocyte-like cells. In contrast, when
cultured in low serum medium (1%) in the presence of EGF
and LIF, rat exocrine cells can transdifferentiate to functional
β-cells . In that particular study, 10% of dedifferentiated
acinar cells expressed insulin, with a total insulin content
of 40 to 90% of primary β-cells, and transplantation of
about 100,000 of these insulin-positive cells was sufficient
to revert hyperglycemia in a diabetic nude mouse model. In
an independent study, Minami et al. showed that suspension
culture in presence of EGF and nicotinamide converted 5%
of adult murine acinar cells to glucose responsive insulin-
tracing study using the acinus-specific amylase or elastase
promoter confirmed the identity of the starting population.
In addition, acinus-to-duct transdifferentiation was shown
to occur, in response to EGF-receptor signalling, through an
intermediate nestin-positive stage in an in vitro culture of
pancreatic explants .
Regarding human cells, there are no data available
about a possible acinus-to-islet cell plasticity. One possible
explanation is the selective death by apoptosis of human
acinar cells when cultured in vitro. In contrast, human
ductal cells can survive, adhere to plastic culture dish, and
proliferate . Nevertheless the possible acinus-to-duct
transdifferentiation suggested by others  cannot be ruled
out in this study.
2.5. Liver Cells. Liver and pancreas have a common embry-
ological origin, both arising from the primitive gut. There-
fore liver cells have been hypothesized to constitute a
potential cell source for β-cell generation .
Ferber and collaborators demonstrated that an adeno-
virus-mediated expression of the key pancreatic transcrip-
tion factor Pdx1 in mouse liver (intravenous infusion)
resulted in transdifferentiation of hepatocytes to β-cell-like
cells . Pdx1 expression found in 60% of hepatocytes
resulted in a 3-fold increase of plasma insulin levels, 59%
as insulin, and 41% as proinsulin. These data indicate
that proinsulin was processed which was substantiated by
expression of the prohormone convertase 1/3. The amount
a diabetic mouse model. A similar approach was successfully
reported with adenovirus-mediated expression of Pdx1
or Ngn3  and the coexpression of NeuroD1/Beta2
and betacellulin  in the liver. Of note, although the
overexpression of Pdx1 was able to revert the elevated
blood glucose of diabetic mice, the animals died from liver
inflammation most likely due to the exocrine-differentiating
activity of Pdx1. Interestingly Yechoor et al. showed that
gene transfer of a pancreatic key transcription factor (Ngn3
in this study) in liver leads to long-term diabetes reversal in
mice. Howevertheauthorsdemonstratedthat, although
insulin expression was transiently induced in terminally
differentiated hepatocytes, the long-term diabetes reversal
obtained in these mice was resulting from the differentiation
of hepatic progenitors able to generate islet-like clusters. The
phenomenon was described as “transdetermination,” that is,
lineage switching in lineage-determined, but not terminally
Human adult liver cells were shown to expand in vitro
and to transdifferentiate towards an endocrine pancreatic
lineage after Pdx1 overexpression . Pdx1-expressing
human liver cells were found to express insulin that
is stored in secretory granules, which are released in a
glucose-regulated manner. When transplanted under the
kidney capsule of diabetic immunodeficient mice, these
cells ameliorated hyperglycemia for prolonged periods of
time. Similar studies using human fetal progenitor liver cells
were reported [88, 89]. Since harvesting and propagating
significant numbers of primary hepatocytes from patients
with diabetes would be theoretically feasible, the liver can be
considered as an interesting extrapancreatic source for β-cell
Mesenchymal stem cells (MSC) were originally identified
in bone marrow by Friedenstein in 1976 as a rare, het-
erogeneous, non hematopoietic, and multipotent stromal
population able to differentiate to mesenchymal lineages
including bone, fat, and cartilage. MSC are virtually present
in all organs, including the pancreas and the islets of
of these cells in the body . Interestingly, MSC can be
obtained from live donors (and potentially from the patient
itself) and are easily expandable in vitro. Therefore, despite
the fact that their identity and their exact role in vivo have
Journal of Transplantation7
Table 2: Potential sources of de novo β-cells among differentiated
adult MSC: BM: bone marrow, AT: adipose tissue; UCB: umbilical
in vitroin vivoin vitro in vivo
not been clearly defined yet, mesenchymal stem cells have
been envisaged for a broad range of therapeutic applications
including type 1 diabetes [91, 92]. The data of this part are
summarized in Table 2.
