Discovery and 3D imaging of a novel ΔNp63-expressing basal cell type in human pancreatic ducts with implications in disease.

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.

Retraction Note: Transient cytokine treatment induces acinar cell reprogramming and regenerates functional beta cell mass in diabetic mice.

This article has been retracted; see accompanying Retraction Note, which can be accessed via a link at the top of the paper.

Evidence of Tissue Repair in Human Donor Pancreas After Prolonged Duration of Stay in Intensive Care.

M2 macrophages play an important role in tissue repair and regeneration. They have also been found to modulate ß-cell replication in mouse models of pancreatic injury and disease. We previously reported that ß-cell replication is strongly increased in a subgroup of human organ donors characterized by prolonged duration of stay in an intensive care unit (ICU) and increased number of leukocytes in the pancreatic tissue. In the present study we investigated the relationship between duration of stay in the ICU, M2 macrophages, vascularization, and pancreatic cell replication. Pancreatic organs from 50 donors without diabetes with different durations of stay in the ICU were analyzed by immunostaining and digital image analysis. The number of CD68+CD206+ M2 macrophages increased three- to sixfold from ≥6 days' duration of stay in the ICU onwards. This was accompanied by a threefold increased vascular density and a four- to ninefold increase in pancreatic cells positive for the replication marker Ki67. A strong correlation was observed between the number of M2 macrophages and ß-cell replication. These results show that a prolonged duration of stay in the ICU is associated with an increased M2 macrophage number, increased vascular density, and an overall increase in replication of all pancreatic cell types. Our data show evidence of marked levels of tissue repair in the human donor pancreas.

Vegf-A mRNA transfection as a novel approach to improve mouse and human islet graft revascularisation.

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 < 0.05] vs 0.063 ± 0.02 [n = 4] in Gfp mRNA transfected group (GFP) [p < 0.05]); EndoC-bH3 grafts (2.85- and 2.48-fold. respectively) (0.085 ± 0.02 [n = 4] in VEGF vs 0.030 ± 0.004 [n = 4] in no RNA [p < 0.05] vs 0.034 ± 0.01 [n = 5] in GFP [p < 0.05]); and human islet grafts (3.17- and 3.80-fold, respectively) (0.048 ± 0.013 [n = 3] in VEGF vs 0.015 ± 0.0051 [n = 4] in no RNA [p < 0.01] vs 0.013 ± 0.0046 [n = 4] in GFP [p < 0.01]). At day 30 post transplantation, human islet grafts maintained a vascularisation benefit (1.70- and 1.82-fold, respectively) (0.049 ± 0.0042 [n = 8] in VEGF vs 0.029 ± 0.0052 [n = 5] in no RNA [p < 0.05] vs 0.027 ± 0.0056 [n = 4] in GFP [p < 0.05]) and a higher beta cell volume (1.64- and 2.26-fold, respectively) (0.0292 ± 0.0032 µl [n = 7] in VEGF vs 0.0178 ± 0.0021 µl [n = 5] in no RNA [p < 0.01] vs 0.0129 ± 0.0012 µl [n = 4] in GFP [p < 0.001]). CONCLUSIONS/INTERPRETATION: Vegf-A mRNA transfection before transplantation provides a promising and safe strategy to improve engraftment of islets and other cell-based implants.

Semi-automated digital measurement as the method of choice for beta cell mass analysis.

Pancreas injury by partial duct ligation (PDL) activates beta cell differentiation and proliferation in adult mouse pancreas but remains controversial regarding the anticipated increase in beta cell volume. Several reports unable to show beta cell volume augmentation in PDL pancreas used automated digital image analysis software. We hypothesized that fully automatic beta cell morphometry without manual micrograph artifact remediation introduces bias and therefore might be responsible for reported discrepancies and controversy. However, our present results prove that standard digital image processing with automatic thresholding is sufficiently robust albeit less sensitive and less adequate to demonstrate a significant increase in beta cell volume in PDL versus Sham-operated pancreas. We therefore conclude that other confounding factors such as quality of surgery, selection of samples based on relative abundance of the transcription factor Neurogenin 3 (Ngn3) and tissue processing give rise to inter-laboratory inconsistencies in beta cell volume quantification in PDL pancreas.

Inhibition of Cdk5 Promotes ß-Cell Differentiation From Ductal Progenitors.

