The SDF-1a/CXCR4 Axis is Required for Proliferation and
Maturation of Human Fetal Pancreatic Endocrine
Ayse G. Kayali, Ana D. Lopez, Ergeng Hao, Andrew Hinton, Alberto Hayek, Charles C. King*
Department of Pediatrics, Pediatric Diabetes Research Center, University of California San Diego, San Diego, California, United States of America
The chemokine receptor CXCR4 and ligand SDF-1a are expressed in fetal and adult mouse islets. Neutralization of CXCR4
has previously been shown to diminish ductal cell proliferation and increase apoptosis in the IFNc transgenic mouse model
in which the adult mouse pancreas displays islet regeneration. Here, we demonstrate that CXCR4 and SDF-1a are expressed
in the human fetal pancreas and that during early gestation, CXCR4 colocalizes with neurogenin 3 (ngn3), a key
transcription factor for endocrine specification in the pancreas. Treatment of islet like clusters (ICCs) derived from human
fetal pancreas with SDF-1a resulted in increased proliferation of epithelial cells in ICCs without a concomitant increase in
total insulin expression. Exposure of ICCs in vitro to AMD3100, a pharmacological inhibitor of CXCR4, did not alter
expression of endocrine hormones insulin and glucagon, or the pancreatic endocrine transcription factors PDX1, Nkx6.1,
Ngn3 and PAX4. However, a strong inhibition of b cell genesis was observed when in vitro AMD3100 treatment of ICCs was
followed by two weeks of in vivo treatment with AMD3100 after ICC transplantation into mice. Analysis of the grafts for
human C-peptide found that inhibition of CXCR4 activity profoundly inhibits islet development. Subsequently, a model
pancreatic epithelial cell system (CFPAC-1) was employed to study the signals that regulate proliferation and apoptosis by
the SDF-1a/CXCR4 axis. From a selected panel of inhibitors tested, both the PI 3-kinase and MAPK pathways were identified
as critical regulators of CFPAC-1 proliferation. SDF-1a stimulated Akt phosphorylation, but failed to increase
phosphorylation of Erk above the high basal levels observed. Taken together, these results indicate that SDF-1a/CXCR4
axis plays a critical regulatory role in the genesis of human islets.
Citation: Kayali AG, Lopez AD, Hao E, Hinton A, Hayek A, et al. (2012) The SDF-1a/CXCR4 Axis is Required for Proliferation and Maturation of Human Fetal
Pancreatic Endocrine Progenitor Cells. PLoS ONE 7(6): e38721. doi:10.1371/journal.pone.0038721
Editor: Kathrin Maedler, University of Bremen, Germany
Received March 27, 2012; Accepted May 14, 2012; Published June 22, 2012
Copyright: ? 2012 Kayali et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a grant from the Larry L. Hillblom Foundation and the California Institute for Regenerative Medicine (CIRM). The funders
had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have the following conflicts: The authors note that Genentech (San Francisco, CA), graciously provided HGF/SF for these
experiments. This commercial source did not provide any funding, financial assistance employment, consultancy, patents, products in development or marketed
products for this project. This does not alter our adherence to all the PLoS ONE policies on sharing data and materials.
* E-mail: firstname.lastname@example.org
The need to find b-cell sources independent of human cadaveric
sources useful for the development of cell-based therapies for
patients with type 1 diabetes depends to a great extent on
enhanced understanding of the molecular mechanisms that
regulate human endocrine pancreas maturation. These insights
will help the derivation of new protocols for both differentiation of
human embryonic stem cells (hESCs) and regeneration of the
compromised endocrine pancreas either from sources such as
acinar tissue, other endocrine hormone expressing cells, or the
Chemokines are a superfamily of small secreted (8–10 kD)
cytokines that bind and activate heptahelical transmembrane G-
protein coupled receptors (reviewed in ) that are involved in a
number of diverse biological processes, including leukocyte
trafficking [2,3], regulation of HIV infection , mobilization of
hematopoietic stem cells , regulation of angiogenesis ,
metastasis and fetal development . Although a number of
chemokines play critical roles in organogenesis , SDF-1a and
CXCR4 comprise the only chemokine/chemokine receptor pair
that individually results in embryonic lethality in mouse knock-
outs. Mice with genetic disruption of either the CXCR4 receptor
or SDF-1a ligand display abnormal gastrointestinal vasculature,
aberrant migration of cerebellar neurons, impaired B-lymphopoi-
esis, cardiac ventricular septal defects, and failure of bone marrow
hematopietic colonization [9,10,11,12]. Identical phenotypes of
the knockouts for SDF-1a and CXCR4 suggest that CXCR4 is the
only receptor for SDF-1a, although recent studies have demon-
strated that SDF-1a can also bind and activate CXCR7 .
