Here we demonstrate that both the heterotrimeric G-protein coupled receptor CXCR4 and its ligand SDF-1α 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-1α 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-1α 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 β-cell specific genes in vitro after 5 days. However, after transplantion of AMD3100 treated ICCs into nude mice, there was a profound inhibition of β-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 β-cell growth and proliferation.
Expression of CXCR4 is a well-established cell surface marker for human embryonic stem cells lineage restricted to definitive endoderm 
, but expression is subsequently diminished rapidly after this stage 
. The co-localization of CXCR4 positive cells with ngn3, a transcription factor expressed in cells 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-1α 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 SDF-1α 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-1α resulted in augmented cell proliferation.
SDF-1α stimulated proliferation has been documented in CD34(+) hematopoietic progenitors 
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 IFNγ 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-1α/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 maturation of ICCs transplanted into nude mice 
. Liu et al have recently postulated a connection between SDF-1α secretion and GLP-1 production in injured islets 
. According to this recent report, injury to the islets results in SDF-1α release. In response to SDF-1α GLP-1 is produced by the alpha cells and feeds back to induce the growth and survival of β-cells. In the present study, the SDF-1α 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 β-cell growth in human fetal ICCs 
. The ability of SDF-1α to induce the proliferation of pancreatic epithelial cells may provide an increased number of precursors for subsequent differentiation into β-cells in vivo
In vitro studies demonstrated a clear capacity of SDF-1α to enhance replication of human fetal ICCs (); however, RT-PCR analysis revealed no significant increase in endocrine cell markers, including PDX-1, Ngn3, Pax-4, Nkx 6.1, or insulin ( and ). 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 ( and ). Long-term treatment of fetal ICCs with the CXCR4 specific inhibitor AMD3100 blocks both cell growth and C-peptide release, suggesting that the SDF-1α/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 experiment.
To examine the dynamics of CXCR4/SDF-1α 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 Giα-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-1α treatment. However, inhibition of PI 3-kinase or canonical MAP kinase signaling attenuated proliferation ( 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-1α stimulates the phosphorylation of Akt and mitogen-activated protein (MAP) kinase in pancreatic duct cells 
In mouse islets, SDF-1α expression was restricted to cells surrounding the ducts and the microvasculature both around the ducts and in the islets 
. SDF-1α expression was also observed in the β-cells of the pancreas in neonatal mice up to 21 days of age, after which SDF-1α was no longer expressed in the β-cells 
. We have also observed SDF-1α expression the ducts and cells immediately surrounding the ducts in the human adult pancreas () with no apparent expression in the islets, suggesting similarities between the two systems. Further characterization of the SDF-1α expression in the human pancreas will be addressed in future studies.
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-1α under the control of the insulin promoter (RIP-SDF-1α mice) are somewhat protected against STZ-induced diabetes. Their studies also showed SDF-1α induced promotion of β-cell survival by Akt activation 
. Subsequent studies by the same group have suggested that the activation of the WNT pathway by SDF-1α may be part of the mechanism that promotes β-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-1α effect on proliferation in this context appears to be Akt dependent. SDF-1α 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 β-cells, an effect that involves GSK3 and Cyclins D1 and D2 
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 
. CXCR4 has been has been shown to be involved in the stimulation of apoptosis by HIV in both T cells and neurons 
. SDF-1α has also been shown to directly stimulate apoptosis in neural cells 
. Frequently, SDF-1α stimulation is associated with enhanced survival as reported in hematopoietic progenitor cells 
, fetal thymus 
and in bone marrow myelopoiesis 
. Previous studies have shown that SDF-1α has a pro-survival effect in mouse pancreas, MIN-6 cells, and INS-1 cells 
. Our observation of SDF-1α stimulation of apoptosis in CFPAC-1 cells was unexpected. It is conceivable that the stimulatory effect of SDF-1α 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-1α 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 β-cells.