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Reciprocal signalling between the endothelium and the pancreatic epithelium is crucial for coordinated differentiation of the embryonic endocrine and exocrine pancreas. In the adult pancreas, islets depend on their dense capillary network to adequately respond to changes in plasma glucose levels. Vascular changes contribute to the onset and progression of both type 1 and type 2 diabetes. Impaired revascularisation of islets transplanted in individuals with type 1 diabetes is linked to islet graft failure and graft loss. This review summarises our understanding of the role of vascular endothelial growth factor-A (VEGF-A) and endothelial cells in beta cell development, physiology and disease. In addition, the therapeutic potential of modulating VEGF-A levels in beta and beta-like cells for transplantation is discussed.

Diabetes mellitus is a pandemic metabolic disorder that results from either the autoimmune destruction or the dysfunction of insulin-producing pancreatic beta cells. A promising cure is beta cell replacement through the transplantation of islets of Langerhans. However, donor shortage hinders the widespread implementation of this therapy. Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, represent an attractive alternative beta cell source for transplantation. Although major advances over the past two decades have led to the generation of stem cell-derived beta-like cells that share many features with genuine beta cells, producing fully mature beta cells remains challenging. Here, we review the current status of beta cell differentiation protocols and highlight specific challenges that are associated with producing mature beta cells. We address the challenges and opportunities that are offered by monogenic forms of diabetes. Finally, we discuss the remaining hurdles for clinical application of stem cell-derived beta cells and the status of ongoing clinical trials., SCOPUS: re.j, info:eu-repo/semantics/published

Objective: The aggressive basal-like molecular subtype of pancreatic ductal adenocarcinoma (PDAC) harbours a ΔNp63 (p40) gene expression signature reminiscent of a basal cell type. Distinct from other epithelia with basal tumours, ΔNp63+ basal cells reportedly do not exist in the normal pancreas. Design: We evaluated ΔNp63 expression in human pancreas, chronic pancreatitis (CP) and PDAC. We further studied in depth the non-cancerous tissue and developed a three-dimensional (3D) imaging protocol (FLIP-IT, Fluorescence Light sheet microscopic Imaging of Paraffin-embedded or Intact Tissue) to study formalin-fixed paraffin-embedded samples at single cell resolution. Pertinent mouse models and HPDE cells were analysed. Results: In normal human pancreas, rare ΔNp63+ cells exist in ducts while their prevalence increases in CP and in a subset of PDAC. In non-cancer tissue, ΔNp63+ cells are atypical KRT19+ duct cells that overall lack SOX9 expression while they do express canonical basal markers and pertain to a niche of cells expressing gastrointestinal stem cell markers. 3D views show that the basal cells anchor on the basal membrane of normal medium to large ducts while in CP they exist in multilayer dome-like structures. In mice, ΔNp63 is not found in adult pancreas nor in selected models of CP or PDAC, but it is induced in organoids from larger Sox9low ducts. In HPDE, ΔNp63 supports a basal cell phenotype at the expense of a classical duct cell differentiation programme. Conclusion: In larger human pancreatic ducts, basal cells exist. ΔNp63 suppresses duct cell identity. These cells may play an important role in pancreatic disease, including PDAC ontogeny, but are not present in mouse models., SCOPUS: ar.j, info:eu-repo/semantics/published

SUMMARYObjectiveAn aggressive basal-like molecular subtype of pancreatic ductal adenocarcinoma (PDAC) exists, driven by ΔNp63. In other epithelia, ΔNp63+basal cells have stem cell capacity and can be at the origin of tumors. In the pancreas, basal cells have not been identified.DesignWe assessed basal cell markers in human and mouse pancreas, chronic pancreatitis and PDAC, and developed a 3D imaging protocol (FLIP-IT) to study sizeable samples at single cell resolution. We generated organoid cultures of ducts from Sox9-eGFP reporter mice.ResultsIn normal human pancreas, rare ΔNp63+cells exist in ducts that expand in chronic pancreatitis. ΔNp63+cells express KRT19 and canonical basal markers (KRT5, KRT14 and S100A2) but lack markers of duct cells such as CA19.9 and SOX9. In addition, ΔNp63+cells pertain to a niche of cells expressing gastrointestinal stem cell markers. 3D views of the ductal tree in formalin fixed paraffin embedded samples show that basal cells are localized on the basal membrane of medium to large ducts and expand as multilayer dome-like structures in chronic pancreatitis. In mice, ΔNp63 expression is induced when culturing organoids from Sox9-low ductal cells but could not be found in normal pancreas nor in models of pancreatitis or pancreatic cancer.ConclusionWe discovered a novel ductal cell population in normal human pancreas similar to basal cells in other tissues. Using FLIP-IT, we provide unprecedented 3D visualization of these cells in archival clinical specimens. ΔNp63+cells may play an important role in pancreatic tissue regeneration and cancer.SUMMARY BOXWhat is already known about this subject?ΔNp63 has a central role in determining the basal-like subtype of pancreatic ductal adenocarcinoma (PDAC).Different to other tissues with basal cancers, the normal pancreas reportedly does not contain (ΔNp63-expressing) basal cells.Current protocols face severe limitations for marker-based identification and 3D imaging of individual (rare) cells in archival pancreatic samples.What are the new findings?We report a rare and atypical pancreatic duct cell that expresses ΔNp63, other basal cell markers and g.i. stem cell markers.The number of these basal cells increases in diseases such as chronic pancreatitis and pancreatic cancer.We provide an easy to implement protocol for 3D clearing and high-resolution imaging of sizeable samples of (fresh or FFPE) human pancreas or an entire mouse pancreas.Except after culturing medium to large ducts as organoids, we fail to detect basal cells in mouse experimental pancreatic models.How might it impact on clinical practice in the foreseeable future?Extrapolating from knowledge in other organs, basal cells in the pancreas may have a stem cell/progenitor role, including in diseases such as (basal) pancreatic cancer.Use of the 3D imaging protocol in archival clinical specimens will allow unprecedented insights in pancreatic histopathology.For above mentioned diseases, we caution for findings in experimental mouse models that may not (fully) recapitulate the etiopathogenesis.

