EndoC-ßH5 cells are storable and ready-to-use human pancreatic beta cells with physiological insulin secretion.

OBJECTIVES: Readily accessible human pancreatic beta cells that are functionally close to primary adult beta cells are a crucial model to better understand human beta cell physiology and develop new treatments for diabetes. We here report the characterization of EndoC-ßH5 cells, the latest in the EndoC-ßH cell family. METHODS: EndoC-ßH5 cells were generated by integrative gene transfer of immortalizing transgenes hTERT and SV40 large T along with Herpes Simplex Virus-1 thymidine kinase into human fetal pancreas. Immortalizing transgenes were removed after amplification using CRE activation and remaining non-excized cells eliminated using ganciclovir. Resulting cells were distributed as ready to use EndoC-ßH5 cells. We performed transcriptome, immunological and extensive functional assays. RESULTS: Ready to use EndoC-ßH5 cells display highly efficient glucose dependent insulin secretion. A robust 10-fold insulin secretion index was observed and reproduced in four independent laboratories across Europe. EndoC-ßH5 cells secrete insulin in a dynamic manner in response to glucose and secretion is further potentiated by GIP and GLP-1 analogs. RNA-seq confirmed abundant expression of beta cell transcription factors and functional markers, including incretin receptors. Cytokines induce a gene expression signature of inflammatory pathways and antigen processing and presentation. Finally, modified HLA-A2 expressing EndoC-ßH5 cells elicit specific A2-alloreactive CD8 T cell activation. CONCLUSIONS: EndoC-ßH5 cells represent a unique storable and ready to use human pancreatic beta cell model with highly robust and reproducible features. Such cells are thus relevant for the study of beta cell function, screening and validation of new drugs, and development of disease models.

Transient regulation of focal adhesion via Tensin3 is required for nascent oligodendrocyte differentiation.

The differentiation of oligodendroglia from oligodendrocyte precursor cells (OPCs) to complex and extensive myelinating oligodendrocytes (OLs) is a multistep process that involves large-scale morphological changes with significant strain on the cytoskeleton. While key chromatin and transcriptional regulators of differentiation have been identified, their target genes responsible for the morphological changes occurring during OL myelination are still largely unknown. Here, we show that the regulator of focal adhesion, Tensin3 (Tns3), is a direct target gene of Olig2, Chd7, and Chd8, transcriptional regulators of OL differentiation. Tns3 is transiently upregulated and localized to cell processes of immature OLs, together with integrin-ß1, a key mediator of survival at this transient stage. Constitutive <i>Tns3</i> loss of function leads to reduced viability in mouse and humans, with surviving knockout mice still expressing Tns3 in oligodendroglia. Acute deletion of <i>Tns3</i> in vivo, either in postnatal neural stem cells (NSCs) or in OPCs, leads to a twofold reduction in OL numbers. We find that the transient upregulation of Tns3 is required to protect differentiating OPCs and immature OLs from cell death by preventing the upregulation of p53, a key regulator of apoptosis. Altogether, our findings reveal a specific time window during which transcriptional upregulation of Tns3 in immature OLs is required for OL differentiation likely by mediating integrin-ß1 survival signaling to the actin cytoskeleton as OL undergo the large morphological changes required for their terminal differentiation.

The HASTER lncRNA promoter is a cis-acting transcriptional stabilizer of HNF1A.

The biological purpose of long non-coding RNAs (lncRNAs) is poorly understood. Haploinsufficient mutations in HNF1A homeobox A (HNF1A), encoding a homeodomain transcription factor, cause diabetes mellitus. Here, we examine HASTER, the promoter of an lncRNA antisense to HNF1A. Using mouse and human models, we show that HASTER maintains cell-specific physiological HNF1A concentrations through positive and negative feedback loops. Pancreatic ß cells from Haster mutant mice consequently showed variegated HNF1A silencing or overexpression, resulting in hyperglycaemia. HASTER-dependent negative feedback was essential to prevent HNF1A binding to inappropriate genomic regions. We demonstrate that the HASTER promoter DNA, rather than the lncRNA, modulates HNF1A promoter-enhancer interactions in cis and thereby regulates HNF1A transcription. Our studies expose a cis-regulatory element that is unlike classic enhancers or silencers, it stabilizes the transcription of its target gene and ensures the fidelity of a cell-specific transcription factor program. They also show that disruption of a mammalian lncRNA promoter can cause diabetes mellitus.