The recent finding that CXCR4 is a marker for definitive
endoderm (DE) during the differentiation of human embryonic
stem cells (hESCs) led us to investigate the fate of this receptor
between DE formation and the generation of hormone producing
endocrine cells. While the mechanism of action of CXCR4 in this
context has not been studied, we have previously documented
SDF-1a/CXCR4 receptor pair expression in fetal mouse pancreas
and its obligatory function in an adult mouse model of pancreatic
regeneration . In these transgenic mice in which IFNc is
expressed under the control of the insulin promoter, the pancreas
displays ductal proliferation and islets exhibit regeneration
[15,16,17,18]. In this system, SDF-1a stimulated migration and
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activation of the signaling molecules MAPK, Akt, and Src in
pancreatic ductal cells. A protective effect on ductal cell apoptosis
and a parallel induction of ductal proliferation was observed in vivo
[14,18]. Other studies have also explored the role of CXCR4 in
development. In zebrafish lacking CXCR4 receptor, duplication
of endodermal organs including liver and pancreas was observed
. Additionally, in a study of the second stage epithelial
transition in embryos from SDF-1a and CXCR4 knockout mice,
Hick et al reported transient defective morphogenensis in the
ventral and dorsal pancreas, suggesting an important role of this
pathway in the branching morphogenesis of endocrine pancreas
In the present study, we identify the SDF-1a/CXCR4 signaling
axis as an important component of human fetal b-cell develop-
ment and begin to uncover the downstream signaling events that
are critical for this process. Using Immunofluorescence, CXCR4
expression is tracked through human fetal pancreas development
and demonstrated to exclusively co-localize with insulin positive
cells during later stages of development. Furthermore, CXCR4
activity is demonstrated to be essential for the in vivo differentiation
of islet-like clusters into b-cells and that SDF-1a is required for the
proliferation of epithelial endocrine precursors through activation
of PI 3-kinase and Akt. Taken together, these data identify SDF-
1a/CXCR4 signaling as a critical component of islet genesis.
Localization of CXCR4 Expression in Human Fetal and
Our laboratory and others had previously identified SDF-1a/
CXCR4 expression and signaling in mouse islets , . Given
that the CXCR4 receptor is also used as a marker of definitive
endoderm in human embryonic stem cells , we performed
immunofluorescence to explore the relationship between CXCR4
expression and endocrine specification. In 11.6-week human fetal
pancreas, cells expressing CXCR4 also expressed neurogenin 3
(ngn3), a transcription factor necessary for endocrine commitment
(Fig. 1). Therefore, in the epithelial migration in the early stages of
formation of islet-like clusters in the human pancreas, the ngn3
positive cells that are destined to differentiate into endocrine cells
are all marked by CXCR4.
Using immunofluorescence microscopy, we next explored
CXCR4 expression in human fetal islets at various stages of
development (Fig. 2). At week 10.5 of gestation, CXCR4 was
diffusely expressed throughout the pancreatic epithelium, includ-
ing co-expression with the few cells that were insulin positive
(Fig. 2A). Glucagon-positive cells, however, did not express
CXCR4 at the same gestational age (Fig. 2B). In islet-like clusters
(ICCs), at both 15.3 and 22 weeks of gestation, CXCR4 expression
became restricted primarily to insulin (Fig. 2C and E), but not
glucagon positive cells (Fig. 2D and F). Furthermore, CXCR4 was
expressed both in the islets and the ducts of human adult pancreas,
but not in acinar tissue (Fig. 3A and B). CXCR4 mRNA levels in
all ICC samples were much lower than CXCR4 levels found in
definitive endoderm, the first committed stage of endocrine cell
development characterized by very high CXCR4 levels (Fig. 3C).
However, compared to pluripotent human embryonic stem cells,
the four different human fetal pancreatic samples and human
adult islets displayed significantly higher CXCR4 expression
We next undertook studies to determine whether expression of
SDF-1a, the CXCR4 ligand, could be detected in human adult
pancreas. Expression of SDF-1a was restricted to the ducts of the
human adult pancreas (Fig. 4A–B). RT-PCR analysis showed that
SDF-1a is expressed in heterogeneous cell populations of human
fetal and adult islet samples, as well as pluripotent human
embryonic stem cells and definitive endoderm (Fig. 4C). Taken
together, these data reveal that CXCR4 is expressed in widely in
human fetal pancreatic endoderm, but later in development is
selectively expressed in ductal tissue and insulin expressing cells in
the human fetal and adult pancreas; whereas SDF-1a is expressed
and localized to ducts in human adult pancreas.
Effect of SDF-1a/CXCR4 Axis on b Cell Maturation
In vitro study.
Human fetal ICCs can be maintained in
suspension in vitro for up to 5 days without compromising their
integrity . Previous studies from our group have shown that
in vitro treatment of ICCs with Exendin 4  and keratinocyte
growth factor (KGF)  followed by transplantation under the
kidney capsule of immunocompromised mice and continued in vivo
Exendin 4 or KGF treatment resulted in accelerated maturation
and proliferation of b-cells. We first wanted to determine whether
treatment of the ICCs with SDF-1a would accelerate in vitro
differentiation of the b-cells. ICCs derived from human fetal islets
(12 to 16.5 week gestational-age) were treated with SDF-1a
(100 ng/ml) or hepatocyte growth factor (HGF; 10 ng/ml) in vitro
every other day for a period of five days. HGF is a growth factor
that is the primary component of the mesenchyme induced b-cell
growth in fetal ICCs . In vitro treatment with SDF-1a or HGF
did not result in a significant change in insulin content as
measured by ELISA (Fig. 5).
In these early gestation ICCs, incubation with human serum
was sufficient to induce epithelial cell proliferation (Fig. 6A).
Treatment with HGF or SDF-1a further stimulated the prolifer-
ation of epithelial cells (Fig. 6B and C, respectively). SDF-1a was
as efficient as HGF at inducing cell proliferation, indicating a role
for this receptor in cell growth and development. These data
suggest that during the earlier stages of human pancreatic b-cell
development, both HGF and SDF-1a can act as potent mitogens.
To determine whether SDF-1a expression in ICCs regulates
proliferation or maturation in a paracrine manner, we next asked
whether pharmacologic inhibition of CXCR4 by AMD3100 could
impact the expression of transcription factors important in
pancreatic endocrine differentiation. RT-PCR analysis of PDX-
1, ngn3, Pax4, MAFA, Nkx6.1, insulin, and glucagon was
performed in RNA from control and AMD3100 treated ICCs
from 13 and 17 week-gestational human fetal pancreata (Fig. 7).