Reprogramming of pancreatic exocrine cells into cells resembling beta cells may provide a strategy for treating diabetes. Here we show that transient administration of epidermal growth factor and ciliary neurotrophic factor to adult mice with chronic hyperglycemia efficiently stimulates the conversion of terminally differentiated acinar cells to beta-like cells. Newly generated beta-like cells are epigenetically reprogrammed, functional and glucose responsive, and they reinstate normal glycemic control for up to 248 d. The regenerative process depends on Stat3 signaling and requires a threshold number of Neurogenin 3 (Ngn3)-expressing acinar cells. In contrast to previous work demonstrating in vivo conversion of acinar cells to beta-like cells by viral delivery of exogenous transcription factors, our approach achieves acinar-to-beta-cell reprogramming through transient cytokine exposure rather than genetic modification.

Ductal cells have been proposed as a source of adult β cell neogenesis, but this has remained controversial. By combining lineage tracing, 3D imaging, and single-cell RNA sequencing (scRNA-seq) approaches, we show that ductal cells contribute to the β cell population over time. Lineage tracing using the Neurogenin3 (Ngn3)-CreERT line identified ductal cells expressing the endocrine master transcription factor Ngn3 that were positive for the δ cell marker somatostatin and occasionally co-expressed insulin. The number of hormone-expressing ductal cells was increased in Akita+/- diabetic mice, and ngn3 heterozygosity accelerated diabetes onset. scRNA-seq of Ngn3 lineage-traced islet cells indicated that duct-derived somatostatin-expressing cells, some of which retained expression of ductal markers, gave rise to β cells. This study identified Ngn3-expressing ductal cells as a source of adult β cell neogenesis in homeostasis and diabetes, suggesting that this mechanism, in addition to β cell proliferation, maintains the adult islet β cell population.

AIMS/HYPOTHESIS: Endothelial-endocrine cell interactions and vascular endothelial growth factor (VEGF)-A signalling are deemed essential for maternal islet vascularisation, glucose control and beta cell expansion during mouse pregnancy. The aim of this study was to assess whether pregnancy-associated beta cell expansion was affected under conditions of islet hypovascularisation. METHODS: Soluble fms-like tyrosine kinase 1 (sFLT1), a VEGF-A decoy receptor, was conditionally overexpressed in maternal mouse beta cells from 1.5 to 14.5 days post coitum. Islet vascularisation, glycaemic control, beta cell proliferation, individual beta cell size and total beta cell volume were assessed in both pregnant mice and non-pregnant littermates. RESULTS: Conditional overexpression of sFLT1 in beta cells resulted in islet hypovascularisation and glucose intolerance in both pregnant and non-pregnant mice. In contrast to non-pregnant littermates, glucose intolerance in pregnant mice was transient. sFLT1 overexpression did not affect pregnancy-associated changes in beta cell proliferation, individual beta cell size or total beta cell volume. CONCLUSIONS/INTERPRETATION: Reduced intra-islet VEGF-A signalling results in maternal islet hypovascularisation and impaired glycaemic control but does not preclude beta cell expansion during mouse pregnancy.