The Amyotrophic Lateral Sclerosis M114T PFN1 Mutation Deregulates Alternative Autophagy Pathways and Mitochondrial Homeostasis.

Mutations in profilin 1 (PFN1) have been identified in rare familial cases of Amyotrophic Lateral Sclerosis (ALS). PFN1 is involved in multiple pathways that could intervene in ALS pathology. However, the specific pathogenic role of PFN1 mutations in ALS is still not fully understood. We hypothesized that PFN1 could play a role in regulating autophagy pathways and that PFN1 mutations could disrupt this function. We used patient cells (lymphoblasts) or tissue (post-mortem) carrying PFN1 mutations (M114T and E117G), and designed experimental models expressing wild-type or mutant PFN1 (cell lines and novel PFN1 mice established by lentiviral transgenesis) to study the effects of PFN1 mutations on autophagic pathway markers. We observed no accumulation of PFN1 in the spinal cord of one E117G mutation carrier. Moreover, in patient lymphoblasts and transfected cell lines, the M114T mutant PFN1 protein was unstable and deregulated the RAB9-mediated alternative autophagy pathway involved in the clearance of damaged mitochondria. In vivo, motor neurons expressing M114T mutant PFN1 showed mitochondrial abnormalities. Our results demonstrate that the M114T PFN1 mutation is more deleterious than the E117G variant in patient cells and experimental models and suggest a role for the RAB9-dependent autophagic pathway in ALS.

Does the Expression and Epigenetics of Genes Involved in Monogenic Forms of Parkinson's Disease Influence Sporadic Forms?

Parkinson's disease (PD) is a disorder characterized by a triad of motor symptoms (akinesia, rigidity, resting tremor) related to loss of dopaminergic neurons mainly in the Substantia nigra pars compacta. Diagnosis is often made after a substantial loss of neurons has already occurred, and while dopamine replacement therapies improve symptoms, they do not modify the course of the disease. Although some biological mechanisms involved in the disease have been identified, such as oxidative stress and accumulation of misfolded proteins, they do not explain entirely PD pathophysiology, and a need for a better understanding remains. Neurodegenerative diseases, including PD, appear to be the result of complex interactions between genetic and environmental factors. The latter can alter gene expression by causing epigenetic changes, such as DNA methylation, post-translational modification of histones and non-coding RNAs. Regulation of genes responsible for monogenic forms of PD may be involved in sporadic PD. This review will focus on the epigenetic mechanisms regulating their expression, since these are the genes for which we currently have the most information available. Despite technical challenges, epigenetic epidemiology offers new insights on revealing altered biological pathways and identifying predictive biomarkers for the onset and progression of PD.

Using Inhibitory DREADDs to Silence LC Neurons in Monkeys.

Understanding the role of the noradrenergic nucleus locus coeruleus (LC) in cognition and behavior is critical: It is involved in several key behavioral functions such as stress and vigilance, as well as in cognitive processes such as attention and decision making. In recent years, the development of viral tools has provided a clear insight into numerous aspects of brain functions in rodents. However, given the specificity of primate brains and the key benefit of monkey research for translational applications, developing viral tools to study the LC in monkeys is essential for understanding its function and exploring potential clinical strategies. Here, we describe a pharmacogenetics approach that allows to selectively and reversibly inactivate LC neurons using Designer Receptors Exclusively Activated by Designer Drugs (DREADD). We show that the expression of the hM4Di DREADD can be restricted to noradrenergic LC neurons and that the amount of LC inhibition can be adjusted by adapting the dose of the specific DREADD activator deschloroclozapine (DCZ). Indeed, even if high doses (>0.3 mg/kg) induce a massive inhibition of LC neurons and a clear decrease in vigilance, smaller doses (<0.3 mg/kg) induce a more moderate decrease in LC activity, but it does not affect vigilance, which is more compatible with an assessment of subtle cognitive functions such as decision making and attention.

