Bone marrow chimerism breaks the barrier to pancreatic islet transplantation.

In their recent Cell Reports paper, Chang and colleagues report on a successful strategy to achieve durable mixed hematopoietic chimerism that promotes the engraftment and long-term function of pancreatic islet allotransplants in fully immunocompetent mice without immunosuppression.

Pancreatic Cancer Cells Require the Transcription Factor MYRF to Maintain ER Homeostasis.

Many tumors of endodermal origin are composed of highly secretory cancer cells that must adapt endoplasmic reticulum (ER) activity to enable proper folding of secreted proteins and prevent ER stress. We found that pancreatic ductal adenocarcinomas (PDACs) overexpress the myelin regulatory factor (MYRF), an ER membrane-associated transcription factor (TF) released by self-cleavage. MYRF was expressed in the well-differentiated secretory cancer cells, but not in the poorly differentiated quasi-mesenchymal cells that coexist in the same tumor. MYRF expression was controlled by the epithelial identity TF HNF1B, and it acted to fine-tune the expression of genes encoding highly glycosylated, cysteine-rich secretory proteins, thus preventing ER overload. MYRF-deficient PDAC cells showed signs of ER stress, impaired proliferation, and an inability to form spheroids in vitro, while in vivo they generated highly secretory but poorly proliferating and hypocellular tumors. These data indicate a role of MYRF in the control of ER homeostasis in highly secretory PDAC cells.

Cyb5r3 links FoxO1-dependent mitochondrial dysfunction with ß-cell failure.

OBJECTIVE: Diabetes is characterized by pancreatic ß-cell dedifferentiation. Dedifferentiating ß cells inappropriately metabolize lipids over carbohydrates and exhibit impaired mitochondrial oxidative phosphorylation. However, the mechanism linking the ß-cell's response to an adverse metabolic environment with impaired mitochondrial function remains unclear. METHODS: Here we report that the oxidoreductase cytochrome b5 reductase 3 (Cyb5r3) links FoxO1 signaling to ß-cell stimulus/secretion coupling by regulating mitochondrial function, reactive oxygen species generation, and nicotinamide actin dysfunction (NAD)/reduced nicotinamide actin dysfunction (NADH) ratios. RESULTS: The expression of Cyb5r3 is decreased in FoxO1-deficient ß cells. Mice with ß-cell-specific deletion of Cyb5r3 have impaired insulin secretion, resulting in glucose intolerance and diet-induced hyperglycemia. Cyb5r3-deficient ß cells have a blunted respiratory response to glucose and display extensive mitochondrial and secretory granule abnormalities, consistent with altered differentiation. Moreover, FoxO1 is unable to maintain expression of key differentiation markers in Cyb5r3-deficient ß cells, suggesting that Cyb5r3 is required for FoxO1-dependent lineage stability. CONCLUSIONS: The findings highlight a pathway linking FoxO1 to mitochondrial dysfunction that can mediate ß-cell failure.

Connexin 43 Functions as a Positive Regulator of Stem Cell Differentiation into Definitive Endoderm and Pancreatic Progenitors.

Efficient stem cell differentiation into pancreatic islet cells is of critical importance for the development of cell replacement therapies for diabetes. Here, we identify the expression pattern of connexin 43 (Cx43), a gap junction (GJ) channel protein, in human embryonic stem cell (hESC)-derived definitive endoderm (DE) and primitive gut tube cells, representing early lineages for posterior foregut (PF), pancreatic progenitors (PP), pancreatic endocrine progenitors (PE), and islet cells. As the function of GJ channels is dependent on their gating status, we tested the impact of supplementing hESC-derived PP cell cultures with AAP10, a peptide that promotes Cx43 GJ channel opening. We found that this treatment promotes the expression of DE markers FoxA2 and Sox17, leads to a more efficient derivation of DE, and improves the yield of PF, PP, and PE cells. These results demonstrate a functional involvement of GJ channels in the differentiation of embryonic stem cells into pancreatic cell lineages.