We report a recurrent CNOT1 de novo missense mutation, GenBank: NM_016284.4; c.1603C>T (p.Arg535Cys), resulting in a syndrome of pancreatic agenesis and abnormal forebrain development in three individuals and a similar phenotype in mice. CNOT1 is a transcriptional repressor that has been suggested as being critical for maintaining embryonic stem cells in a pluripotent state. These findings suggest that CNOT1 plays a critical role in pancreatic and neurological development and describe a novel genetic syndrome of pancreatic agenesis and holoprosencephaly.

Aims/hypothesis Hypoxia in the initial days after islet transplantation leads to considerable loss of islet mass and contributes to disappointing outcomes in the clinical setting. The aim of the present study was to investigate whether co-transplantation of human non-endothelial bone marrow-derived multipotent adult progenitor cells (MAPCs), which are non-immunogenic and can secrete angiogenic growth factors during the initial days after implantation, could improve islet engraftment and survival. Methods Islets (150) were co-transplanted, with or without human MAPCs (2.5 × 105) as separate or composite pellets, under the kidney capsule of syngeneic alloxan-induced diabetic C57BL/6 mice. Blood glucose levels were frequently monitored and IPGTTs were carried out. Grafts and serum were harvested at 2 and 5 weeks after transplantation to assess outcome. Results Human MAPCs produced high amounts of angiogenic growth factors, including vascular endothelial growth factor, in vitro and in vivo, as demonstrated by the induction of neo-angiogenesis in the chorioallantoic membrane assay. Islet–human MAPC co-transplantation as a composite pellet significantly improved the outcome of islet transplantation as measured by the initial glycaemic control, diabetes reversal rate, glucose tolerance and serum C-peptide concentration compared with the outcome following transplantation of islets alone. Histologically, a higher blood vessel area and density in addition to a higher vessel/islet ratio were detected in recipients of islet–human MAPC composites. Conclusions/interpretation The present data suggest that co-transplantation of mouse pancreatic islets with human MAPCs, which secrete high amounts of angiogenic growth factors, enhance islet graft revascularisation and subsequently improve islet graft function. Electronic supplementary material The online version of this article (doi:10.1007/s00125-016-4120-3) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

AIMS/HYPOTHESIS: The initial avascular period following islet transplantation seriously compromises graft function and survival. Enhancing graft revascularisation to improve engraftment has been attempted through virus-based delivery of angiogenic triggers, but risks associated with viral vectors have hampered clinical translation. In vitro transcribed mRNA transfection circumvents these risks and may be used for improving islet engraftment. METHODS: Mouse and human pancreatic islet cells were transfected with mRNA encoding the angiogenic growth factor vascular endothelial growth factor A (VEGF-A) before transplantation under the kidney capsule in mice. RESULTS: At day 7 post transplantation, revascularisation of grafts transfected with Vegf-A (also known as Vegfa) mRNA was significantly higher compared with non-transfected or Gfp mRNA-transfected controls in mouse islet grafts (2.11- and 1.87-fold, respectively) (vessel area/graft area, mean ± SEM: 0.118 ± 0.01 [n = 3] in Vegf-A mRNA transfected group (VEGF) vs 0.056 ± 0.01 [n = 3] in no RNA [p

Summary Generating an unlimited source of human insulin-producing cells is a prerequisite to advance β cell replacement therapy for diabetes. Here, we describe a 3D culture system that supports the expansion of adult human pancreatic tissue and the generation of a cell subpopulation with progenitor characteristics. These cells display high aldehyde dehydrogenase activity (ALDHhi), express pancreatic progenitors markers (PDX1, PTF1A, CPA1, and MYC), and can form new organoids in contrast to ALDHlo cells. Interestingly, gene expression profiling revealed that ALDHhi cells are closer to human fetal pancreatic tissue compared with adult pancreatic tissue. Endocrine lineage markers were detected upon in vitro differentiation. Engrafted organoids differentiated toward insulin-positive (INS+) cells, and circulating human C-peptide was detected upon glucose challenge 1 month after transplantation. Engrafted ALDHhi cells formed INS+ cells. We conclude that adult human pancreatic tissue has potential for expansion into 3D structures harboring progenitor cells with endocrine differentiation potential., Highlights • A 3D culture system can support the expansion of adult human pancreatic tissue • An ALDHhi cell subpopulation is identified in these organoids • ALDHhi cells, and not ALDHlo cells, are capable of forming new organoids • ALDHhi cells show endocrine differentiation potential, In the context of β cell replacement therapy for diabetes, de Koning and colleagues describe a 3D culture platform that supports ex vivo expansion of human pancreatic tissue as organoids. These organoids harbor a subpopulation of ALDHhi cells that display proliferative capacity and can differentiate to an endocrine fate.

