Human hypoimmune primary pancreatic islets avoid rejection and autoimmunity and alleviate diabetes in allogeneic humanized mice.

Transplantation of allogeneic pancreatic donor islets has successfully been performed in selected patients with difficult-to-control insulin-dependent diabetes and impaired awareness of hypoglycemia (IAH). However, the required systemic immunosuppression associated with this procedure prevents this cell replacement therapy from more widespread adoption in larger patient populations. We report the editing of primary human islet cells to the hypoimmune HLA class I- and class II-negative and CD47-overexpressing phenotype and their reaggregation into human HIP pseudoislets (p-islets). Human HIP p-islets were shown to survive, engraft, and ameliorate diabetes in immunocompetent, allogeneic, diabetic humanized mice. HIP p-islet cells were further shown to avoid autoimmune killing in autologous, diabetic humanized autoimmune mice. The survival and endocrine function of HIP p-islet cells were not impaired by contamination of unedited or partially edited cells within the p-islets. HIP p-islet cells were eliminated quickly and reliably in this model using a CD47-targeting antibody, thus providing a safety strategy in case HIP cells exert toxicity in a future clinical setting. Transplantation of human HIP p-islets for which no immunosuppression is required has the potential to lead to wider adoption of this therapy and help more diabetes patients with IAH and history of severe hypoglycemic events to achieve insulin independence.

Activation of pancreatic-duct-derived progenitor cells during pancreas regeneration in adult rats.

The adult pancreas has considerable capacity to regenerate in response to injury. We hypothesized that after partial pancreatectomy (Px) in adult rats, pancreatic-duct cells serve as a source of regeneration by undergoing a reproducible dedifferentiation and redifferentiation. We support this hypothesis by the detection of an early loss of the ductal differentiation marker Hnf6 in the mature ducts, followed by the transient appearance of areas composed of proliferating ductules, called foci of regeneration, which subsequently form new pancreatic lobes. In young foci, ductules express markers of the embryonic pancreatic epithelium - Pdx1, Tcf2 and Sox9 - suggesting that these cells act as progenitors of the regenerating pancreas. The endocrine-lineage-specific transcription factor Neurogenin3, which is found in the developing embryonic pancreas, was transiently detected in the foci. Islets in foci initially resemble embryonic islets in their lack of MafA expression and lower percentage of beta-cells, but with increasing maturation have increasing numbers of MafA(+) insulin(+) cells. Taken together, we provide a mechanism by which adult pancreatic duct cells recapitulate aspects of embryonic pancreas differentiation in response to injury, and contribute to regeneration of the pancreas. This mechanism of regeneration relies mainly on the plasticity of the differentiated cells within the pancreas.

Snail2, a mediator of epithelial-mesenchymal transitions, expressed in progenitor cells of the developing endocrine pancreas.

The mammalian pancreas develops by the expansion and morphogenesis of the epithelial cells of the foregut endoderm via the sequential activation of transcription factors that direct differentiation into the various pancreatic lineages. Implicit in this growth and differentiation are the temporal and spatial processes of cell migration and three-dimensional organization, which cooperate to form a properly functioning organ. In many organ systems, such as the kidney, heart, and neural crest derivatives, migration and tissue morphogenesis is accomplished by the transient conversion of stationary epithelial cells to migratory mesenchymal-like cells in a process known as epithelial-mesenchymal transition (EMT). We report the identification of the expression of the transcription factor Snail2/Slug, a known inducer of EMT and cell movement, in both the endocrine and exocrine cells of the developing mouse pancreas. Snail2 is expressed in Neurogenin3-positive endocrine progenitor cells, and expression is maintained during endocrine cell differentiation where it becomes increasingly restricted to the insulin-producing beta cells and somatostatin-producing delta cells. In the adult pancreas, the expression of Snail2 is maintained at low but detectable levels in all beta cells, indicating a latent role for Snail2 in the mature islet. These findings of Snail2 expression during endocrine pancreas development are relevant to the recent evidence demonstrating the involvement of EMT in the expansion of human islet tissue in vitro. EMT-like events appear to be involved in the development of the mammalian pancreas in vivo.