Nrf2/antioxidant pathway mediates ß cell self-repair after damage by high-fat diet-induced oxidative stress.

Many theories have been advanced to better understand why ß cell function and structure relentlessly deteriorate during the course of type 2 diabetes (T2D). These theories include inflammation, apoptosis, replication, neogenesis, autophagy, differentiation, dedifferentiation, and decreased levels of insulin gene regulatory proteins. However, none of these have considered the possibility that endogenous self-repair of existing ß cells may be an important factor. To examine this hypothesis, we conducted studies with female Zucker diabetic fatty rats fed a high-fat diet (HFD) for 1, 2, 4, 7, 9, 18, or 28 days, followed by a return to regular chow for 2-3 weeks. Repair was defined as reversal of elevated blood glucose and of inappropriately low blood insulin levels caused by a HFD, as well as reversal of structural damage visualized by imaging studies. We observed evidence of functional ß cell damage after a 9-day exposure to a HFD and then repair after 2-3 weeks of being returned to normal chow (blood glucose [BG] = 348 ± 30 vs. 126 ± 3; mg/dl; days 9 vs. 23 day, P < 0.01). After 18- and 28-day exposure to a HFD, damage was more severe and repair was less evident. Insulin levels progressively diminished with 9-day exposure to a HFD; after returning to a regular diet, insulin levels rebounded toward, but did not reach, normal values. Increase in ß cell mass was 4-fold after 9 days and 3-fold after 18 days, and there was no increase after 28 days of a HFD. Increases in ß cell mass during a HFD were not different when comparing values before and after a return to regular diet within the 9-, 18-, or 28-day studies. No changes were observed in apoptosis or ß cell replication. Formation of intracellular markers of oxidative stress, intranuclear translocation of Nrf2, and formation of intracellular antioxidant proteins indicated the participation of HFD/oxidative stress induction of the Nrf2/antioxidant pathway. Flow cytometry-based assessment of ß cell volume, morphology, and insulin-specific immunoreactivity, as well as ultrastructural analysis by transmission electron microscopy, revealed that short-term exposure to a HFD produced significant changes in ß cell morphology and function that are reversible after returning to regular chow. These results suggest that a possible mechanism mediating the ability of ß cells to self-repair after a short-term exposure to a HFD is the activation of the Nrf2/antioxidant pathway.

A CCR2+ myeloid cell niche required for pancreatic ß cell growth.

Organ-specific patterns of myeloid cells may contribute tissue-specific growth and/or regenerative potentials. The perinatal stage of pancreas development marks a time characterized by maximal proliferation of pancreatic islets, ensuring the maintenance of glucose homeostasis throughout life. Ontogenically distinct CX3CR1+ and CCR2+ macrophage populations have been reported in the adult pancreas, but their functional contribution to islet cell growth at birth remains unknown. Here, we uncovered a temporally restricted requirement for CCR2+ myeloid cells in the perinatal proliferation of the endocrine pancreatic epithelium. CCR2+ macrophages are transiently enriched over CX3CR1+ subsets in the neonatal pancreas through both local expansion and recruitment of immature precursors. Using CCR2-specific depletion models, we show that loss of this myeloid population leads to a striking reduction in ß cell proliferation, dysfunctional islet phenotypes, and glucose intolerance in newborns. Replenishment of pancreatic CCR2+ myeloid compartments by adoptive transfer rescues these defects. Gene profiling identifies pancreatic CCR2+ myeloid cells as a prominent source of IGF2, which contributes to IGF1R-mediated islet proliferation. These findings uncover proproliferative functions of CCR2+ myeloid subsets and identify myeloid-dependent regulation of IGF signaling as a local cue supporting pancreatic proliferation.

αE-Catenin Is a Positive Regulator of Pancreatic Islet Cell Lineage Differentiation.