3.1. Islet(-Derived) MSC. We and others reported on
the expansion of MSC-like cell population from isolated
human islets: human islet-derived precursor cells (IPC)
[93–95], NIP/Nestin-positive Islet-derived Progenitors ,
PIDM/Pancreatic Islet-Derived Mesenchymal cells ,
PHID/Proliferating Human Islet-Derived cells , and
more [99, 100]. These cells are able to proliferate ex vivo and
can be passaged. Interestingly, most of the groups reported
a common characteristic, which is aggregation into clusters
ranging in sizes between 50 and 200μm (similar range as
primary islets) under serum starvation and a subsequent
increase in expression of endocrine markers. However others
failed to reproduce these data of partial differentiation
[101, 102]. In any case, the endocrine markers remained
at very low level when compared to freshly isolated human
islets. Davani et al. reported a further capacity of IPC to
differentiate into functional β-cells that secrete human C-
peptide in response to glucose after reimplantation of 4-day-
old aggregates in mice .
The origin of these cells remains elusive and very
controversial. Initially Gershengorn and colleagues proposed
an Epithelial-to-Mesenchymal Transition (EMT) to occur
from human β-cells . However this idea was rapidly
refuted by the same group and others after lineage tracing
experiments performed in transgenic mouse models that
failed to show any EMT of murine β-cells [103–106].
The overall conclusion of these four studies was that the
proliferative cell population derived from cultured murine
islets was not originating from β-cells,since no β-cellspecific
markers were identified in these cells. Efrat and collaborators
recently developed a lineage tracing system similar to the
techniques applied in transgenic mouse models, but now
applied to human cells in vitro using lentiviral vectors .
The dual viral system relies on the β-cell specific expression
of the CRE recombinase in one vector and a CMV-GFP
reporter vector in another vector in which GFP expression is
restricted by a “floxed” intermediate sequence. As lentiviral
vectors integrate into the genome of the transduced cells, the
reporter gene will remain expressed in all cells originating
from the initial pool of labeled cells. In their follow-up study,
Russ et al. slightly modified the system by using a tamoxifen-
inducible CRE/ER recombinase, restricting the labeling
. Human β-cells were efficiently (50%) and specifically
provided evidence that human β-cells can dedifferentiate to
an MSC-like cell population and proliferate when cultured
ex vivo, in contrast to mouse β-cells, as revealed earlier from
the transgenic mice studies [103–106]. Intriguingly, 40% of
cells exhibiting MSC markers in culture (likely to be the
cell population previously named IPC, NIP, PIDM, or PHID
by others) resulted from an EMT of β-cells. On the other
hand, several groups suggested, but never unequivocally
demonstrated, the presence of a mesenchymal stem cell
we investigated the presence of MSC(-like cells) in freshly
isolated human islets, and we identified a double-positive
CD90/CD105 population representing approximately 2%
of the total islet cell population. The presence of these
cells inside freshly isolated human islets was confirmed
by confocal microscopy . An independent study val-
idated the presence of pancreatic MSC in the periacinar,
perivascular, and periductal space of human pancreas .
The functional significance of the presence of these cells
in the islets and the possible interplay between islet-MSC,
endocrine cells, and the vascular system in human islets
remain to be further clarified. Altogether these data suggest
that the MSC-like cell population derived from human
islets in culture results from subpopulations of at least
two origins: proliferation of islet-MSC and proliferation of
dedifferentiated β-cells. More studies, in particular detailed
lineage-tracing experiments will be needed to confirm this
At the moment it remains unclear whether all mesenchy-
mal stem(-like) cells are equal and whether MSC originating
from islets would be more prone to differentiate toward
the endocrine lineage. Two groups determined the gene
expression profiles in human islet-derived MSC [110, 111].
of the population with the archetypical bone marrow-
MSC. Remarkably cultured islet-derived IPCs are different
from bone marrow-MSC (BM-MSC) in that they express
a set of islet-specific genes, although at low level. Upon
differentiation, following the rather basic differentiation
protocols developed so far for these cells, gene expression
data showed that IPCs are able to go further along the
endocrine pathways than BM-MSC.