Inhibition of notch signaling is known to induce differentiation of endocrine cells in zebrafish and mouse. After performing an unbiased in vivo screen of ∼2,200 small molecules in zebrafish, we identified an inhibitor of Cdk5 (roscovitine), which potentiated the formation of β-cells along the intrapancreatic duct during concurrent inhibition of notch signaling. We confirmed and characterized the effect with a more selective Cdk5 inhibitor, (R)-DRF053, which specifically increased the number of duct-derived β-cells without affecting their proliferation. By duct-specific overexpression of the endogenous Cdk5 inhibitors Cdk5rap1 or Cdkal1 (which previously have been linked to diabetes in genome-wide association studies), as well as deleting cdk5, we validated the role of chemical Cdk5 inhibition in β-cell differentiation by genetic means. Moreover, the cdk5 mutant zebrafish displayed an increased number of β-cells independently of inhibition of notch signaling, in both the basal state and during β-cell regeneration. Importantly, the effect of Cdk5 inhibition to promote β-cell formation was conserved in mouse embryonic pancreatic explants, adult mice with pancreatic ductal ligation injury, and human induced pluripotent stem (iPS) cells. Thus, we have revealed a previously unknown role of Cdk5 as an endogenous suppressor of β-cell differentiation and thereby further highlighted its importance in diabetes.

Background:Commonly used islet transplantation protocols experiencesignificant islet injury and graft loss; therefore new methods to support thelong-term benefits of this treatment are needed. Recent studies have shownthat non-endothelial bone marrow-derived multipotent adult progenitor cells(MAPC) possess significant immune-modulating abilities and can secreteangiogenic molecules, which could make them ideal in islet transplantationprocedures to improve graft outcome.Methods:Islets (150) were co-transplanted with or without human MAPC(250 000), as separate or composite pellets, under the kidney capsule ofsyngeneic alloxan-induced diabetic C57BL/6 mice. Blood glucose levels werefrequently monitored and intraperitoneal glucose tolerance tests were carriedout. Grafts and serum were harvested at 2 and 5 weeks after transplantation.Results:Human MAPC were able to produce several angiogenic growthfactors, among which vascular endothelial growth factor (VEGF) is one of themost important, and to induce angiogenesis in thein vivochorioallantoicmembrane (CAM) assay. Islet-human MAPC co-transplantation particularly asa composite pellet significantly improved the outcome of islet transplantation asmeasured by the initial glycemic control, diabetes reversal rate, glucosetolerance, and serum C-peptide concentration compared with transplantationof islets alone. Likewise islet-human MAPC recipients had increased intra-graftCD31 gene expression levels compared to islet alone recipients, suggestingstable blood vessel formation. Indeed, significantly more blood vessel area anddensity in addition to higher vessel/islet ratio were detected with islets-humanMAPC composites, which suggests that direct contact with human MAPC ismore beneficial than indirect contact for mouse islets.Conclusions:The present data encourage the use of human MAPC in islettransplantation protocols. Our results demonstrate the improvement of isletgraft function. (Less)

A comparative analysis of mouse and human pancreatic development may reveal common mechanisms that control key steps as organ morphogenesis and cell proliferation and differentiation. More specifically, understanding beta cell development remains an issue, despite recent progress related to their generation from human embryonic and induced pluripotent stem cells. In this study, we use an integrated approach, including prospective isolation, organ culture, and characterization of intermediate stages, and report that cells from human and mouse fetal pancreas can be expanded in the long term and give rise to hollow duct-like structures in 3D cultures. The expanded cells express a combination of markers (E-cadherin, PDX1, NKX6-1, SOX9, and HNF1β) that reveals pancreatic progenitor identity. Proliferation of embryonic progenitors was stimulated by the Wnt agonist R-spondin1 (RSPO1), FGF10, and EGF. This combination of growth factors allowed maintaining human fetal pancreatic progenitors in culture for many passages, a finding not reported previously. Importantly, in the absence of EGF, proliferation was reduced, while endocrine differentiation was significantly enhanced. We conclude that modulation of EGF signaling affects in vitro expansion and differentiation of progenitors from embryonic pancreas of both mice and man.

Identifying pathways for beta cell generation is essential for cell therapy in diabetes. Here we investigate the potential of 17β-estradiol (E2) and estrogen receptor (ER) signaling for stimulating beta cell generation during embryonic development and in severely injured adult pancreas. Estradiol concentration, ER activity and number of ERα transcripts were enhanced in pancreas injured by partial duct ligation (PDL), along with nuclear localization of ERα in beta cells. PDL-induced proliferation of beta cells depended on aromatase activity. The activation of Neurogenin3 (Ngn3) gene expression and beta cell growth in PDL pancreas were impaired when ERα was turned off chemically or genetically (ERα(-/-)), while in situ delivery of E2 promoted beta cell formation. In embryonic pancreas, beta cell replication, number of Ngn3(+) progenitor cells and expression of key transcription factors of the endocrine lineage were decreased by ERα inactivation. The present study reveals that E2 and ERα signaling can drive betacell replication and formation in mouse pancreas.