An in vivo Caenorhabditis elegans model for therapeutic research in human prion diseases.

Human prion diseases are fatal neurodegenerative disorders that include sporadic, infectious and genetic forms. Inherited Creutzfeldt-Jakob disease due to the E200K mutation of the prion protein-coding gene is the most common form of genetic prion disease. The phenotype resembles that of sporadic Creutzfeldt-Jakob disease at both the clinical and pathological levels, with a median disease duration of 4 months. To date, there is no available treatment for delaying the occurrence or slowing the progression of human prion diseases. Existing in vivo models do not allow high-throughput approaches that may facilitate the discovery of compounds targeting pathological assemblies of human prion protein or their effects on neuronal survival. Here, we generated a genetic model in the nematode Caenorhabditis elegans, which is devoid of any homologue of the prion protein, by expressing human prion protein with the E200K mutation in the mechanosensitive neuronal system. Expression of E200K prion protein induced a specific behavioural pattern and neurodegeneration of green fluorescent protein-expressing mechanosensitive neurons, in addition to the formation of intraneuronal inclusions associated with the accumulation of a protease-resistant form of the prion protein. We demonstrated that this experimental system is a powerful tool for investigating the efficacy of anti-prion compounds on both prion-induced neurodegeneration and prion protein misfolding, as well as in the context of human prion protein. Within a library of 320 compounds that have been approved for human use and cross the blood-brain barrier, we identified five molecules that were active against the aggregation of the E200K prion protein and the neurodegeneration it induced in transgenic animals. This model breaks a technological limitation in prion therapeutic research and provides a key tool to study the deleterious effects of misfolded prion protein in a well-described neuronal system.

The Host Protein Aquaporin-9 is Required for Efficient Plasmodium falciparum Sporozoite Entry into Human Hepatocytes.

Hepatocyte invasion by Plasmodium sporozoites represents a promising target for innovative antimalarial therapy, but the molecular events mediating this process are still largely uncharacterized. We previously showed that Plasmodium falciparum sporozoite entry into hepatocytes strictly requires CD81. However, CD81-overexpressing human hepatoma cells remain refractory to P. falciparum infection, suggesting the existence of additional host factors necessary for sporozoite entry. Here, through differential transcriptomic analysis of human hepatocytes and hepatoma HepG2-CD81 cells, the transmembrane protein Aquaporin-9 (AQP9) was found to be among the most downregulated genes in hepatoma cells. RNA silencing showed that sporozoite invasion of hepatocytes requires AQP9 expression. AQP9 overexpression in hepatocytes increased their permissiveness to P. falciparum. Moreover, chemical disruption with the AQP9 inhibitor phloretin markedly inhibited hepatocyte infection. Our findings identify AQP9 as a novel host factor required for P. falciparum sporozoite hepatocyte-entry and indicate that AQP9 could be a potential therapeutic target.

Neonatal diabetes mutations disrupt a chromatin pioneering function that activates the human insulin gene.

Despite the central role of chromosomal context in gene transcription, human noncoding DNA variants are generally studied outside of their genomic location. This limits our understanding of disease-causing regulatory variants. INS promoter mutations cause recessive neonatal diabetes. We show that all INS promoter point mutations in 60 patients disrupt a CC dinucleotide, whereas none affect other elements important for episomal promoter function. To model CC mutations, we humanized an ∼3.1-kb region of the mouse Ins2 gene. This recapitulated developmental chromatin states and cell-specific transcription. A CC mutant allele, however, abrogated active chromatin formation during pancreas development. A search for transcription factors acting through this element revealed that another neonatal diabetes gene product, GLIS3, has a pioneer-like ability to derepress INS chromatin, which is hampered by the CC mutation. Our in vivo analysis, therefore, connects two human genetic defects in an essential mechanism for developmental activation of the INS gene.