There was no significant difference in expression of any of the
transcription factors measured upon treatment with AMD3100.
Insulin and glucagon expression increased with gestation but did
not change with AMD3100 treatment (data not shown).
In vivo study.
The five day in vitro studies suggested that
proliferation of epithelial cells in the ICCs may ultimately be a
source for b-cells. However, there was no observed increase in the
number of b-cells present in response to SDF-1a treatment in the
time frame of the in vitro assay as measured by insulin content. RT-
PCR studies also indicated that inhibition of CXCR4 in vitro did
not alter the expression of transcription factors involved in the
maturation of b-cells. Therefore, we next wanted to provide an
extended differentiation period in vivo in the absence of SDF-1a for
the ICCs. Both, control and AMD3100 treated ICCs were
transplanted under the kidney capsule of immunodeficient mice.
Post transplantation, mice were injected intraperitoneally with
saline or AMD 3100 (5 mg/g) every other day for two weeks.
Starting at eight weeks after transplantation, human C-peptide in
serum from the mice was assayed. Mice that received control ICCs
began to release human C-peptide after approximately four
months, while mice treated with the CXCR4 inhibitor did not
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produce any human C-peptide (Fig. 8). The grafts of control mice
showed extensive human insulin and glucagon positive cells
(Fig. 9A and B), but the AMD3100 treated animals did not
generate any insulin or glucagon staining at the transplant site
(Fig. 9C). Beads grafted with the ICCs under the kidney capsule
allowed us to definitively identify the transplant site in the absence
of hormone positive cells (Fig. 9C). These studies underscore the
importance of the SDF-1a/CXCR4 signaling axis in the
development of insulin producing cells.
The Effect of SDF-1a Stimulation on Proliferation of
Treatment of ICCs in vitro showed that SDF-1a stimulates
proliferation of epithelial cells in ICCs. We next addressed the
mechanistic aspects of CXCR4 function. Considering that ICCs
contain a heterogeneous cell population, we used the pancreatic
ductal carcinoma cell line, CFPAC-1, that expresses CXCR4 (data
not shown) and shares adhesion and migration characteristics with
those of ICCs . Initially, we addressed proliferation of
CFPAC-1 cells following stimulation with SDF-1a for 24 hrs.
Under basal conditions, 13% of the cells were proliferating.
Treatment with SDF-1a increased proliferation in a dose
dependent manner (Fig. 10). At 100 ng/ml, SDF-1a increased
proliferation by 26.6%, while 300 ng/ml of SDF-1a augmented
proliferation by 71.6%.
The Effect of Inhibitors of PI 3-kinase, MAPK, PLC, and
PKA Pathways on Basal and SDF-1a Stimulated
Proliferation in CFPAC-1 Cells
CFPAC-1 cells were treated with a panel of pharmacological
inhibitors to identify which signaling pathways downstream of
CXCR4 were involved in proliferation (Fig. 11). Inhibition of the
PI 3-kinase with LY294002 or MAPK with U0126 inhibited SDF-
1a stimulated proliferation by 50% and 48% respectively. The
PLC inhibitor, edelfosine, and the PKA inhibitor H89 inhibited
SDF-1a stimulated proliferation to a lesser extent (,20%), while
IBMX/forskolin, and JNK inhibitor II had no effect (data not
Effect of SDF-1a on Apoptosis in CFPAC-1 Cells
Over the course of the proliferation studies, we noticed that
SDF-1a induced cell death. To explore this, we compared SDF-1a
mediated apoptosis in CFPAC-1 cells to a combination of TNFa,
IL1b and IFNc that has been shown to cause apoptosis in islets
. This approach also allowed us to determine whether SDF-1a
can counteract or synergize with the apoptotic effect of the
Figure 1. CXCR4 and Ngn3 are co-expressed in the branching epithelia of 11-week gestational human fetal pancreas.
Photomicrographs (20X) of two representative areas depict Ngn3 (green) in nuclei and CXCR4 (red) in membranes. The composite images (A, B)
are resolved into their green (C, D) and red (E, F) channels for optimal visualization.
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cytokines specified above. In CFPAC-1 cells, a TNFa, IL1b and
IFNc cocktail induced apoptosis four-fold as measured by the
percentage of TUNEL positive cells (Fig. 12). At low SDF-1a
concentrations (100 ng/ml) apoptosis was unchanged; however, at
high SDF-1a levels (300 ng/ml) an approximately two-fold
increase in TUNEL positive cells was observed. The TNF cocktail
in combination with SDF-1a (100 ng/ml) increased apoptosis
approximately 5 fold. This increase was not statistically significant
compared to TNF cocktail alone. Furthermore, combining the
cytokine cocktail with SDF-1a at 300 ng/ml caused excessive cell
death (data not shown).
SDF-1a Stimulates Akt Phosphorylation in the Cell Line
Inhibitor studies underscored the importance of PI 3-kinase and
MAPK signaling pathway activation in SDF-1a-mediated prolif-
eration. At least two distinct processes could be responsible for
CFPAC-1 proliferation: decreased apoptosis through activation of
PI 3-kinase signaling or increased proliferation through activation
of MAPK. To determine the relative input of these downstream
signaling pathways in CFPAC-1 proliferation, we next assessed the
SDF-1a stimulated phosphorylation of MAPK and Akt in vitro.
MAPK was phosphorylated in serum starved CFPAC-1 cells;
addition of SDF-1a did not further stimulate MAPK phosphor-
ylation significantly in at least four independent experiments
(Fig. 13A). However, Akt was robustly phosphorylated at serine
473 in response to SDF-1a (100 ng/ml and 300 ng/ml) (Fig. 13B).