Pancreas injury by partial duct ligation (PDL) activates a healing response, encompassing β-cell neogenesis and proliferation. Macrophages (MΦs) were recently shown to promote β-cell proliferation after PDL, but they remain poorly characterized. We assessed myeloid cell diversity and the factors driving myeloid cell dynamics following acute pancreas injury by PDL. In naive and sham-operated pancreas, the myeloid cell compartment consisted mainly of two distinct tissue-resident MΦ types, designated MHC-II(lo) and MHC-II(hi) MΦs, the latter being predominant. MHC-II(lo) and MHC-II(hi) pancreas MΦs differed at the molecular level, with MHC-II(lo) MΦs being more M2-activated. After PDL, there was an early surge of Ly6C(hi) monocyte infiltration in the pancreas, followed by a transient MHC-II(lo) MΦ peak and ultimately a restoration of the MHC-II(hi) MΦ-dominated steady-state equilibrium. These intricate MΦ dynamics in PDL pancreas depended on monocyte recruitment by C-C chemokine receptor 2 and macrophage-colony stimulating factor receptor as well as on macrophage-colony stimulating factor receptor-dependent local MΦ proliferation. Functionally, MHC-II(lo) MΦs were more angiogenic. We further demonstrated that, at least in C-C chemokine receptor 2-KO mice, tissue MΦs, rather than Ly6C(hi) monocyte-derived MΦs, contributed to β-cell proliferation. Together, our study fully characterizes the MΦ subsets in the pancreas and clarifies the complex dynamics of MΦs after PDL injury.

Background Pancreatic agenesis is an extremely rare cause of neonatal diabetes mellitus and has enabled the discovery of several key transcription factors essential for normal pancreas and beta cell development. Case presentation We report a case of a Caucasian female with complete pancreatic agenesis occurring together with semilobar holoprosencephaly (HPE), a more common brain developmental disorder. Clinical findings were later confirmed by autopsy, which also identified agenesis of the gallbladder. Although the sequences of a selected set of genes related to pancreas agenesis or HPE were wild-type, the patient’s phenotype suggests a genetic defect that emerges early in embryonic development of brain, gallbladder and pancreas. Conclusions Developmental defects of the pancreas and brain can occur together. Identifying the genetic defect may identify a novel key regulator in beta cell development.

International audience; The recent discovery that genetically modified α cells can regenerate and convert into β-like cells in vivo holds great promise for diabetes research. However, to eventually translate these findings to human, it is crucial to discover compounds with similar activities. Herein, we report the identification of GABA as an inducer of α-to-β-like cell conversion in vivo. This conversion induces α cell replacement mechanisms through the mobilization of duct-lining precursor cells that adopt an α cell identity prior to being converted into β-like cells, solely upon sustained GABA exposure. Importantly, these neo-generated β-like cells are functional and can repeatedly reverse chemically induced diabetes in vivo. Similarly, the treatment of transplanted human islets with GABA results in a loss of α cells and a concomitant increase in β-like cell counts, suggestive of α-to-β-like cell conversion processes also in humans. This newly discovered GABA-induced α cell-mediated β-like cell neogenesis could therefore represent an unprecedented hope toward improved therapies for diabetes.

Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5+ stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) that expand five-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality., Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis The establishment of conditions for long-term culture and expansion of adult, bi-potent pancreas progenitors may facilitate novel and tailored therapeutic approaches.

Creating an abundant source of β(-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 β(-like)-cells through transdifferentiation. While β(-like)-cells that are in vitro differentiated from human embryonic stem cells are already being grafted in patients, β(-like)-cells generated by transdifferentiation are not yet ready for clinical application. These cells would however offer several advantages over the current β(-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 β-cells.

AIMS/HYPOTHESIS: When the beta cell mass or function declines beyond a critical point, hyperglycaemia arises. Little is known about the potential pathways involved in beta cell rescue. As two cytokines, epidermal growth factor (EGF) and ciliary neurotrophic factor (CNTF), restored a functional beta cell mass in mice with long-term hyperglycaemia by reprogramming acinar cells that transiently expressed neurogenin 3 (NGN3), the current study assesses the effect of these cytokines on the functional beta cell mass after an acute chemical toxic insult. METHODS: Glycaemia and insulin levels, pro-endocrine gene expression and beta cell origin, as well as the role of signal transducer and activator of transcription 3 (STAT3) signalling, were assessed in EGF+CNTF-treated mice following acute hyperglycaemia. RESULTS: The mice were hyperglycaemic 1 day following i.v. injection of the beta cell toxin alloxan, when the two cytokines were applied. One week later, 68.6 ± 4.6% of the mice had responded to the cytokine treatment and increased their insulin(+) cell number to 30% that of normoglycaemic control mice, resulting in restoration of euglycaemia. Although insulin(-) NGN3(+) cells appeared following acute EGF+CNTF treatment, genetic lineage tracing showed that the majority of the insulin(+) cells originated from pre-existing beta cells. Beta cell rescue by EGF+CNTF depends on glycaemia rather than on STAT3-induced NGN3 expression in acinar cells. CONCLUSIONS/INTERPRETATION: In adult mice, EGF+CNTF allows the rescue of beta cells in distress when treatment is given shortly after the diabetogenic insult. The rescued beta cells restore a functional beta cell mass able to control normal blood glucose levels. These findings may provide new insights into compensatory pathways activated early after beta cell loss.