In summary, islet-(derived) MSC could be of a valuable
therapeutic significance since they appear to retain some
genetic characteristic making them closer to endocrine
cells than other sources of MSC such as bone-marrow-
derived MSC. Further studies will be needed to verify this
can dedifferentiate ex vivo into a mesenchymal phenotype in
contrast to murine β-cells illustrates again the discrepancy in
plasticity of these cells between rodents and humans.
8 Journal of Transplantation
3.2. Exocrine/Islet Depleted Tissue(-Derived) MSC. Similarly
to human islet cells cultured ex vivo, two independent
studies reported that a population of MSC(-like) cells could
be derived from human pancreatic exocrine tissue. Under
defined conditions, these cells could show some sign of
differentiation toward the endocrine lineage [112, 113].
The exact origin of these cells remains also unclear.
On the one hand, epithelial-to-mesenchymal transition
from exocrine cells has been proposed by Seeberger et
al. . Along the same line, Fanjul et al. described
coexpression of ductal markers and mesenchymal markers
cells in one pancreas from a nondiabetic subject and three
pancreata from patients with type 2 diabetes . Shin
et al. demonstrated that the transdifferentiation capacity of
human ductal cells was reduced after EMT . On the
other hand, Sordi et al. suggested that the mesenchymal stem
cell population growing out of cultured pancreatic tissue
(both endocrine and exocrine fractions were tested) would
be mostly originating from proliferating “pancreatic MSC”
that are partly derived from the bone marrow . Total
bone marrow transplantations from donor GFP transgenic
mice in lethally irradiated recipient mice were performed.
Surprisingly, after 12 weeks, GFP-labeled bone-marrow-
derived cells were found to be localized preferentially in two
organs, pancreas (4.82 ± 4% of total) and lung (4.43 ± 2.3%
of total). 18.5 ± 4% of MSC derived in culture from these
pancreata were found to be GFP positive.
However once again, in absence of data on pancreatic
tissues after bone marrow transplantation in humans, we
can wonder how these results would be translatable to the
human situation. In a study of a pancreas allograft removed
8 months after transplantation, it was found that part of
pancreatic MSC expressed recipient HLA, suggesting an
extrapancreatic origin of these cells .
Besides their putative differentiation capacity, MSC dis-
play very interesting additional characteristics. Sordi et al.
and exocrine tissue and cotransplanted in mice with a
minimal pancreatic islet mass facilitated the restoration of
normoglycemia and neovascularization of the islet graft
tissue but show limited capacity of differentiation towards
the endocrine pathway. Similarly to islet(-derived) MSC, the
origin and role of these cells are unclear.
3.3. Extrapancreatic Sources of MSC. Bone marrow, adipose
tissue, and umbilical cord blood are the main sources of
BM-MSC reported so far. Hess et al. reported that trans-
plantation of murine bone marrow cells in streptozotocin-
treated mice was able to reduce hyperglycemia by initiating
endogenous pancreatic regeneration. A majority of bone-
marrow-derived cells were found to be localized near ductal
and islet structures. Quantitative analysis of the pancreas
revealed a very low frequency of donor insulin-positive cells.
However the presence of donor cells was accompanied by
a rapid proliferation of recipient pancreatic β-cells and by
neogenesis of insulin-positive cells of recipient origin within
a week after transplantation. The mechanism behind this
regeneration process remains to be clarified. The authors
suggested that bone-marrow-derived endothelial cells could
be involved by secreting factors that enhance tissue repair
. This model was confirmed by others . Along
the same line, Lechner et al. observed no evidence for
significant transdifferentiation of labeled (GFP transgenic
mice) murine bone marrow into pancreatic β-cells in vivo
. By using a mouse model for impaired bone-marrow-
derived cell mobilization (Nos3 −/− mice), Hasegawa et
al. demonstrated that homing of donor bone-marrow-
derived cells in recipient bone marrow and subsequent
mobilization into the injured periphery were required for
β-cell regeneration. Interestingly, simple bone marrow cell
infusion without preirradiation had no effects, suggesting
that injury signals are involved in triggering this process
. Finally, in similar transplantation experiments, Urban
et al. revealed that murine-bone-marrow derived mesenchy-
bone marrow cells nor MSC transplantation was effective
alone . In contrast to all these studies and using a
lineage tracing system (Ins/CRE-LoxP/EGFP), Ianus et al.
