Making ß(-like)-cells from exocrine pancreas.

Creating an abundant source of ß(-like)-cells has been a major goal in diabetes research for many decades. The concept of cell plasticity has inspired many strategies towards regenerative medicine, but its successes have been limited until very recently. Today, most cell types in the pancreas are considered candidates for the generation of ß(-like)-cells through transdifferentiation. While ß(-like)-cells that are in vitro differentiated from human embryonic stem cells are already being grafted in patients, ß(-like)-cells generated by transdifferentiation are not yet ready for clinical application. These cells would however offer several advantages over the current ß(-like)-cells generated by directed differentiation, especially concerning safety issues. In addition, perfect control of the transdifferentiation efficiency would through targeted drug delivery support a non-invasive cell therapy for diabetes. Lastly, focusing on the exocrine pancreas as prime candidate makes sense in view of their abundance and high plasticity. Keeping these hopeful perspectives in mind, it is worth to continue focused research on the mechanisms that control transdifferentiation from pancreas exocrine to ß-cells.

STAT3 modulates ß-cell cycling in injured mouse pancreas and protects against DNA damage.

Partial pancreatic duct ligation (PDL) of mouse pancreas induces a doubling of the ß-cell mass mainly through proliferation of pre-existing and newly formed ß-cells. The molecular mechanism governing this process is still largely unknown. Given the inflammatory nature of PDL and inflammation-induced signaling via the signal transducer and activator of transcription 3 (STAT3), the activation and the role of STAT3 in PDL-induced ß-cell proliferation were investigated. Duct ligation stimulates the expression of several cytokines that can act as ligands inducing STAT3 signaling and phosphorylation in ß-cells. ß-Cell cycling increased by conditional ß-cell-specific Stat3 knockout and decreased by STAT3 activation through administration of interleukin-6. In addition, the level of DNA damage in ß-cells of PDL pancreas increased after deletion of Stat3. These data indicate a role for STAT3 in maintaining a steady state in the ß-cell, by modulating its cell cycle and protection from DNA damage.

Accessory cells for ß-cell transplantation.

Despite recent advances, insulin therapy remains a treatment, not a cure, for diabetes mellitus with persistent risk of glycaemic alterations and life-threatening complications. Restoration of the endogenous ß-cell mass through regeneration or transplantation offers an attractive alternative. Unfortunately, signals that drive ß-cell regeneration remain enigmatic and ß-cell replacement therapy still faces major hurdles that prevent its widespread application. Co-transplantation of accessory non-islet cells with islet cells has been shown to improve the outcome of experimental islet transplantation. This review will highlight current travails in ß-cell therapy and focuses on the potential benefits of accessory cells for islet transplantation in diabetes.

Inducible VEGF expression by human embryonic stem cell-derived mesenchymal stromal cells reduces the minimal islet mass required to reverse diabetes.

Islet transplantation has been hampered by loss of function due to poor revascularization. We hypothesize that co-transplantation of islets with human embryonic stem cell-derived mesenchymal stromal cells that conditionally overexpress VEGF (hESC-MSC:VEGF) may augment islet revascularization and reduce the minimal islet mass required to reverse diabetes in mice. HESC-MSCs were transduced by recombinant lentiviruses that allowed conditional (Dox-regulated) overexpression of VEGF. HESC-MSC: VEGF were characterized by tube formation assay. After co-transplantation of hESC-MSC:VEGF with murine islets in collagen-fibrin hydrogel in the omental pouch of diabetic nude mice, we measured blood glucose, body weight, glucose tolerance and serum C-peptide. As control, islets were transplanted alone or with non-transduced hESC-MSCs. Next, we compared functional parameters of 400 islets alone versus 200 islets co-transplanted with hESC-MSC:VEGF. As control, 200 islets were transplanted alone. Metabolic function of islets transplanted with hESC-MSC:VEGF significantly improved, accompanied by superior graft revascularization, compared with control groups. Transplantation of 200 islets with hESC-MSC:VEGF showed superior function over 400 islets alone. We conclude that co-transplantation of islets with VEGF-expressing hESC-MSCs allowed for at least a 50% reduction in minimal islet mass required to reverse diabetes in mice. This approach may contribute to alleviate the need for multiple donor organs per patient.

Reprogramming of human pancreatic exocrine cells to ß-like cells.

Rodent acinar cells exhibit a remarkable plasticity as they can transdifferentiate to duct-, hepatocyte- and islet ß-like cells. We evaluated whether exocrine cells from adult human pancreas can similarly respond to proendocrine stimuli. Exocrine cells from adult human pancreas were transduced directly with lentiviruses expressing activated MAPK (mitogen-activated protein kinase) and STAT3 (signal transducer and activator of transcription 3) and cultured as monolayers or as 3D structures. Expression of STAT3 and MAPK in human exocrine cells activated expression of the proendocrine factor neurogenin 3 in 50% to 80% of transduced exocrine cells. However, the number of insulin-positive cells increased only in the exocrine cells grown initially in suspension before 3D culture. Lineage tracing identified human acinar cells as the source of Ngn3- and insulin-expressing cells. Long-term engraftment into immunocompromised mice increased the efficiency of reprogramming to insulin-positive cells. Our data demonstrate that exocrine cells from human pancreas can be reprogrammed to transplantable insulin-producing cells that acquire functionality. Given the large number of exocrine cells in a donor pancreas, this approach presents a novel strategy to expand cell therapy in type 1 diabetes.