The development and function of epithelia depend on the establishment and maintenance of cell-cell adhesion and intercellular junctions, which operate as mechanosensor hubs for the transduction of biochemical signals regulating cell proliferation, differentiation, survival, and regeneration. Here, we show that αE-catenin, a key component of adherens junctions, functions as a positive regulator of pancreatic islet cell lineage differentiation by repressing the sonic hedgehog pathway (SHH). Thus, deletion of αE-catenin in multipotent pancreatic progenitors resulted in (1) loss of adherens junctions, (2) constitutive activation of SHH, (3) decrease in islet cell lineage differentiation, and (4) accumulation of immature Sox9+ progenitors. Pharmacological blockade of SHH signaling in pancreatic organ cultures and in vivo rescued this defect, allowing αE-catenin-null Sox9+ pancreatic progenitors to differentiate into endocrine cells. The results uncover crucial functions of αE-catenin in pancreatic islet development and harbor significant implications for the design of ß cell replacement and regeneration therapies in diabetes.

Pancreatic ß cell identity requires continual repression of non-ß cell programs.

Loss of ß cell identity, the presence of polyhormonal cells, and reprogramming are emerging as important features of ß cell dysfunction in patients with type 1 and type 2 diabetes. In this study, we have demonstrated that the transcription factor NKX2.2 is essential for the active maintenance of adult ß cell identity as well as function. Deletion of Nkx2.2 in ß cells caused rapid onset of a diabetic phenotype in mice that was attributed to loss of insulin and downregulation of many ß cell functional genes. Concomitantly, NKX2.2-deficient murine ß cells acquired non-ß cell endocrine features, resulting in populations of completely reprogrammed cells and bihormonal cells that displayed hybrid endocrine cell morphological characteristics. Molecular analysis in mouse and human islets revealed that NKX2.2 is a conserved master regulatory protein that controls the acquisition and maintenance of a functional, monohormonal ß cell identity by directly activating critical ß cell genes and actively repressing genes that specify the alternative islet endocrine cell lineages. This study demonstrates the highly volatile nature of the ß cell, indicating that acquiring and sustaining ß cell identity and function requires not only active maintaining of the expression of genes involved in ß cell function, but also continual repression of closely related endocrine gene programs.

A method for high-throughput functional imaging of single cells within heterogeneous cell preparations.

Functional characterization of individual cells within heterogeneous tissue preparations is challenging. Here, we report the development of a versatile imaging method that assesses single cell responses of various endpoints in real time, while identifying the individual cell types. Endpoints that can be measured include (but are not limited to) ionic flux (calcium, sodium, potassium and hydrogen), metabolic responsiveness (NAD(P)H, mitochondrial membrane potential), and signal transduction (H2O2 and cAMP). Subsequent to fluorescent imaging, identification of cell types using immunohistochemistry allows for mapping of cell type to their respective functional real time responses. To validate the utility of this method, NAD(P)H responses to glucose of islet alpha versus beta cells generated from dispersed pancreatic islets, followed by the construction of frequency distributions characterizing the variability in the magnitude of each individual cell responses were compared. As expected, no overlap between the glucose response frequency distributions for beta cells versus alpha cells was observed, thereby establishing both the high degree of fidelity and low rate of both false-negatives and false-positives in this approach. This novel method has the ability not only to resolve single cell level functional differences between cell types, but also to characterize functional heterogeneity within a given cell type.

Essential Role of Lyn in Fibrosis.

Fibrotic disorders involve replacement of normal parenchyma with myofibroblasts, which deposit connective tissue, leading to obliteration of the function of the underlying organ. The treatment options are inadequate and reflect the fact that signaling targets in myofibroblasts are unknown. Here we identify the hyperactive Lyn signaling in myofibroblasts of patients with chronic pancreatitis-induced fibrosis. Lyn activation coexpress with markers of activated myofibroblasts, and is increased ~11-fold in chronic pancreatitis compared to normal tissue. Inhibition of Lyn with siRNA or INNO-406 leads to the substantial decrease of migration and proliferation of human chronic pancreatitis myofibroblasts in vitro, while leaving migration and proliferation of normal myofibroblasts only slightly affected. Furthermore, inhibition of Lyn prevents synthesis of procollagen and collagen in myofibroblasts in a mouse model of chronic pancreatitis-induced fibrosis. We conclude that Lyn, as a positive regulator of myofibroblast migration, proliferation, and collagen production, is a key target for preventing fibrosis.