claimed that transplanted murine bone marrow cells were
able to differentiate themselves into functional β-cells .
Four to six weeks after transplantation from male mice
into lethally irradiated recipient female mice, recipient mice
contained Y chromosome and EGFP double-positive cells in
their pancreatic islets. Of note, β-cell specificity was verified
since neither bone marrow cells nor circulating peripheral
blood nucleated cells of donor or recipient mice had any
detectable EGFP. Cell fusion was also ruled out in this
Regarding human cells, Prockop and colleagues evalu-
ated the competence of bone marrow MSC in a similar type
of experiment. Human bone marrow MSC were delivered
via intracardiac infusions in diabetic NOD/SCID mice.
Although rare β-cells were found to be of human origin
(i.e., BM-MSC derived), blood glucose levels were found
to be decreased after some weeks. The authors suggested
that human BM-MSC were able to home to and promote
repair of pancreatic islets and renal glomeruli in a dia-
betic mouse model . In a parallel study, Sordi et al.
showed that human BM-MSC express a restricted set of
functionally active chemokine receptors (CXCR4, CX3CR1,
CXCR6, CCR1, CCR7) capable of promoting migration to
pancreatic islets . Butler and collaborators studied 31
human pancreases obtained at autopsy from patients who
had received a bone marrow-derived graft (26 cases), a
peripheral blood-derived graft (4 cases), or a combination
of both peripheral blood- and bone marrow-derived stem
cells (1 case) . More than 4000 islets were examined
in this relatively large cohort, and no pancreatic β-cells
were found to be derived from donor cells (including
two cases of patients with type 2 diabetes). Therefore, it
appears that in humans the bone marrow compartment does
not contribute to pancreatic β-cell mass maintenance in
healthy individuals. Nevertheless promising clinical results
on glucose metabolism were recently reported. A clinical
trial in 11 patients with type 1 diabetes was designed to
Journal of Transplantation9
test the safety and efficacy of intraportal coinfusion of
insulin-producing adipose tissue-derived MSC and bone
marrow cells. Differentiation of adipose tissue-derived MSC
was initiated in vitro by a 3-day culture period in a
defined medium described earlier . A mean follow-
up of about two years showed significant improvements
of all clinical parameters related to diabetes (a decrease
in insulin requirements, an increase in C-peptide levels,
and absence of diabetic ketoacidosis) . In a clinical
trial involving 25 patients with type 2 diabetes, Ricordi
and collaborators observed reduced insulin requirements
and significant improvements of all metabolic variables
(12-month follow-up) after an intrapancreatic infusion of
autologous bone marrow cells . However no data are
available about the possible differentiation of MSC after
implantation, and it is more likely that improved glucose
metabolism is related to paracrine effects of MSC in this last
In vitro, several groups reported the successful derivation
of functional β-cells from rodent bone marrow cells in the
presence or absence of serum  and with addition of
growth factors like nicotinamide and β-mercaptoethanol
 and conophylline and betacellulin-delta4 . After
transplantation these cells can (partly) revert hyperglycemia
in diabetic mice. Also murine adipose tissue-derived MSC
could be efficiently converted reaching up to 48% of
cells that expressed c-peptide, following a 3-stage and 10-
day differentiation protocol involving activin A, sodium
butyrate, β-mercaptoethanol, taurine, GLP-1, nicotinamide,
and nonessential amino acids .
The approach is slightly different in human cells, involv-
ing the combination of defined medium and virus-mediated
ectopic expression of proendocrine transcription factors.