Human blood outgrowth endothelial cells improve islet survival and function when co-transplanted in a mouse model of diabetes.

AIMS/HYPOTHESIS: As current islet-transplantation protocols suffer from significant graft loss and dysfunction, strategies to sustain the long-term benefits of this therapy are required. Rapid and adequate oxygen and nutrient delivery by blood vessels improves islet engraftment and function. The present report evaluated a potentially beneficial effect of adult human blood outgrowth endothelial cells (BOEC) on islet graft vascularisation and function. METHODS: Human BOEC, 5 × 10(5), were co-transplanted with a rat marginal-islet graft under the kidney capsule of hyperglycaemic NOD severe combined immunodeficiency (SCID) mice, and the effect on metabolic outcome was evaluated. RESULTS: Although vessel density remained unaffected, co-transplantation of islets with BOEC resulted in a significant and specific improvement of glycaemia and increased plasma C-peptide. Moreover, in contrast to control mice, BOEC recipients displayed reduced beta cell death and increases in body weight, beta cell proliferation and graft-vessel and beta cell volume. In vivo cell tracing demonstrated that BOEC remain at the site of transplantation and do not expand. The potential clinical applicability was underscored by the observed metabolic benefit of co-transplanting islets with BOEC derived from a type 1 diabetes patient. CONCLUSIONS/INTERPRETATION: The present data support the use of autologous BOEC in translational studies that aim to improve current islet-transplantation protocols for the treatment of brittle type 1 diabetes.

Ectopic expression of neurogenin 3 in neonatal pig pancreatic precursor cells induces (trans)differentiation to functional alpha cells.

AIMS/HYPOTHESIS: Neurogenin 3 (NEUROG3), a basic helix-loop-helix transcription factor that is needed for endocrine cell development in the embryonic pancreas, has been shown to induce transdifferentiation of duct cells from adult pancreas towards a neuro-endocrine phenotype. Our study explored the endocrine transdifferentiation potential of NEUROG3 in neonatal pancreatic precursor cells. MATERIALS AND METHODS: A replication-deficient adenovirus expressing Neurog3 and green fluorescent protein (GFP) (Ad-NEUROG3) was used to infect neonatal pig pancreatic cell preparations enriched for endocrine islet and cytokeratin-positive precursor cells. GFP-positive cells were sorted using flow cytometry on days 3 and 8 after infection and characterised at the transcript and protein level. For in vivo experiments, the total population of Ad-NEUROG3-infected pancreatic cells was transplanted, then later removed for determination of graft hormone content and immunohistochemistry. RESULTS: Among the GFP-positive cells, the fraction of precursor cells decreased by more than 85% at day 8 after infection, while the fraction of glucagon-positive cells increased 2.5-fold and the beta cell number remained the same. Transplantation of the Ad-NEUROG3-infected pancreatic cell preparation failed to reverse streptozotocin-induced hyperglycaemia, while non-infected cells and a control cell preparation infected with replication-deficient adenovirus expressing only GFP were able to do so. At day 109 after transplantation, kidneys grafted with Ad-NEUROG3-infected pancreatic cells contained significantly decreased insulin and increased glucagon levels. Abundant glucagon-immunopositive cells were seen in Ad-NEUROG3-infected grafts, which were virtually devoid of proliferating insulin-positive cells. CONCLUSIONS/INTERPRETATION: In summary, adenoviral delivery of NEUROG3 to pancreatic precursor cells from neonatal pig pancreas promotes alpha cell differentiation in vitro and in vivo.

A comprehensive analysis of cytokine-induced and nuclear factor-kappa B-dependent genes in primary rat pancreatic beta-cells.

Type 1 diabetes mellitus results from an autoimmune destruction of pancreatic beta-cells. Cytokines, such as interleukin-1 beta and interferon-gamma, are putative mediators of immune-induced beta-cell death and, under in vitro conditions, cause beta-cell apoptosis. We have recently shown that interleukin-1 beta + interferon-gamma modifies the expression of >200 genes in beta-cells. Several of these genes are putative targets for the transcription factor nuclear factor-kappa B (NF-kappa B), and in subsequent experiments we showed that NF-kappa B activation is mostly pro-apoptotic in beta-cells. To identify cytokine-induced and NF-kappa B-regulated genes in primary rat beta-cells, we presently combined two experimental approaches: 1) blocking of NF-kappa B activation in cytokine-exposed beta-cells by a recombinant adenovirus (AdI kappa B((SA)2)) containing an inhibitor of NF-kappa B alpha (I kappa Bac) super-repressor (S32A/S36A) and 2) study of gene expression by microarray analysis. We identified 66 cytokine-modified and NF-kappa B-regulated genes in beta-cells. Cytokine-induced NF-kappa B activation decreased Pdx-1 and increased c-Myc expression. This, together with NF-kappa B-dependent inhibition of Glut-2, pro-hormone convertase-1, and Isl-1 expression, probably contributes to the loss of differentiated beta-cell functions. NF-kappa B also regulates several genes encoding for chemokines and cytokines in beta-cells. The present data suggest that NF-kappa B is a key "switch regulator" of transcription factors and gene networks controlling cytokine-induced beta-cell dysfunction and death.