Culture, differentiation, and transduction of mouse E12.5 pancreatic spheres: an in vitro model for the secondary transition of pancreas development.

During the secondary transition of rodent pancreatic development, mainly between E12.5 and E15.5 in mice, exocrine and endocrine populations differentiate from pancreatic progenitors. Here we describe an experimental system for its study in vitro. First, we show that spheres derived from dissociated E12.5 mouse pancreases differentiate within 7 days into most pancreatic exocrine and endocrine cell types, including beta cells. The proportion and spatial repartition of the different endocrine populations mirror those observed during normal development. Thus, dissociation and culture do not impair the developmental events affecting pancreatic progenitors during the secondary transition. Moreover, dissociated cells from mouse E12.5 pancreas were transduced with ecotropic MLV-based retroviral vectors or, though less efficiently, with a mixture of ALV(A)-based retroviral vectors and gesicles containing the TVA (Tumor Virus A) receptor. As an additional improvement, we also created a transgenic mouse line expressing TVA under the control of the 4.5 kB pdx1 promoter (pdx1-TVA). We demonstrate that pancreatic progenitors from dissociated pdx1-TVA pancreas can be specifically transduced by ALV(A)-based retroviral vectors. Using this model, we expressed an activated mutant of the YAP transcriptional co-activator in pancreatic progenitors. These experiments indicate that deregulated YAP activity reduces endocrine and exocrine differentiation in the resulting spheres, confirming and extending previously published data. Thus, our experimental model recapitulates in vitro the crucial developmental decisions arising at the secondary transition and provides a convenient tool to study their genetic control.

G-protein coupled receptor 88 knockdown in the associative striatum reduces psychiatric symptoms in a translational male rat model of Parkinson disease.

Background: In addition to motor disability, another characteristic feature of Parkinson disease is the early appearance of psychiatric symptoms, including apathy, depression, anxiety and cognitive deficits; treatments for these symptoms are limited by the development of adverse effects such as impulse-control disorders. In this context, we investigated the orphan G protein-coupled receptor 88 (GPR88) as a novel therapeutic target. Methods: We used lentiviral-mediated expression of specifically designed microRNA to knock down Gpr88 in a translational male rat model of early Parkinson disease obtained by dopamine loss in the dorsolateral striatum as a result of 6-hydroxydopamine lesions. We evaluated the impact of Gpr88 knockdown on the Parkinson disease model using behavioural, immunohistochemical and in situ hybridization studies. Results: Knockdown of Gpr88 in associative territories of the dorsal striatum efficiently reduced alterations in mood, motivation and cognition through modulation of the regulator of the G-protein signalling 4 and of the truncated splice variant of the FosB transcription factor. Knockdown of Gpr88 also reduced allostatic changes in striatal activity markers that may be related to patterns observed in patients and that provide support for an "overload" hypothesis for the etiology of the psychiatric symptoms of Parkinson disease. Limitations: Behavioural tests assessing specific cognitive and motivational parameters are needed to further characterize the effects of the lesion and of Gpr88 knockdown in early-stage and advanced Parkinson disease models, presenting more extensive dopamine loss. Additional studies focusing on the direct and indirect striatal output pathways are also required, because little is known about the signalling pathways regulated by GPR88 in different striatal cell types. Conclusion: GPR88 may constitute a highly relevant target for the treatment of the psychiatric symptoms of Parkinson disease.

Patient-Derived Midbrain Organoids to Explore the Molecular Basis of Parkinson's Disease.