ECM Signaling Regulates Collective Cellular Dynamics to Control Pancreas Branching Morphogenesis.

During pancreas development, epithelial buds undergo branching morphogenesis to form an exocrine and endocrine gland. Proper morphogenesis is necessary for correct lineage allocation of pancreatic progenitors; however, the cellular events underlying pancreas morphogenesis are unknown. Here, we employed time-lapse microscopy and fluorescent labeling of cells to analyze cell behaviors associated with pancreas morphogenesis. We observed that outer bud cells adjacent to the basement membrane are pleomorphic and rearrange frequently; additionally, they largely remain in the outer cell compartment even after mitosis. These cell behaviors and pancreas branching depend on cell contacts with the basement membrane, which induce actomyosin cytoskeleton remodeling via integrin-mediated activation of FAK/Src signaling. We show that integrin signaling reduces E-cadherin-mediated cell-cell adhesion in outer cells and provide genetic evidence that this regulation is necessary for initiation of branching. Our study suggests that regulation of cell motility and adhesion by local niche cues initiates pancreas branching morphogenesis.

MMTV/LTR Promoter-Driven Transgenic Expression of EpCAM Leads to the Development of Large Pancreatic Islets.

Our previous work demonstrated an important role of EpCAM in the regulation of pancreatic cell adhesion, growth and differentiation. Here we investigated the consequences of human EpCAM (hEpCAM) overexpression under the control of the MMTV-LTR promoter, known to drive robust gene expression in a number of ductal epithelia, including the pancreas. In this animal model (MMTV-hEpCAM) we uncovered a striking pancreatic phenotype exhibiting a 12-fold increase in the islet cell mass, with normal expression patterns of insulin and the transcription factor PDX-1. Intriguingly, these large islet clusters revealed an altered architectural organization of α- and δ-cells that appeared interspersed with ß-cells in the islet cores. This suggests an effect of the hEpCAM transgene on the function of other cell adhesion molecules that we have previously shown to regulate islet cell type segregation. Consistent with this finding, we show that the pancreatic epithelium in MMTV-hEpCAM transgenic mice exhibits a redistribution of ß-catenin, a known regulator of E-cadherin-mediated adhesions. Collectively, these results provide an important in vivo validation of hEpCAM signaling properties in normal epithelia and offer unique opportunities to further explore the function of this glycoprotein in select pancreatic cell lineages to elicit islet cell expansion, and/or regeneration in diabetes.

Controlled induction of human pancreatic progenitors produces functional beta-like cells in vitro.

Directed differentiation of human pluripotent stem cells into functional insulin-producing beta-like cells holds great promise for cell replacement therapy for patients suffering from diabetes. This approach also offers the unique opportunity to study otherwise inaccessible aspects of human beta cell development and function in vitro. Here, we show that current pancreatic progenitor differentiation protocols promote precocious endocrine commitment, ultimately resulting in the generation of non-functional polyhormonal cells. Omission of commonly used BMP inhibitors during pancreatic specification prevents precocious endocrine formation while treatment with retinoic acid followed by combined EGF/KGF efficiently generates both PDX1(+) and subsequent PDX1(+)/NKX6.1(+) pancreatic progenitor populations, respectively. Precise temporal activation of endocrine differentiation in PDX1(+)/NKX6.1(+) progenitors produces glucose-responsive beta-like cells in vitro that exhibit key features of bona fide human beta cells, remain functional after short-term transplantation, and reduce blood glucose levels in diabetic mice. Thus, our simplified and scalable system accurately recapitulates key steps of human pancreas development and provides a fast and reproducible supply of functional human beta-like cells.