To assess whether the signaling mechanisms activated by SDF-1a
in the CFPAC-1s reflect those in the combination of epithelial, islet
progenitor, and b-cells in ICCs derived from fetal pancreas, we
performed parallel experiments in ICCs (from a 15.7 wk gestation
human fetus) grown in suspension culture for four days. Consistent
with the CFPAC-1 model system, basal MAPK phosphorylation
was elevated despite serum starvation overnight and persisted at
high levels in the presence of SDF-1a (Fig. 13C). SDF-1a
stimulation of the ICCs resulted in the increased Akt phosphor-
ylation at serine 473 (Fig. 13D). Taken together, our observations
Figure 2. Time course of CXCR4, insulin, and glucagon expression in human fetal pancreas. At 10.5 weeks of gestation CXCR4 (red) is
expressed throughout the pancreatic epithelia (A and B). Most of the insulin-expressing cells (green) in islet-like structure appear yellow because they
co-express CXCR4 (A). B. The few glucagon (green) expressing cells at 10.5 weeks of gestation do not express CXCR4. At 15.3 weeks of gestation
CXCR4 expression is restricted to the islet-like clusters, which also express insulin (green), again appearing yellow C. The glucagon (green) expressing
cells in the islet like structures are contiguous to the CXCR4 expressing cells and no co-expression is detected D. By 22 weeks, the glucagon (green)
positive cells, also located in the islet-like cluster, surround the CXCR4 (red) positive cells (F), which co-express with insulin.
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Figure 3. CXCR4 is expressed in human adult islets and ducts. Panel A illustrates CXCR4 (green) expression in human adult islets; note the
absence of CXCR4 staining in exocrine tissue surrounding the islet. Panel B reveals that in the human adult islet CXCR4 expressing cells coexpress
insulin (yellow). Human adult ducts also exhibit CXCR4 (green) staining. C. RT-qPCR analysis of CXCR4 mRNA in human embryonic stem cells (hESCs),
hESC derived definitive endoderm (DE), fetal islet cell clusters at 11, 12 and 18 weeks gestation (11 WK, 12 WK, 18 WK), and adult islets. Expression
levels expressed as DCt values relative to Cyclophilin G. D. Due to extremely high expression in DE, CXCR4 mRNA levels shown comparing only hESCs,
fetal ICCs, and adult islets.
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Figure 4. SDF-1 expression in human adult pancreas. Panels A and B. Double immunofluorescent staining of SDF-1a (red) and insulin (green)
in human adult islets demonstrates ductal enrichment of SDF-1a, but no expression in islets. Panel C. SDF-1a mRNA expression in hESCs, definitive
endoderm (DE), 12 week (12 WK) and 18 week (18 WK) human fetal ICCs and adult islets. mRNA levels are expressed as DCt values relative to
Figure 5. HGF or SDF-1 treatment of human ICCs in vitro does not alter insulin content. Insulin content expressed as picomoles/mg DNA of
fetal ICCs after 5-day culture in medium containing 10% human serum or serum plus HGF or SDF-1a was assessed. Data represents the average of five
separate sets of experiments. Values are expressed as mean 6 SEM and were not significant by Student’s t-test.
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are consistent with a SDF-1a mediated effect on proliferation in
fetal endocrine pancreas through Akt signaling.
Here we demonstrate that both the heterotrimeric G-protein
coupled receptor CXCR4 and its ligand SDF-1a are expressed in
the human fetal pancreas. CXCR4 co-localizes with Ngn3 positive
cells in early gestation, and gradually becomes restricted to cells
expressing insulin, but not glucagon. SDF-1a expression, on the
other hand, does not overlap with hormone positive cells; rather its
expression appears to be restricted to ductal structures in the
pancreatic architecture. Treatment of human fetal ICCs with
SDF-1a had no effect on insulin content, but it led to increased cell
proliferation. Attenuation of CXCR4 activity by treatment with
the inhibitor AMD3100 had no effect on expression of b-cell
specific genes in vitro after 5 days. However, after transplantion of
AMD3100 treated ICCs into nude mice, there was a profound
inhibition of b-cell genesis. The survival and proliferative signals
could be directly modulated downstream of CXCR4 by Akt in the
cell line CFPAC-1. These results provide the first example of
CXCR4 mediated signaling in the modulation of human b-cell
growth and proliferation.
Expression of CXCR4 is a well-established cell surface marker
for human embryonic stem cells lineage restricted to definitive
endoderm [22,29], but expression is subsequently diminished
rapidly after this stage . The co-localization of CXCR4
positive cells with ngn3, a transcription factor expressed in cells
Figure 6. SDF-1 stimulates proliferation of epithelial cells in ICCs from early gestational human fetal pancreas. ICCs isolated from fetal
pancreas of 12 to 14 weeks were grown in suspension culture, treated with 10% human serum alone, with HGF (10 ng/ml) or with SDF-1a (100 ng/
ml) for six days. ICCs were fixed and analyzed by immunofluorescence staining. PanCytokeratin (green) is an epithelial marker. Ki67 (red) is used as a
marker of proliferation.
Figure 7. Comparison of mRNA levels for endocrine markers in control and AMD3100 treated ICCs derived from human fetal
pancreas by RT-PCR. The values of mRNA for endocrine markers are expressed as mean 6 SEM of DCTs of the transcription factors compared to
Cyclophilin A. Ngn3 (Panel A), PAX-4 (Panel B) and MAF-A (data not shown) were detected at approximately 35 to 37 PCR cycles, PDX-1 (Panel C) and
Nkx6.1 (Panel D) were detected at 30 to 31 PCR cycles both in control and AMD3100 treated ICCs. Four different preps from gestational ages 13 to 17
weeks were analyzed. N.S. = not significant by Student’s t-test.