Partial pancreatic duct ligation (PDL) of mouse pancreas induces a doubling of the β-cell mass mainly through proliferation of pre-existing and newly formed β-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 β-cell proliferation were investigated. Duct ligation stimulates the expression of several cytokines that can act as ligands inducing STAT3 signaling and phosphorylation in β-cells. β-Cell cycling increased by conditional β-cell-specific Stat3 knockout and decreased by STAT3 activation through administration of interleukin-6. In addition, the level of DNA damage in β-cells of PDL pancreas increased after deletion of Stat3. These data indicate a role for STAT3 in maintaining a steady state in the β-cell, by modulating its cell cycle and protection from DNA damage.

Aims/hypothesis Pw1 or paternally-expressed gene 3 (Peg3) encodes a zinc finger transcription factor that is widely expressed during mouse embryonic development and later restricted to multiple somatic stem cell lineages in the adult. The aim of the present study was to define Pw1 expression in the embryonic and adult pancreas and investigate its role in the beta cell cycle in Pw1 wild-type and mutant mice. Methods We analysed PW1 expression by immunohistochemistry in pancreas of nonpregant and pregnant mice and following injury by partial duct ligation. Its role in the beta cell cycle was studied in vivo using a novel conditional knockout mouse and in vitro by lentivirus-mediated gene knockdown. Results We showed that PW1 is expressed in early pancreatic progenitors at E9.5 but becomes progressively restricted to fully differentiated beta cells as they become established after birth and withdraw from the cell cycle. Notably, PW1 expression declines when beta cells are induced to proliferate and loss of PW1 function activates the beta cell cycle. Conclusions/interpretation These results indicate that PW1 is a co-regulator of the beta cell cycle and can thus be considered a novel therapeutic target in diabetes. Electronic supplementary material The online version of this article (doi:10.1007/s00125-016-3954-z) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

The generation of beta(-like) cells to compensate for their absolute or relative shortage in type 1 and type 2 diabetes is an obvious therapeutic strategy. Patients first received grafts of donor islet cells over 25 years ago, but this procedure has not become routine in clinical practice because of a donor cell shortage and (auto)immune problems. Transplantation of differentiated embryonic and induced pluripotent stem cells may overcome some but not all the current limitations. Reprogramming exocrine cells towards functional beta(-like) cells would offer an alternative abundant and autologous source of beta(-like) cells. This review focuses on work by our research group towards achieving such a source of cells. It summarises a presentation given at the 'Can we make a better beta cell?' symposium at the 2015 annual meeting of the EASD. It is accompanied by two other reviews on topics from this symposium (by Amin Ardestani and Kathrin Maedler, DOI: 10.1007/s00125-016-3892-9 , and by Heiko Lickert and colleagues, DOI: 10.1007/s00125-016-3949-9 ) and a commentary by the Session Chair, Shanta Persaud (DOI: 10.1007/s00125-016-3870-2 ).

Despite recent advances, insulin therapy remains a treatment, not a cure, for diabetes mellitus with persistent risk of glycemic alterations and life-threatening complications. Restoration of the endogenous beta cell mass through regeneration or transplantation offers an attractive alternative. Unfortunately, signals that drive beta cell regeneration remain enigmatic and beta 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 beta cell therapy and focuses on the potential benefits of accessory cells for islet transplantation in diabetes. ispartof: Diabetes, Obesity & Metabolism vol:18 issue:2 pages:115-124 ispartof: location:England status: published