Inhibition of Mist1 homodimer formation induces pancreatic acinar-to-ductal metaplasia.

The pancreas consists of three main cell lineages (endocrine, exocrine, and duct) that develop from common primitive foregut precursors. The transcriptional network responsible for endocrine cell development has been studied extensively, but much less is known about the transcription factors that maintain the exocrine and duct cell lineages. One transcription factor that may be important to exocrine cell function is Mist1, a basic helix-loop-helix (bHLH) factor that is expressed in acinar cells. In order to perform a molecular characterization of this protein, we employed coimmunoprecipitation and bimolecular fluorescence complementation assays, coupled with electrophoretic mobility shift assay studies, to show that Mist1 exists in vivo as a homodimer complex. Analysis of transgenic mice expressing a dominant-negative Mist1 transgene (Mist1(mutant basic) [Mist1(MB)]) revealed the cell autonomous effect of inhibiting endogenous Mist1. Mist1(MB) cells become disorganized, exhibit a severe depletion of intercellular gap junctions, and express high levels of the glycoprotein clusterin, which has been shown to demarcate immature acinar cells. Inhibition of Mist1 transcriptional activity also leads to activation of duct-specific genes, such as cytokeratin 19 and cytokeratin 20, suggesting that alterations in the bHLH network produce a direct acinar-to-ductal phenotypic switch in mature cells. We propose that Mist1 is a key transcriptional regulator of exocrine pancreatic cells and that in the absence of functional Mist1, acinar cells do not maintain their normal identity.

Transcription factor snail modulates hormone expression in established endocrine pancreatic cell lines.

The development of differentiated cells from undifferentiated progenitor cells is one of the central tenets of developmental biology. However, under conditions of tissue morphogenesis, regeneration, and cancer, this process of development is reversed and fully differentiated cells transition to an undifferentiated phenotype. Here we present evidence that the zinc-finger transcription factor Snail modulates this transition in differentiated pancreatic endocrine cell lines. During passage and growth of these cell lines, Snail expression is induced in a subset of cells within the culture, concomitant with a decrease in insulin and/or glucagon expression. As the cells cluster and exit the cell division cycle, nuclear levels of Snail are reduced and hormone expression is resumed. Snail represses proinsulin and proglucagon gene transcription, and reduction of Snail levels by small interfering RNA treatment increases proinsulin gene expression. We propose that Snail modulates the dynamic balance between differentiated and dedifferentiated cells allowing their migration and proliferation. These findings may be relevant to providing approaches for the enhancement of beta-cell growth in individuals with diabetes mellitus.

Novel pancreatic endocrine maturation pathways identified by genomic profiling and causal reasoning.

We have used a previously unavailable model of pancreatic development, derived in vitro from human embryonic stem cells, to capture a time-course of gene, miRNA and histone modification levels in pancreatic endocrine cells. We investigated whether it is possible to better understand, and hence control, the biological pathways leading to pancreatic endocrine formation by analysing this information and combining it with the available scientific literature to generate models using a casual reasoning approach. We show that the embryonic stem cell differentiation protocol is highly reproducible in producing endocrine precursor cells and generates cells that recapitulate many aspects of human embryonic pancreas development, including maturation into functional endocrine cells when transplanted into recipient animals. The availability of whole genome gene and miRNA expression data from the early stages of human pancreatic development will be of great benefit to those in the fields of developmental biology and diabetes research. Our causal reasoning algorithm suggested the involvement of novel gene networks, such as NEUROG3/E2F1/KDM5B and SOCS3/STAT3/IL-6, in endocrine cell development We experimentally investigated the role of the top-ranked prediction by showing that addition of exogenous IL-6 could affect the expression of the endocrine progenitor genes NEUROG3 and NKX2.2.

Neurogenin3: a master regulator of pancreatic islet differentiation and regeneration.