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 ß-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 ß-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 ß-cell differentiation by genetic means. Moreover, the cdk5 mutant zebrafish displayed an increased number of ß-cells independently of inhibition of notch signaling, in both the basal state and during ß-cell regeneration. Importantly, the effect of Cdk5 inhibition to promote ß-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 ß-cell differentiation and thereby further highlighted its importance in diabetes.

Conditional islet hypovascularisation does not preclude beta cell expansion during pregnancy in mice.

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.

Long-Term GABA Administration Induces Alpha Cell-Mediated Beta-like Cell Neogenesis.

The recent discovery that genetically modified α cells can regenerate and convert into ß-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-ß-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 ß-like cells, solely upon sustained GABA exposure. Importantly, these neo-generated ß-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 ß-like cell counts, suggestive of α-to-ß-like cell conversion processes also in humans. This newly discovered GABA-induced α cell-mediated ß-like cell neogenesis could therefore represent an unprecedented hope toward improved therapies for diabetes.

Human multipotent adult progenitor cells enhance islet function and revascularisation when co-transplanted as a composite pellet in a mouse model of diabetes.

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.

The zinc finger transcription factor PW1/PEG3 restrains murine beta cell cycling.

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.

Sources of beta cells inside the pancreas.

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 ).

Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation.

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide new expandable sources of beta cells to alleviate the donor scarcity in human islet transplantation as therapy for diabetes. Although recent advances have been made towards this aim, mechanisms that regulate beta cell expansion and differentiation from a stem/progenitor cell remain to be characterized. Here, we describe a protocol for an injury model in the adult mouse pancreas that can function as a tool to study mechanisms of tissue remodeling and beta cell proliferation and differentiation. Partial duct ligation (PDL) is an experimentally induced injury of the rodent pancreas involving surgical ligation of the main pancreatic duct resulting in an obstruction of drainage of exocrine products out of the tail region of the pancreas. The inflicted damage induces acinar atrophy, immune cell infiltration and severe tissue remodeling. We have previously reported the activation of Neurogenin (Ngn) 3 expressing endogenous progenitor-like cells and an increase in beta cell proliferation after PDL. Therefore, PDL provides a basis to study signals involved in beta cell dynamics and the properties of an endocrine progenitor in adult pancreas. Since, it still remains largely unclear, which factors and pathways contribute to beta cell neogenesis and proliferation in PDL, a standardized protocol for PDL will allow for comparison across laboratories.

Estrogen Receptor α Regulates ß-Cell Formation During Pancreas Development and Following Injury.

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

Concise Review: Macrophages: Versatile Gatekeepers During Pancreatic ß-Cell Development, Injury, and Regeneration.

UNLABELLED: Macrophages are classically considered detrimental for pancreatic ß-cell survival and function, thereby contributing to ß-cell failure in both type 1 (T1D) and 2 (T2D) diabetes mellitus. In addition, adipose tissue macrophages negatively influence peripheral insulin signaling and promote obesity-induced insulin resistance in T2D. In contrast, recent data unexpectedly uncovered that macrophages are not only able to protect ß cells during pancreatitis but also to orchestrate ß-cell proliferation and regeneration after ß-cell injury. Moreover, by altering their activation state, macrophages are able to improve insulin resistance in murine models of T2D. This review will elaborate on current insights in macrophage heterogeneity and on the evolving role of pancreas macrophages during organogenesis, tissue injury, and repair. Additional identification of macrophage subtypes and of their secreted factors might ultimately translate into novel therapeutic strategies for both T1D and T2D. SIGNIFICANCE: Diabetes mellitus is a pandemic disease, characterized by severe acute and chronic complications. Macrophages have long been considered prime suspects in the pathogenesis of both type 1 and 2 diabetes mellitus. In this concise review, current insights in macrophage heterogeneity and on the, as yet, underappreciated role of alternatively activated macrophages in insulin sensing and ß-cell development/repair are reported. Further identification of macrophage subtypes and of their secreted factors might ultimately translate into novel therapeutic strategies for diabetes mellitus.

Macrophage dynamics are regulated by local macrophage proliferation and monocyte recruitment in injured pancreas.

Pancreas injury by partial duct ligation (PDL) activates a healing response, encompassing ß-cell neogenesis and proliferation. Macrophages (MΦs) were recently shown to promote ß-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 ß-cell proliferation. Together, our study fully characterizes the MΦ subsets in the pancreas and clarifies the complex dynamics of MΦs after PDL injury.