Derivation of naive human embryonic stem cells.

The naïve pluripotent state has been shown in mice to lead to broad and more robust developmental potential relative to primed mouse epiblast cells. The human naïve ES cell state has eluded derivation without the use of transgenes, and forced expression of OCT4, KLF4, and KLF2 allows maintenance of human cells in a naïve state [Hanna J, et al. (2010) Proc Natl Acad Sci USA 107(20):9222-9227]. We describe two routes to generate nontransgenic naïve human ES cells (hESCs). The first is by reverse toggling of preexisting primed hESC lines by preculture in the histone deacetylase inhibitors butyrate and suberoylanilide hydroxamic acid, followed by culture in MEK/ERK and GSK3 inhibitors (2i) with FGF2. The second route is by direct derivation from a human embryo in 2i with FGF2. We show that human naïve cells meet mouse criteria for the naïve state by growth characteristics, antibody labeling profile, gene expression, X-inactivation profile, mitochondrial morphology, microRNA profile and development in the context of teratomas. hESCs can exist in a naïve state without the need for transgenes. Direct derivation is an elusive, but attainable, process, leading to cells at the earliest stage of in vitro pluripotency described for humans. Reverse toggling of primed cells to naïve is efficient and reproducible.

Macrophage/epithelium cross-talk regulates cell cycle progression and migration in pancreatic progenitors.

Macrophages populate the mesenchymal compartment of all organs during embryogenesis and have been shown to support tissue organogenesis and regeneration by regulating remodeling of the extracellular microenvironment. Whether this mesenchymal component can also dictate select developmental decisions in epithelia is unknown. Here, using the embryonic pancreatic epithelium as model system, we show that macrophages drive the epithelium to execute two developmentally important choices, i.e. the exit from cell cycle and the acquisition of a migratory phenotype. We demonstrate that these developmental decisions are effectively imparted by macrophages activated toward an M2 fetal-like functional state, and involve modulation of the adhesion receptor NCAM and an uncommon "paired-less" isoform of the transcription factor PAX6 in the epithelium. Over-expression of this PAX6 variant in pancreatic epithelia controls both cell motility and cell cycle progression in a gene-dosage dependent fashion. Importantly, induction of these phenotypes in embryonic pancreatic transplants by M2 macrophages in vivo is associated with an increased frequency of endocrine-committed cells emerging from ductal progenitor pools. These results identify M2 macrophages as key effectors capable of coordinating epithelial cell cycle withdrawal and cell migration, two events critical to pancreatic progenitors' delamination and progression toward their differentiated fates.

SEL1L regulates adhesion, proliferation and secretion of insulin by affecting integrin signaling.

SEL1L, a component of the endoplasmic reticulum associated degradation (ERAD) pathway, has been reported to regulate the (i) differentiation of the pancreatic endocrine and exocrine tissue during the second transition of mouse embryonic development, (ii) neural stem cell self-renewal and lineage commitment and (iii) cell cycle progression through regulation of genes related to cell-matrix interaction. Here we show that in the pancreas the expression of SEL1L is developmentally regulated, such that it is readily detected in developing islet cells and in nascent acinar clusters adjacent to basement membranes, and becomes progressively restricted to the islets of Langherans in post-natal life. This peculiar expression pattern and the presence of two inverse RGD motifs in the fibronectin type II domain of SEL1L protein indicate a possible interaction with cell adhesion molecules to regulate islets architecture. Co-immunoprecipitation studies revealed SEL1L and ß1-integrin interaction and, down-modulation of SEL1L in pancreatic ß-cells, negatively influences both cell adhesion on selected matrix components and cell proliferation likely due to altered ERK signaling. Furthermore, the absence of SEL1L protein strongly inhibits glucose-stimulated insulin secretion in isolated mouse pancreatic islets unveiling an important role of SEL1L in insulin trafficking. This phenotype can be rescued by the ectopic expression of the ß1-integrin subunit confirming the close interaction of these two proteins in regulating the cross-talk between extracellular matrix and insulin signalling to create a favourable micro-environment for ß-cell development and function.