Karnieli et al. reported that overexpression of Pdx1 in BM-
MSC from 9 of 14 donors can trigger their differentiation to
a β-cell-like phenotype displaying about 1% of the regular
insulin content and able to control the insulin release
in a glucose-dependent manner in vitro . The cells
lacked expression of Beta2/NeuroD1. However transplan-
tation into streptozotocin-treated mice resulted in further
differentiation, including induction of Beta2/NeuroD1 and
reduction of hyperglycemia. Similar results were obtained in
a parallel study . Human MSC from other sources than
bone marrow were also evaluated. MSC isolated from the
Wharton’s jelly of the umbilical cord were differentiated to
islet-like cell clusters through stepwise culturing in neuron-
conditioned medium . The clusters were found to
express islet-specific genes and to be glucose responsive in
vitro. Functionality was further verified after transplantation
into the liver of streptozotocin-induced diabetic rats via
was observed by electron microscopy 12 weeks after trans-
In summary, the capacity of extrapancreatic mesenchy-
mal stem cells to differentiate to β-cells in vivo appears
to be very limited. Nevertheless, several groups reported
a successful differentiation to functional β-cell-like cells in
vitro especially from human MSC. MSC appear to display
unique migratory and secretory properties (growth factors,
cytokines) that make them attractive as “helper” cells for
tissue repair (improve engraftment, viability, function) (for
review see [92, 136]).
A crucial aspect common to all putative cell sources will be
to uncover the mechanisms that preserve and control cell
identity in order to enable successful manipulation of adult
cell plasticity in clinical settings. Up to now, most efforts to
sion (overexpression/downregulation of key transcription
factors) and growth-factor-mediated activation of specific
signaling pathways. A new era has started aiming at better
understanding the processes that regulate gene expression.
Chromatin accessibility is a determining factor blocking
or facilitating expression of specific genes. Cell identity
is regulated by epigenetic factors that tightly regulate the
activation or repression of genes including genomic DNA
methylation, histone modifications, and noncoding RNA
regulation (for review please see [137, 138]). Dhawan et
al. recently demonstrated that pancreatic β-cell identity is
maintained by DNA methylation-mediated repression of
Arx . The question whether all MSC(-like cells) are
equal was recently addressed. Mutskov et al. investigated the
patterns of histone modifications over the insulin gene in
human islets and IPC (the MSC-like population derived ex
vivo from human islets) compared to HeLa and BM-MSC
. Although neither IPC nor HeLa nor BM-MSC express
insulin, IPC showed significant levels of active chromatin
modifications, similarly to human islets although at a more
moderate level. The probable multiorigin of the IPC might
obscure the interpretation of these results. However these
epigenetic marks absent in the unrelated cell types (HeLa
and BM-MSC) might be part of a general mechanism
whereby tissue-derived precursor/stem cells are committed
to a distinct specification. Non-coding RNA (such as siRNA
(long non-coding RNA)) are emerging as key players in
regulation of development . Joglekar et al. demon-
strated that the miR-30 family of miRNAs contribute to the
regulation of the dedifferentiation of human fetal β-cells
through epithelial-to-mesenchymal transition by negatively
regulating the translation of mesenchymal genes .
Epigenetic reprogramming of cell types with shared
developmental history could be an effective strategy for
pancreatic β-cell replacement therapies. The cells may
display some intrinsic commitment to become islets even
during adulthood and might thus require fewer triggers
to differentiate/transdifferentiate towards a β-cell lineage.
Along this line, in the field of reprogramming to iPS, the
notion of epigenetic “memory” inherited from the parental
cell is coming forward. Bar-Nur et al. observed a preferential
lineage-specific differentiation in iPS derived from human
β-cells . These new insights in gene regulation should
help to exploit the full potential of adult cell plasticity in
the perspective of cell replacement therapies to treat diseases
such as type 1 diabetes.