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destined to become pancreatic endocrine cells indicates that
CXCR4 expression is important in the further lineage restriction
from endocrine progenitors to mature endocrine cells.
Important clues about the role of the CXCR4/SDF-1a
during the transition from definitive endoderm to an endocrine
precursor cell expressing the pancreatic precursor transcription
factor PDX1 have recently been described by Katsumoto et al.,
who found that this signaling pathway plays an important role
in establishing the fate of pancreatic progenitors in chick
embryos . These authors observed that overexpression of
Figure 8. Serum Human C-peptide levels in nu/nu mice transplanted with human fetal ICCs treated with AMD3100. Sixteen weeks
after transplantation, circulating human C-peptide levels were measured 30 minutes after a glucose challenge in fasted nu/nu mice that had been
treated with saline or AMD3100. For both the saline and AMD3100 groups n=5. For the control group, C-peptide levels were 517.36198.83
(mean6SEM). C-peptide levels in the AMD3100 treatment group were undetectable. P,0.05 by Student’s t-test.
Figure 9. Expression of insulin and glucagon in grafts from control and AMD3100 treated ICCs transplanted into nu/nu mice. Double
immunofluorescent staining of insulin (green) and glucagon (red) in ICCs transplanted under the kidney capsule of nu/nu athymic mice treated with
saline (A, B) or AMD3100 every two days during two weeks following transplantation. Insulin and glucagon expression is extensive in the control ICCs.
C. Insulin or glucagon was not detectable in the grafts from the mice treated with AMD3100. Note the presence of the round beads that were
inserted to identify the transplant site.
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SDF-1a attracted cells expressing Lmo2, which subsequently
induced Pdx1-expressing pancreatic progenitors and enhanced
differentiation into insulin-expressing cells. Similar to our
studies, treatment of cells with AMD3100 reduced pancreatic
bud formation and treatment of cells with SDF-1a resulted in
augmented cell proliferation.
SDF-1a stimulated proliferation has been documented in
CD34(+) hematopoietic progenitors [31,32] cerebellar granule
cells  and astrocytes  from neonatal rodents. Conditional
inactivation of CXCR4 in osteoprecursors has been shown to
result in reduced postnatal bone formation, which was partially
due to decreased osteoblast proliferation . Our previous study
using the IFNc mouse, which displays regeneration in the ducts of
the adult pancreas showed that inhibition of CXCR4 function by
in vivo CXCR4 antibody administration inhibited ductal cell
proliferation. In vivo the SDF-1a/CXCR4 axis can exert its effects
at multiple levels , including adherance to the extracellular
matrix, migration, further proliferation, and differentiation in
response to endogenous growth factors. In a previous study, we
reported that parenterally administered Exendin-4, a long-lasting
analogue of Glucagon-Like-Peptide-1 (GLP-1), induces matura-
tion of ICCs transplanted into nude mice . Liu et al have
recently postulated a connection between SDF-1a secretion and
GLP-1 production in injured islets [24,35]. According to this
recent report, injury to the islets results in SDF-1a release. In
response to SDF-1a GLP-1 is produced by the alpha cells and
feeds back to induce the growth and survival of b-cells. In the
present study, the SDF-1a stimulated proliferation in ICCs
derived from the human embryonic pancreas was comparable to
the effect of HGF, a growth factor and primary component of the
mesenchyme induced b-cell growth in human fetal ICCs . The
ability of SDF-1a to induce the proliferation of pancreatic
epithelial cells may provide an increased number of precursors
for subsequent differentiation into b-cells in vivo.
In vitro studies demonstrated a clear capacity of SDF-1a to
enhance replication of human fetal ICCs (Fig. 6C); however, RT-
PCR analysis revealed no significant increase in endocrine cell
markers, including PDX-1, Ngn3, Pax-4, Nkx 6.1, or insulin (Fig. 5
Figure 10. SDF-1 stimulates proliferation in CFPAC-1 cells.
CFPAC-1 cells were seeded in 12 well plates and grown to 50–70%
confluence. Following 24 hr serum starvation, cells were stimulated
with SDF-1a (100 ng/ml or 300 ng/ml) for 16 hrs. BrdU was added 4 hrs
before fixation. BrdU incorporation was determined as described under
Materials and Methods. BrdU incorporation was stimulated 26.6% and
71.6% by 100 ng/ml and 300 ng/ml SDF-1a, respectively. Each bar
represents the average of three experiments (mean 6 SEM); P,0.03 by
analysis of variance.
Figure 11. The effect of the inhibitors of PI 3-kinase, MAPK, PLC, and PKA on SDF-1a stimulated proliferation in CFPAC-1 cells.