Transcription factor mediated lineage reprogramming of human pancreatic exocrine tissue could conceivably provide an unlimited supply of islets for transplantation in the treatment of diabetes. Exocrine tissue can be efficiently reprogrammed to islet-like cells using a cocktail of transcription factors: Pdx1, Ngn3, MafA and Pax4 in combination with growth factors. We show here that overexpression of exogenous Pax4 in combination with suppression of the endogenous transcription factor ARX considerably enhances the production of functional insulin-secreting β-like cells with concomitant suppression of α-cells. The efficiency was further increased by culture on laminin-coated plates in media containing low glucose concentrations. Immunocytochemistry revealed that reprogrammed cultures were composed of ~45% islet-like clusters comprising >80% monohormonal insulin+ cells. The resultant β-like cells expressed insulin protein levels at ~15-30% of that in adult human islets, efficiently processed proinsulin and packaged insulin into secretory granules, exhibited glucose responsive insulin secretion, and had an immediate and prolonged effect in normalising blood glucose levels upon transplantation into diabetic mice. We estimate that approximately 3 billion of these cells would have an immediate therapeutic effect following engraftment in type 1 diabetes patients and that one pancreas would provide sufficient tissue for numerous transplants.

AIMS/HYPOTHESIS: Efficient stimulation of cycling activity in cultured beta cells would allow the design of new strategies for cell therapy in diabetes. Neural crest stem cells (NCSCs) play a role in beta cell development and maturation and increase the beta cell number in co-transplants. The mechanism behind NCSC-induced beta cell proliferation and the functional capacity of the new beta cells is not known. METHODS: We developed a new in vitro co-culture system that enables the dissection of the elements that control the cellular interactions that lead to NCSC-dependent increase in islet beta cells. RESULTS: Mouse NCSCs were cultured in vitro, first in medium that stimulated their proliferation, then under conditions that supported their differentiation. When mouse islet cells were cultured together with the NCSCs, more than 35% of the beta cells showed cycle activity. This labelling index is more than tenfold higher than control islets cultured without NCSCs. Beta cells that proliferated under these culture conditions were fully glucose responsive in terms of insulin secretion. NCSCs also induced beta cell proliferation in islets isolated from 1-year-old mice, but not in dissociated islet cells isolated from human donor pancreas tissue. To stimulate beta cell proliferation, NCSCs need to be in intimate contact with the beta cells. CONCLUSIONS/INTERPRETATION: Culture of islet cells in contact with NCSCs induces highly efficient beta cell proliferation. The reported culture system is an excellent platform for further dissection of the minimal set of factors needed to drive this process and explore its potential for translation to diabetes therapy.

OBJECTIVE Generating functional β-cells by inducing their proliferation may provide new perspectives for cell therapy in diabetes. Transcription factor E2F1 controls G1- to S-phase transition during the cycling of many cell types and is required for pancreatic β-cell growth and function. However, the consequences of overexpression of E2F1 in β-cells are unknown. RESEARCH DESIGN AND METHODS The effects of E2F1 overexpression on β-cell proliferation and function were analyzed in isolated rat β-cells and in transgenic mice. RESULTS Adenovirus AdE2F1-mediated overexpression of E2F1 increased the proliferation of isolated primary rat β-cells 20-fold but also enhanced β-cell death. Coinfection with adenovirus AdAkt expressing a constitutively active form of Akt (protein kinase B) suppressed β-cell death to control levels. At 48 h after infection, the total β-cell number and insulin content were, respectively, 46 and 79% higher in AdE2F1+AdAkt-infected cultures compared with untreated. Conditional overexpression of E2F1 in mice resulted in a twofold increase of β-cell proliferation and a 70% increase of pancreatic insulin content, but did not increase β-cell mass. Glucose-challenged insulin release was increased, and the mice showed protection against toxin-induced diabetes. CONCLUSIONS Overexpression of E2F1, either in vitro or in vivo, can stimulate β-cell proliferation activity. In vivo E2F1 expression significantly increases the insulin content and function of adult β-cells, making it a strategic target for therapeutic manipulation of β-cell function.

AIMS/HYPOTHESIS: Prolonged exposure of rat beta cells to the insulin secretagogue glibenclamide has been found to induce a sustained increase in basal insulin synthesis. This effect was calcium-dependent and localised in cells that had been degranulated by the drug. Since it was blocked by the translation inhibitor cycloheximide, we examined whether sustained exposure to glibenclamide activates translational factors by calcium-dependent signalling pathways. METHODS: Purified rat beta cells were cultured with and without glibenclamide in the presence or absence of inhibitors of calcium-dependent signalling pathways before measurement of basal and stimulated protein and insulin synthesis, and assessment of abundance of (phosphorylated) translation factors. RESULTS: A 24 h exposure to glibenclamide induced activation of four translation factors, i.e. phosphorylation of eukaryotic initiation factor (eIF) 4e binding protein 1 and ribosomal protein S6 (rpS6), and dephosphorylation of eIF-2alpha and eukaryotic elongation factor 2. The rise in phospho-rpS6 intensity was localised to a subpopulation of beta cells with low insulin content. This activation of translational factors and the associated elevation of insulin synthesis were completely blocked by the calcium channel blocker verapamil and partially blocked by the mammalian target of rapamycin (mTOR) inhibitor rapamycin, the protein kinase A (PKA) inhibitor Rp-8-Br-cAMPs and the mitogen-activated protein kinase/ extracellular signal-regulated kinase kinase (MEK) inhibitor U0126; a combination of inhibitors exhibited additive effects. CONCLUSIONS/INTERPRETATION: Prolonged exposure to glibenclamide activates protein translation in pancreatic beta cells through the calcium-regulated mTOR, PKA and MEK signalling pathways. The observed intercellular differences in translation activation are proposed as underlying mechanism for functional heterogeneity in the pancreatic beta cell population.