Inhibition of cytokine-induced NF-kappaB activation by adenovirus-mediated expression of a NF-kappaB super-repressor prevents beta-cell apoptosis.

Cytokine-induced beta-cell death is an important event in the pathogenesis of type 1 diabetes. The transcription factor nuclear factor-kappaB (NF-kappaB) is activated by interleukin-1beta (IL-1beta), and its activity promotes the expression of several beta-cell genes, including pro- and anti-apoptotic genes. To elucidate the role of cytokine (IL-1beta + gamma-interferon [IFN-gamma])-induced expression of NF-kappaB in beta-cell apoptosis, rat beta-cells were infected with the recombinant adenovirus AdIkappaB((SA)2), which contained a nondegradable mutant form of inhibitory kappaB (IkappaB((SA)2), with S32A and S36A) that locks NF-kappaB in a cytosolic protein complex, preventing its nuclear action. Expression of IkappaB((SA)2) inhibited cytokine-stimulated nuclear translocation and DNA-binding of NF-kappaB. Cytokine-induced gene expression of several NF-kappaB targets, namely inducible nitric oxide synthase, Fas, and manganese superoxide dismutase, was prevented by AdIkappaB((SA)2), as established by reverse transcriptase-polymerase chain reaction, protein blot, and measurement of nitrite in the medium. Finally, beta-cell survival after IL-1beta + IFN-gamma treatment was significantly improved by IkappaB((SA)2) expression, mostly through inhibition of the apoptotic pathway. Based on these findings, we conclude that NF-kappaB activation, under in vitro conditions, has primarily a pro-apoptotic function in beta-cells.

Modulation of rat pancreatic acinoductal transdifferentiation and expression of PDX-1 in vitro.

AIMS/HYPOTHESIS: In adult pancreatic regeneration models exocrine acini are found to transdifferentiate to duct-like complexes. This has also been associated with the formation of new endocrine islet cells. We aimed to establish an in vitro model in which this transdifferentiation process is characterised and can be modulated. METHODS: Purified rat pancreatic acini were cultured in suspension. Differentiation was analysed by immunocytochemistry, electron microscopy, western blotting and RT-PCR. RESULTS: During culture acinar cells directly transdifferentiated without dividing, the cells lost their acinar phenotype and started to express cytokeratins 20 and 7 and fetal liver kinase-1 (Flk-1) receptors for vascular endothelial growth factor. Expression of the acinar pancreatic exocrine transcription factor (PTF-1) remained and the pancreatic duodenal homeobox-containing transcription factor (PDX-1) was induced. When transdifferentiation was completed, the cells started to express protein gene product 9.5, a panneuroendocrine marker. By combining these features, the transdifferentiated cells show similar characteristics to precursor cells during active beta-cell neogenesis. We were able to modulate the differentiation state by addition of nicotinamide or sodium butyrate, agents which are known to stimulate endocrine differentiation in other models. CONCLUSION/INTERPRETATION: Here, we present an in vitro system in which the cellular differentiation of putative pancreatic endocrine precursor cells and their PDX-1 expression can be modulated, thereby providing a possible model for the study of beta-cell transdifferentiation.

Adult human pancreatic duct and islet cells exhibit similarities in expression and differences in phosphorylation and complex formation of the homeodomain protein Ipf-1.

The homeodomain transcription factor encoded by the pancreatic and duodenal homeobox gene-1 (Ipf-1) is essential for pancreatic ontogenesis. Whether Ipf-1 is also involved in the neogenesis of beta-cells in the adult pancreas is unknown. We examined whether Ipf-1 is expressed in adult human pancreatic ducts, which are thought to generate new beta-cells. In tissue sections, virtually all duct cells were immunopositive for Ipf-1, as were the islet beta-cells but not the acinar cells. After isolation and culture, both duct and islet cell preparations contained the Ipf-1 immunoreactive proteins p42 and p45 (42 and 45 kDa, respectively) in similar proportions, but the expression levels were twofold lower in duct cells. After 4 h of labeling, the endocrine cells exhibited a sevenfold higher phosphorylation of p42 than the duct cells, whereas p45 was phosphorylated only in endocrine cells. Homeobox binding transcription factor complexes with Ipf-1 in duct cells differed from those in endocrine cells in terms of gel mobility, sequence specificity, and affinity. The observed similarities in Ipf-1 expression by adult human pancreatic duct cells and endocrine cells may reflect their common ontogenic origin, whereas the differences in Ipf-1 phosphorylation and complex formation may correlate with their divergent differentiation.