CFPAC-1 cells were seeded in 12 well plates and grown to 50–70% confluence. Following 24 hr serum starvation, cells were pre-treated with
LY294002 (30 mM), U0126 (30 mM), Edelfosine (10 mM), or H89 (10 mM) for 30 minutes and stimulated with SDF-1a for 16 hrs. BrdU was added 4 hrs
before fixation. BrdU incorporation was determined as described under Materials and Methods. (*, P,0.05 SDF-1 versus basal); (**, p,0.02 SDF-1/
Edelfosine versus SDF-1 alone); (***, p,0.0001 SDF-1/LY294002 and SDF-1/U0216 versus SDF-1 alone) by Student’s t-test. N.S. = not significant by
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Figure 12. The cytokine cocktail TNFa, IL1b and IFNc and SDF-1a (300 ng/ml) stimulate CFPAC-1 apoptosis. CFPAC-1 cells were seeded
in 12 well plates and grown to 50–70% confluence. Following 24 hr serum starvation, cells were stimulated with either the cytokine cocktail (TNF)
consisting of IL-1b (2 ng/ml), IFN-c (100 ng/ml) and TNF-a (100 ng/ml), or SDF-1a at 100 ng/ml, 300 ng/ml, or the TNF cocktail in combination with
SDF-1a 100/ng/ml for 24 hrs. The cells were fixed at the end of the incubation apoptosis was quantitated using the TUNEL method as described in
the Materials and Methods. The number of TUNEL positive nuclei was expressed as the percentage of the total number cells counted in the acquired
images. Data is expressed as mean 6 SEM. (*, p,0.03 versus basal); (**, p,0.01 versus basal) by Student’s t-test. N.S. = not significant by Student’s t-
Figure 13. SDF-1a stimulates Akt but not MAPK phosphorylation in CFPAC-1 cells and fetal ICCs. Following 48 hr serum starvation,
CFPAC-1 cells were stimulated with 100 ng/ml or 300 ng/ml human recombinant SDF-1a for 10 min at 37uC. Whole cell lystes were analyzed by
western blot, using antibodies raised against dually phosphorylated phospho-MAPK(ERK1/ERK2)(A) or phospho-Akt (Ser473)(B). Following overnight
serum starvation, ICCs from human fetal pancreas of 15 weeks gestation were stimulated with 100 ng/ml or 300 ng/ml SDF-1a for 10 min at 37uC.
Whole cell lystes were analyzed by western blot, using an antibody raised against phospho-MAPK(C) or phospho-Akt (Ser473) (D). All blots were
stripped and reblotted with antibodies to total Akt, Erk, and Hsp90 sequentially to confirm equal loading.
SDF-1a/CXCR4 in b Cell Maturation
PLoS ONE | www.plosone.org 10 June 2012 | Volume 7 | Issue 6 | e38721
and 7). One possible explanation is that the proliferating epithelial
cells are not of endocrine origin. However, the results from the
transplant studies suggest an alternate explanation (Fig. 8 and 9).
Long-term treatment of fetal ICCs with the CXCR4 specific
inhibitor AMD3100 blocks both cell growth and C-peptide
release, suggesting that the SDF-1a/CXCR4 signaling axis is
essential for cells to survive and develop into insulin secreting cells.
The latter observation suggests that a temporal aspect of CXCR4
mediated signaling is critical for development in vivo after cell
transplantation that might not be observed in a short in vitro
To examine the dynamics of CXCR4/SDF-1a expression in
development, signaling related to proliferation and apoptosis was
explored in a cell line to avoid the effects of heterogeneity in a fetal
ICC population. CXCR4 is a Gia-coupled heterotrimeric
heptahelical transmembrane protein with well-defined signaling
outputs . Pharmacological manipulation of these signaling
events allowed us to define pathways important for the observed
effects. Both edelfosine (a PLC inhibitor) and H89 (a PKA
inhibitor) failed to alter the effects of SDF-1a treatment. However,
inhibition of PI 3-kinase or canonical MAP kinase signaling
attenuated proliferation (Fig. 11 and data not shown), suggesting
signaling from CXCR4 through these two pathways was essential
for cell function. These results are consistent with our findings in
the mouse, where we demonstrated that SDF-1a stimulates the
phosphorylation of Akt and mitogen-activated protein (MAP)
kinase in pancreatic duct cells .
In mouse islets, SDF-1a expression was restricted to cells
surrounding the ducts and the microvasculature both around the
ducts and in the islets . SDF-1a expression was also observed
in the b-cells of the pancreas in neonatal mice up to 21 days of age,
after which SDF-1a was no longer expressed in the b-cells .
We have also observed SDF-1a expression the ducts and cells
immediately surrounding the ducts in the human adult pancreas
(Fig. 4B) with no apparent expression in the islets, suggesting
similarities between the two systems. Further characterization of
the SDF-1a expression in the human pancreas will be addressed in
In the stepwise differentiation of human embryonic stem cells
the expression of CXCR4 has been established as a marker of
definitive endoderm . Habener et al have reported that mice
expressing SDF-1a under the control of the insulin promoter
(RIP-SDF-1a mice) are somewhat protected against STZ-
induced diabetes. Their studies also showed SDF-1a induced
promotion of b-cell survival by Akt activation . Subsequent
studies by the same group have suggested that the activation of
the WNT pathway by SDF-1a may be part of the mechanism
that promotes b-cell survival . Therefore, the stimulation of
Akt in the absence of an anti-apoptotic effect is intriguing. Taken
together with the lack of an effect on MAPK, the SDF-1a effect
on proliferation in this context appears to be Akt dependent.
SDF-1a stimulates proliferation in human cortical neural
progenitor cells derived from human fetal brain tissue. This
proliferative effect has been shown to be dependent on Akt
phosphorylation . The Akt pathway, the primary mediator of
PI 3-kinase signaling, has also been shown to regulate the
proliferation of b-cells, an effect that involves GSK3 and Cyclins
D1 and D2 [39,40,41].
Cell growth during embryonic development and disease is a
tightly orchestrated process that ensures the proliferation of
certain cells while allowing the apoptosis of others [42,43].