10 Journal of Transplantation
In conclusion, it emerges that extreme caution should be
taken when translating the findings obtained from rodent
studies to the human situation. Regarding in vivo regener-
ation investigated under physiological conditions or under
injury, it appears that β-cell replication is the predominant
adulthood, neogenesis from (a subpopulation of) ductal
and/or acinar cells seems to be responsible for the increase
of β-cell mass required in physiological situations of higher
insulin demands like obesity or pregnancy. Nevertheless,
the unanswered question remains which cell type could
be involved: progenitor cells present in the ductal area, or
fully differentiated adult cells able to transdifferentiate, or
a distinct subpopulation that is able to dedifferentiate to
a progenitor-like intermediate stage followed by rediffer-
entiation to an endocrine lineage. Remarkably, both the
animal model and the degree of destruction appear to be
key points, as the regeneration processes can be different:
β-cell mass regeneration from α cells has been observed in
models of near total ablation only. Regarding in vitro replica-
tive capacity of β-cells, human β-cells cannot efficiently
replicate, in contrast to rodent cells. However, contrary
to murine β-cells again, human β-cells can dedifferentiate
to a mesenchymal-like phenotype and proliferate. These
discrepancies between findings from human versus rodent
studies are not as unexpected as they seem, given the major
differences observed in islet cell biology field such as timing
of pancreatic developmental stages , islet architecture
and composition [144, 145], and islet innervation .
In the stem cell field, murine and human stem cells are
notoriously dissimilar. Finally, cultured human primary cells
are more prone to replicative aging than murine cells as
telomere shortening limits cell replication and leads to
However, up to now, there is no way to accurately
evaluate the β-cell mass in humans, neither by imaging
techniques nor by physiological measurements. Studies are
limited to histological analysis performed on organs at
autopsy or after pancreatectomy generating static pictures
that could be misinterpreted, even if careful quantification
in strong indications. Therefore, animal models become
essential offering access to a broad range of technologies
obviously not applicable to humans. Among others, the
recently developed genetic approaches (such as lineage
tracing systems, comprising of a tissue-specific promoter
and a (tamoxifen-)inducible recombination system and a
reporter gene) are valuable tools to follow a dynamic
process and reinforce (or sometimes challenge) earlier the-
ories suggested by more descriptive immunohistochemistry
data. Nevertheless several limitations have to be taken into
consideration: a possible leakiness of the recombination
system, the relative specificity of a given promoter that can
display some (transient) activity in nonspecific cells, and a
limited penetrance (usually less than half of the cells are
actually labeled) [147, 148]. Altogether these elements might
contribute to the discrepancies between results obtained by
different labs about the origin of β-cell regeneration for
instance, and hopefully validation of some of the models by
separate labs in different experimental contexts will clarify
the situation in a near future.
Since stimulation of human β-cells replication is still
elusive, other cell sources have been envisaged. Studies from
the last decade revealed an unexpected aspect of plasticity
differentiation. Of interest, it appears that regeneration does
not always require recapitulation of the embryological devel-
opment as needed for efficient differentiation of ES cells. For
instance, the recent discovery of α-to-β-cell plasticity does
not appear to correlate with any developmental process .
Murine and human ductal cells, rodent acinar cells (human
ones cannot be maintained in culture), and human liver cells
could be efficiently converted to functional β-cells able to
revert hyperglycemia in a diabetic mouse model. Although
the potential contribution of MSC to islet regeneration in
a physiological situation remains unclear and their origin
and function are still elusive, islet-derived mesenchymal
stem cells seem to display specific genetic and epigenetic
marks that could make them more prone to differentiation
towards the endocrine compartment than extrapancreatic
sources of MSC. This suggests that all mesenchymal stem
from humans, revealed additional properties (like homing
to injury site and secretion of favorable growth factors)
that may be of clinical use. Therefore, further investigations
will be required to determine if islet-(derived-) MSC can
be stimulated to contribute in any way to β-cell mass
regeneration. Another approach successfully tested has been
to force expression of proendocrine transcription factors in
vivo in the liver and in the exocrine tissue. However no
lineage tracing experiments have been performed in these
studies, and the origin of the newly formed β-cells needs to
Finally, there is currently no unique optimal alternative
cell source for β-cell (re)generation. Therefore a crucial
aspect common to all putative cell sources will be to further
uncover the mechanisms that preserve and control cell
identity in order to enable successful manipulation of adult
cell plasticity for clinical application.
This work has been supported by the Dutch Diabetes Fonds,
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