Background and purpose: Two mechanisms have been proposed to explain the insulin-sensitising properties of metformin in peripheral tissues: (a) inhibition of electron transport chain complex I, and (b) activation of the AMP activated protein kinase (AMPK). However the relationship between these mechanisms and their contribution to β-cell death and dysfunction in vitro, are currently unclear. Experimental approach: The effects of biguanides (metformin and phenformin) were tested on MIN6 β-cells and primary FACS-purified rat β-cells. Cell metabolism was assessed biochemically and by FACS analysis, and correlated with AMPK phosphorylation state and cell viability, with or without fuel substrates. Key results: In MIN6 cells, metformin reduced mitochondrial complex I activity by up to 44% and a 25% net reduction in mitochondrial reducing potential. In rat β-cells, metformin caused NAD(P)H accumulation above maximal glucose-inducible levels, mimicking the effect of rotenone. Drug exposure caused phosphorylation of AMPK on Thr172 in MIN6 cell extracts, indicative of kinase activation. Methyl succinate, a complex II substrate, appeared to bypass metformin blockade of complex I. This resulted in reduced phosphorylation of AMPK, establishing a link between biguanide-induced mitochondrial inhibition and AMPK activation. Corresponding assessment of cell death indicated that methyl succinate decreased biguanide toxicity to β-cells in vitro. Conclusions and implications: AMPK activation can partly be attributed to metformin's inhibitory action on mitochondrial complex I. Anaplerotic fuel metabolism via complex II rescued β-cells from metformin-associated toxicity. We propose that utilisation of anaplerotic nutrients may reconcile in vitro and in vivo effects of metformin on the pancreatic β-cell. British Journal of Pharmacology (2007) 150, 1031–1043. doi:10.1038/sj.bjp.0707189

One of the key promises of regenerative medicine is providing a cure for diabetes. Cell-based therapies are proving their safety and efficiency, but donor beta cell shortages and immunological issues remain major hurdles. Reprogramming of human pancreatic exocrine cells towards beta cells would offer a major advantage by providing an abundant and autologous source of beta cells. Over the past decade our understanding of transdifferentiation processes greatly increased allowing us to design reprogramming protocols that fairly aim for clinical trials.

In vitro cultured pancreatic acinar cells rapidly differentiate. Low temperature exposure prevents this process and improves the efficiency of acinar cell labelling with adenovirus vectors. This may help in tracing β-cell neogenesis from human pancreatic acinar cells., The regenerative medicine field is expanding with great successes in laboratory and preclinical settings. Pancreatic acinar cells in diabetic mice were recently converted into β-cells by treatment with ciliary neurotrophic factor (CNTF) and epidermal growth factor (EGF). This suggests that human acinar cells might become a cornerstone for diabetes cell therapy in the future, if they can also be converted into glucose-responsive insulin-producing cells. Presently, studying pancreatic acinar cell biology in vitro is limited by their high plasticity, as they rapidly lose their phenotype and spontaneously transdifferentiate to a duct-like phenotype in culture. We questioned whether human pancreatic acinar cell phenotype could be preserved in vitro by physico-chemical manipulations and whether this could be valuable in the study of β-cell neogenesis. We found that culture at low temperature (4°C) resulted in the maintenance of morphological and molecular acinar cell characteristics. Specifically, chilled acinar cells did not form the spherical clusters observed in controls (culture at 37°C), and they maintained high levels of acinar-specific transcripts and proteins. Five-day chilled acinar cells still transdifferentiated into duct-like cells upon transfer to 37°C. Moreover, adenoviral-mediated gene transfer evidenced an active Amylase promoter in the 7-day chilled acinar cells, and transduction performed in chilled conditions improved acinar cell labelling. Together, our findings indicate the maintenance of human pancreatic acinar cell phenotype at low temperature and the possibility to efficiently label acinar cells, which opens new perspectives for the study of human acinar-to-β-cell transdifferentiation.

Rodent acinar cells exhibit a remarkable plasticity as they can transdifferentiate to duct-, hepatocyte- and islet β-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.