CXCR4 has been has been shown to be involved in the
stimulation of apoptosis by HIV in both T cells and neurons
[44,45,46,47,48]. SDF-1a has also been shown to directly
stimulate apoptosis in neural cells . Frequently, SDF-1a
stimulation is associated with enhanced survival as reported in
hematopoietic progenitor cells , fetal thymus [51,52] and in
bone marrow myelopoiesis . Previous studies have shown
that SDF-1a has a pro-survival effect in mouse pancreas, MIN-
6 cells, and INS-1 cells [14,21,27]. Our observation of SDF-1a
stimulation of apoptosis in CFPAC-1 cells was unexpected. It is
conceivable that the stimulatory effect of SDF-1a at high
concentrations (300 ng/ml) may be representative of what
happens to ICCs at a particular stage of development. The
in vivo relevance of this apoptotic effect requires further study.
Taken in context, our results suggest that the presence of the
CXCR4/SDF-1a axis in the early pancreas is important for
lineage restriction to endocrine cells, while its persistence in the
adult may indicate either a potential for regeneration or an active
role in proliferation, survival and maintenance of b-cells.
Materials and Methods
to 17 weeks gestation) were provided by Birth Defects Research
Laboratory, University of Washington (Seattle, Wash., USA.)
Informed consent for tissue donation, storage, and use of the
samples was obtained from the donors by the center. The protocol
#081237XT consent statement was in writing. Furthermore, The
University of California, San Diego Human Research Protections
Program approved the whole study (Protocol #081237XT).
The University of California, San Diego Human Research
Protections Program approved the use of human adult tissue for
#071943XT consent statement was in writing. Human adult
pancreas biopsies (block) were collected and processed at The
University of California, San Diego. The Director of the Human
Research Protections Program certified that Project #120578XX
(Mechanisms of Pancreatic Development-2) is exempt from IRB
approval under 45 CFR 46.101(b)), category 4: Research involving
the collection or study of existing data, documents, records,
pathological specimens, or diagnostic specimens, if these sources
are publically available or if the information is recorded by the
investigator in such a manner that subjects cannot be identified
directly or through identifiers linked to the subjects. The samples
The University of California, San Diego Institutional Animal
Care and Use Committee approved the use of athymic mice for
the transplant experiments (Protocol #S00175M). The protocol
#S00175 consent statement was in writing.
Human fetal pancreata used in this study (10
#07943XT). The protocol
Preparation and Treatment of Fetal Pancreatic Islet-like
Cell Clusters (ICCs)
Fetal pancreata were processed as described previously
(Beattie, 1994). Tissue was minced and digested with collage-
nase Type XI (Sigma, St. Louis, Mo., USA) and allowed to
form islet-like clusters in suspension in RPMI-1640 containing
10% human AB serum (Cellgro, Mediatech, Manassas, VA.).
Following the formation of ICCs, the clusters were treated with
HGF (10 ng/ml) (a generous gift by Genentech, San Francisco,
CA.), SDF-1a (100 ng/ml) (PeproTech Inc. Rocky Hill, NJ.) or
AMD 3100 (1 mg/ml) (Sigma, St. Louis, MO.) for 5 days with a
medium changes every two days. At the end of the in vitro
treatment for some experiments the ICCs were processed for
insulin extraction and DNA quantitation as described previously
.SomeICCs were fixed in4%paraformaldehyde,
SDF-1a/CXCR4 in b Cell Maturation
PLoS ONE | www.plosone.org 11June 2012 | Volume 7 | Issue 6 | e38721
embedded first in agarose and then in paraffin for histological
Nu/nu athymic mice were obtained from the in-house
breeding colony at the Animal Care Program, University of
California, San Diego. 500 to 1000 islet-like cell clusters that
were treated in vitro with either phosphate buffered saline or
AMD3100 were placed under the kidney capsule of five athymic
mice each for control and treatment groups using a positive
displacement pipette as described previously . The trans-
planted mice were treated with vehicle (saline) or AMD3100
(5 mg/kg) intraperitoneally every other day for two weeks. Eight
weeks after transplantation, fasted animals were given 3 g/kg
glucose intraperitoneally, and after 30 min; blood samples were
taken for the assay of circulating human C-peptide with a ELISA
kit (Mercodia, Inc.) that is specific for human C-peptide, with no
cross-reactivity with mouse C-peptide. The glucose stimulations
were repeated every four weeks until circulating human C-
peptide was detected. At four to five months, the mice were
sacrificed and serial sections of the kidneys bearing grafts were
examined histologically for insulin and glucagon cells as
described below. Human C-peptide levels in serum of trans-
planted mice were measured with enzyme-linked immunoabsor-
bent assay (ELISA) kits (Mercodia, Inc.).
Human fetal or adult pancreata were fixed in 4% paraformal-
dehyde and embedded in paraffin. Paraffin-embedded tissue was
cut into 4-mm sections and stained with goat polyclonal anti-
CXCR4 antibody raised against the NH2terminal extracellular
domain of the human CXCR4 receptor (Caprologics, Inc.
Gibertville, MA.), mouse monoclonal anti-SDF-1a antibody raised
against recombinant human SDF-1a (R&D Systems, Inc.), sheep
polyclonal anti-insulin antibody (The Binding Site, Inc. San Diego,
CA.), mouse monoclonal anti-insulin antibody (Sigma, St. Louis,
MO.), and mouse monoclonal anti-glucagon (Sigma, St. Louis,
MO.). The visualization of CXCR4 required the use of
biotinylated goat secondary (Jackson Immunoresearch Labs,
Inc.) followed by Streptavidin conjugated Alexa 488 or 536
(Invitrogen, Molecular Probes Inc.). Following the Alexa Flour
incubation, the sections were placed in mounting medium
(Fluorogel with Tris Buffer (Electron Microscopy Sciences,
Hartfield, PA.). For the Ngn3 and CXCR4 co-expression
experiments, human fetal pancreas was cryopreserved in optimal
cutting temperature (Tissue-Tek, Sakura Finetek USA, Torrance,
CA.) and 4 mm sections were cut for staining. Sheep anti-human
Ngn3 antibody raised against recombinant human Ngn3 (R&D
Systems, Inc.) and rabbit anti-CXCR4 antibody raised against the
N-terminal amino acids 1–14 of the human CXCR4 receptor
(Abcam, Cambridge, MA.) were used on the frozen tissue. The
ICCs treated in vitro with HGF, SDF-1a or AMD3100 were
stained with rabbit anti human Ki67 antibody (Neomarkers,
Fremont, CA.) and mouse anti-human (large spectrum) cytoker-
atin (Immunotech, Coulter, Cedex, France).