The basic helix-loop-helix transcription factor neurogenin-3 (Ngn3, Neurog3) is critical for the development of the endocrine cells of the islets. Either disrupted or forced expression of Ngn3 early in mouse pancreas development abrogates the formation of islets. The successive waves of Ngn3 expression that occur during the primary and secondary transitions of endocrine cell development temporally determine the four distinct endocrine cell lineages, α, ß, PP, and δ cells that express glucagon, insulin, pancreatic polypeptide, and somatostatin, respectively. During islet regeneration after injury of the adult mouse pancreas, such as by duct ligation or streptozotocin, Ngn3 is activated in duct-associated stem/progenitor cells that transform into alpha and/or beta cells (Xu et al, Collombat et al). The important role of Ngn3 as a master regulator of endocrine pancreas development directs attention to finding therapeutic approaches to enhance Ngn3 expression in diabetes as a means to increase beta cell mass and functions.

Inhibition of cyclin-dependent kinase 5 activity protects pancreatic beta cells from glucotoxicity.

Type 2 diabetes (T2D) and Alzheimer disease are degenerative diseases that may share common pathophysiologic mechanisms. Neuronal dysfunction in Alzheimer patients has been linked to overactivity of the cyclin-dependent kinase 5 (CDK5) and its activator p35. Both of these proteins are expressed in the insulin-producing beta cells of the pancreas. Further, glucose enhances p35 gene expression, promoting the formation of active p35/CDK5 complexes that regulate the expression of the insulin gene. In T2D, chronic elevations of glucose, glucotoxicity, impair beta cell function. We therefore postulated that CDK5 and p35 may be responsible for this beta cell impairment and that inhibition of CDK5 might have a beneficial effect. To test this hypothesis, the pancreatic cell line INS-1 was selected as a known in vitro model of glucotoxicity, and roscovitine (10 mum) was used as a CDK5 inhibitor. Chronic exposure of INS-1 cells to high glucose (20-30 mm) reduced both insulin mRNA levels and the activity of an insulin promoter reporter gene. Inhibition of CDK5 prevented this decrease of insulin gene expression. We used DNA binding (gel shift) assays and Western immunoblots to demonstrate that cellular levels of the transcription factor PDX-1, normally decreased by glucotoxicity, were preserved with CDK5 inhibition, as was the binding of PDX-1 to the insulin promoter. Analyses of nuclear and cytoplasmic PDX-1 protein levels revealed that CDK5 inhibition restores nuclear PDX-1, without affecting its cytoplasmic concentration, suggesting that CDK5 regulates the nuclear/cytoplasm partitioning of PDX-1. Using a Myc-tagged PDX-1 construct, we showed that the translocation of PDX-1 from the nucleus to the cytoplasm during glucotoxic conditions was prevented when CDK5 was inhibited. These studies indicate that CDK5 plays a role in the loss of beta cell function under glucotoxic conditions and that CDK5 inhibitors could have therapeutic value for T2D.

Exocrine specific expression of Connexin32 is dependent on the basic helix-loop-helix transcription factor Mist1.

Gap junctions are intercellular channels that provide direct passage of small molecules between adjacent cells. In pancreatic acini, the connexin26 (Cx26) and connexin32 (Cx32) proteins form functional channels that coordinate the secretion of digestive enzymes. Although the function of Cx26/Cx32 gap junctions are well characterized, the regulatory circuits that control the spatial and temporal expression patterns of these connexin genes are not known. In an effort to identify the molecular pathways that regulate connexin gene expression, we examined Cx26 and Cx32 gene activities in mice lacking the basic helix-loop-helix transcription factor Mist1 (Mist1KO). Mist1, Cx26 and Cx32 are co-expressed in most exocrine cell types, and acinar cells from Mist1KO mice exhibit a highly disorganized cellular architecture and an altered pattern of expression for several genes involved in regulated exocytosis. Analysis of Mist1KO mice revealed a dramatic decrease in both connexin proteins, albeit through different molecular mechanisms. Cx32 gene transcription was greatly reduced in all Mist1KO exocrine cells, while Cx26 gene expression remained unaffected. However, in the absence of Cx32 protein, Cx26 did not participate in gap junction formation, leading to a complete lack of intercellular communication among Mist1KO acinar cells. Additional studies testing Mist1 gene constructs in pancreatic exocrine cells confirmed that Mist1 transcriptionally regulates expression of the Cx32 gene. We conclude that Mist1 functions as a positive regulator of Cx32 gene expression and, in its absence, acinar cell gap junctions and intercellular communication pathways become disrupted.