ß1 integrin is a crucial regulator of pancreatic ß-cell expansion.

Development of the endocrine compartment of the pancreas, as represented by the islets of Langerhans, occurs through a series of highly regulated events encompassing branching of the pancreatic epithelium, delamination and differentiation of islet progenitors from ductal domains, followed by expansion and three-dimensional organization into islet clusters. Cellular interactions with the extracellular matrix (ECM) mediated by receptors of the integrin family are postulated to regulate key functions in these processes. Yet, specific events regulated by these receptors in the developing pancreas remain unknown. Here, we show that ablation of the ß1 integrin gene in developing pancreatic ß-cells reduces their ability to expand during embryonic life, during the first week of postnatal life, and thereafter. Mice lacking ß1 integrin in insulin-producing cells exhibit a dramatic reduction of the number of ß-cells to only ∼18% of wild-type levels. Despite the significant reduction in ß-cell mass, these mutant mice are not diabetic. A thorough phenotypic analysis of ß-cells lacking ß1 integrin revealed a normal expression repertoire of ß-cell markers, normal architectural organization within islet clusters, and a normal ultrastructure. Global gene expression analysis revealed that ablation of this ECM receptor in ß-cells inhibits the expression of genes regulating cell cycle progression. Collectively, our results demonstrate that ß1 integrin receptors function as crucial positive regulators of ß-cell expansion.

EpCAM: structure and function in health and disease.

Injection of tumor cells in mice more than 30 years ago resulted in the discovery of an epithelial antigen, later defined as a cell adhesion molecule (EpCAM). Although EpCAM has since evoked significant interest as a target in cancer therapy, mechanistic insights on the functions of this glycoprotein have been emerging only very recently. This may have been caused by the multitude of functions attributed to the glycoprotein, its localization at different subcellular sites and complex posttranslational modifications. Here, we review how EpCAM modifies cell-cell contact adhesion strength and tissue plasticity, and how it regulates cell proliferation and differentiation. Major knowledge derived from human diseases will be highlighted: Mutant EpCAM that is absent from the cell surface leads to fatal intestinal abnormalities (congenital tufting enteropathy). EpCAM-mediated cell proliferation in cancer may result from signaling (i) via regulated intramembrane proteolysis and/or (ii) the localization and association with binding partners in specialized membrane microdomains. New insight in EpCAM signaling will help to develop optimized cancer therapies and open new avenues in the field of regenerative medicine.

Anchored phosphatases modulate glucose homeostasis.

Endocrine release of insulin principally controls glucose homeostasis. Nutrient-induced exocytosis of insulin granules from pancreatic ß-cells involves ion channels and mobilization of Ca(2+) and cyclic AMP (cAMP) signalling pathways. Whole-animal physiology, islet studies and live-ß-cell imaging approaches reveal that ablation of the kinase/phosphatase anchoring protein AKAP150 impairs insulin secretion in mice. Loss of AKAP150 impacts L-type Ca(2+) currents, and attenuates cytoplasmic accumulation of Ca(2+) and cAMP in ß-cells. Yet surprisingly AKAP150 null animals display improved glucose handling and heightened insulin sensitivity in skeletal muscle. More refined analyses of AKAP150 knock-in mice unable to anchor protein kinase A or protein phosphatase 2B uncover an unexpected observation that tethering of phosphatases to a seven-residue sequence of the anchoring protein is the predominant molecular event underlying these metabolic phenotypes. Thus anchored signalling events that facilitate insulin secretion and glucose homeostasis may be set by AKAP150 associated phosphatase activity.