Assessment of SDF-1a Effect on Proliferation
The CFPAC-1 cells (ATCC) were grown in monolayer culture
in 12 well plates to 50–70% confluency using Iscove’s Modified
Dulbecco’s medium containing 10% fetal bovine serum. The cells
were then serum starved for 24 hrs and stimulated with SDF-1a
(100 or 300 ng/ml) for 12 hrs, BrdU wad added and the cells were
incubated another 4 hrs and then fixed for 20 min in 4%
paraformaldehyde and stained with rat monoclonal anti-BrdU
antibody (Abcam, Cambridge, MA.) followed by anti-rat Alexa
488 (Invitrogen, Molecular Probes, Inc.) secondary antibody.
DRAQ5 (Cell Signaling Technology) was used to visualize nuclei.
Each treatment was done in triplicate and eight images per well
were acquired on a Zeiss Axiovert microscope (Carl Zeiss
Microimaging, Inc., Thornwood, NY.) using a MicroMax digital
camera (Roper-Princeton Instruments, Acton, MA.) controlled by
MetaFluor software (Universal Imaging, Corp., Sunnyvale, CA.).
The percentage of proliferating cells was calculated by expressing
the number of BrdU positive nuclei divided by the total number of
nuclei in the captured images. LY294002, U0126, Edelfosine,
H89, IBMX, Forskolin, and JNK inhibitor II were obtained from
EMD Biosciences Inc. (San Diego, CA.) The inhibitors were
added following the 24 hr. serum starvation 30 min prior to
stimulation with SDF-1 a.
Total RNA was isolated using an RNeasy PlusMini Kit 50
Qiagen, (Valencia, CA) and cDNA was synthesized using High
Capacity cDNA Reverse Transcription Kit (Applied Biosystems,
Foster City, CA). Quantitative PCR was performed on a StepOne
Plus thermocycler (Life Technologies) with SYBR green master-
mix or Taqman mastermix (Life Technologies). mRNA Ct values
were normalized to either Cyclophilin G or Cyclophilin A. The
PCR primers (Invitrogen, San Diego, CA) and TaqManFAM
probes (Applied Biosystems) used are listed in Table S1.
Assessment of SDF-1a Effect on Apoptosis
The CFPAC-1 cells grown on glass coverslips to 50–70%
confluency 12 well dishes were serum starved for 24 hrs and
stimulated with a cytokine cocktail comprising IL-1b (2 ng/ml),
IFN-c (100 ng/ml) and TNF-a (100 ng/ml) or SDF-1a (100 or
300 ng/ml) or a combination of the cytokine cocktail and SDF-1a
for 24 hrs. Recombinant human IL-1b, IFN-c and TNF-a were
obtained from R&D Systems, Inc. (Minneapolis, MN.) The
concentrations of the cytokines used were based on their apoptotic
effect on human islets . The cells were fixed with 4%
paraformaldehyde at the end of the incubations and TUNEL
staining was performed using the In Situ Cell Death Detection Kit,
POD kit (Roche, Indianapolis, IN.) according to manufacturer’s
instructions. Eight images were captured from each well and each
treatment was performed in triplicate.
Assessment of SDF-1a Stimulation of MAPK and Akt
CFPAC-1 cells were serum starved for 48 hrs and stimulated
with 100 or 300 ng/ml human SDF-1a (PeproTech Inc. Rocky
Hill, NJ.) for ten minutes at 37uC. Cells were lysed with RIPA
buffer containing 20 mmol/liter Tris, pH 7.5, 1 mmol/liter
EDTA, 140 mmol/liter NaCl, 1% NP-40, 1 mmol/liter orthova-
nadate, 1 mmol/liter PMSF, 2 mmol/l sodium pyrophosphate,
25 mmole/l a-glycerophosphate, 10 mmol/l sodium fluoride,
10 mg/ml each of aprotinin, leupeptin, and pepstatin. Equal
amounts of protein were subjected to western blot analysis. Rabbit
polyclonal antibodies to dually phosphorylated phospho-MAPK
(Thr202/Tyr204) and to phospho-Akt (Ser473) were used in
immunodetection (Cell Signaling Technology). The membranes
were stripped and reblotted with mouse monoclonal Erk and
rabbit polyclonal Akt antibodies (Cell Signaling Technology) to
visualize total Erk and Akt expression. Finally the membranes
were stripped and reblotted with a mouse monoclonal Hsp90
antibody to confirm equal protein loading (BD Transduction
Laboratories, San Diego, CA.).
SDF-1a/CXCR4 in b Cell Maturation
PLoS ONE | www.plosone.org 12June 2012 | Volume 7 | Issue 6 | e38721
Primers and probes used in this study.
Conceived and designed the experiments: AK A. Hayek CK. Performed
the experiments: AK AL EH A. Hinton. Analyzed the data: AK A. Hayek
CK. Contributed reagents/materials/analysis tools: AK A. Hinton A.
Hayek CK. Wrote the paper: AK A. Hayek CK.
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