IL-6-dependent proliferation of alpha cells in mice with partial pancreatic-duct ligation.

AIMS/HYPOTHESIS: IL-6 was recently shown to control alpha cell expansion. As beta cells expand following partial pancreatic-duct ligation (PDL) in adult mice, we investigated whether PDL also causes alpha cells to expand and whether IL-6 signalling is involved. As alpha cells can reprogramme to beta cells in a number of beta cell (re)generation models, we examined whether this phenomenon also exists in PDL pancreas. METHODS: Total alpha cell volume, alpha cell size and total glucagon content were evaluated in equivalent portions of PDL- and sham-operated mouse pancreases. Proliferation of glucagon(+) cells was assessed by expression of the proliferation marker Ki67. Inter-conversions between alpha and beta cells were monitored in transgenic mice with conditional cell-type-specific labelling. The role of IL-6 in regulating alpha cell proliferation was evaluated by in situ delivery of an IL-6-inactivating antibody. RESULTS: In response to PDL surgery, alpha cell volume in the ligated tissue was increased threefold, glucagon content fivefold and alpha cell size by 10%. Activation of alpha cell proliferation in PDL pancreas required IL-6 signalling. A minor fraction of alpha cells derived from beta cells, whereas no evidence for alpha to beta cell conversion was obtained. CONCLUSIONS/INTERPRETATION: In PDL-injured adult mouse pancreas, new alpha cells are generated mainly by IL-6-dependent self-duplication and seldom by reprogramming of beta cells.

Transient cytokine treatment induces acinar cell reprogramming and regenerates functional beta cell mass in diabetic mice.

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.

Reversal of hyperglycemia in diabetic mice by a marginal islet mass together with human blood outgrowth endothelial cells is independent of the delivery technique and blood clot-induced processes.

We recently reported that human blood outgrowth endothelial cells (BOEC) are supportive to reverse hyperglycemia in marginal islet mass-transplanted diabetic mice. In this report, we investigated whether the observed effect was evoked by islet packing in a blood clot prior to transplantation or could be mimicked by another method of islet/cell delivery. A marginal islet mass with or without BOEC was grafted underneath the kidney capsule of diabetic recipient mice via a (blood clot-independent) tubing system and compared with previous islet packing in a blood clot. The effect on metabolic outcome of both delivery techniques as well as the additive effect of BOEC was subsequently evaluated. Marginal islet mass transplantation via a tubing system required more islets per recipient than via a blood clot. Using the tubing method, transplantation of a marginal islet mass combined with 5x10 (5) BOEC resulted in reversal of hyperglycemia, improved glucose tolerance and increased kidney insulin content. The present study provides evidence that (1) previous packing in a blood clot results in more effective islet delivery compared with tubing; (2) BOEC exert a beneficial effect on marginal islet transplantation, independent of grafting technique and potential blood clot-induced processes. These data further support the use of BOEC in (pre-) clinical studies that aim to improve current islet transplantation protocols.

The inactivation of Arx in pancreatic α-cells triggers their neogenesis and conversion into functional ß-like cells.

Recently, it was demonstrated that pancreatic new-born glucagon-producing cells can regenerate and convert into insulin-producing ß-like cells through the ectopic expression of a single gene, Pax4. Here, combining conditional loss-of-function and lineage tracing approaches, we show that the selective inhibition of the Arx gene in α-cells is sufficient to promote the conversion of adult α-cells into ß-like cells at any age. Interestingly, this conversion induces the continuous mobilization of duct-lining precursor cells to adopt an endocrine cell fate, the glucagon(+) cells thereby generated being subsequently converted into ß-like cells upon Arx inhibition. Of interest, through the generation and analysis of Arx and Pax4 conditional double-mutants, we provide evidence that Pax4 is dispensable for these regeneration processes, indicating that Arx represents the main trigger of α-cell-mediated ß-like cell neogenesis. Importantly, the loss of Arx in α-cells is sufficient to regenerate a functional ß-cell mass and thereby reverse diabetes following toxin-induced ß-cell depletion. Our data therefore suggest that strategies aiming at inhibiting the expression of Arx, or its molecular targets/co-factors, may pave new avenues for the treatment of diabetes.