Induced pluripotent stem cell-derived organoids offer an unprecedented access to complex human tissues that recapitulate features of architecture, composition and function of in vivo organs. In the context of Parkinson's Disease (PD), human midbrain organoids (hMO) are of significant interest, as they generate dopaminergic neurons expressing markers of Substantia Nigra identity, which are the most vulnerable to degeneration. Combined with genome editing approaches, hMO may thus constitute a valuable tool to dissect the genetic makeup of PD by revealing the effects of risk variants on pathological mechanisms in a representative cellular environment. Furthermore, the flexibility of organoid co-culture approaches may also enable the study of neuroinflammatory and neurovascular processes, as well as interactions with other brain regions that are also affected over the course of the disease. We here review existing protocols to generate hMO, how they have been used so far to model PD, address challenges inherent to organoid cultures, and discuss applicable strategies to dissect the molecular pathophysiology of the disease. Taken together, the research suggests that this technology represents a promising alternative to 2D in vitro models, which could significantly improve our understanding of PD and help accelerate therapeutic developments.

Regulated expression and function of the GABAB receptor in human pancreatic beta cell line and islets.

G protein-coupled receptors are seven transmembrane signaling molecules that are involved in a wide variety of physiological processes. They constitute a large protein family of receptors with almost 300 members detected in human pancreatic islet preparations. However, the functional role of these receptors in pancreatic islets is unknown in most cases. We generated a new stable human beta cell line from neonatal pancreas. This cell line, named ECN90 expresses both subunits (GABBR1 and GABBR2) of the metabotropic GABAB receptor compared to human islet. In ECN90 cells, baclofen, a specific GABAB receptor agonist, inhibits cAMP signaling causing decreased expression of beta cell-specific genes such as MAFA and PCSK1, and reduced insulin secretion. We next demonstrated that in primary human islets, GABBR2 mRNA expression is strongly induced under cAMP signaling, while GABBR1 mRNA is constitutively expressed. We also found that induction and activation of the GABAB receptor in human islets modulates insulin secretion.

MiR-132 controls pancreatic beta cell proliferation and survival through Pten/Akt/Foxo3 signaling.

OBJECTIVE: MicroRNAs (miRNAs) play an integral role in maintaining beta cell function and identity. Deciphering their targets and precise role, however, remains challenging. In this study, we aimed to identify miRNAs and their downstream targets involved in the regeneration of islet beta cells following partial pancreatectomy in mice. METHODS: RNA from laser capture microdissected (LCM) islets of partially pancreatectomized and sham-operated mice were profiled with microarrays to identify putative miRNAs implicated in beta cell regeneration. Altered expression of the selected miRNAs, including miR-132, was verified by RT-PCR. Potential targets of miR-132 were selected through bioinformatic data mining. Predicted miR-132 targets were validated for their changed RNA, protein expression levels, and signaling upon miR-132 knockdown and/or overexpression in mouse MIN6 and human EndoC-ßH1 insulinoma cells. The ability of miR-132 to foster beta cell proliferation in vivo was further assessed in pancreatectomized miR-132-/- and control mice. RESULTS: Partial pancreatectomy significantly increased the number of BrdU+/insulin+ islet cells. Microarray profiling revealed that 14 miRNAs, including miR-132 and -141, were significantly upregulated in the LCM islets of the partially pancreatectomized mice compared to the LCM islets of the control mice. In the same comparison, miR-760 was the only downregulated miRNA. The changed expression of these miRNAs in the islets of the partially pancreatectomized mice was confirmed by RT-PCR only in the case of miR-132 and -141. Based on previous knowledge of its function, we focused our attention on miR-132. Downregulation of miR-132 reduced the proliferation of MIN6 cells while enhancing the levels of pro-apoptotic cleaved caspase-9. The opposite was observed in miR-132 overexpressing MIN6 cells. Microarray profiling, RT-PCR, and immunoblotting of the latter cells demonstrated their downregulated expression of Pten with concomitant increased levels of pro-proliferative factors phospho-Akt and phospho-Creb and inactivation of pro-apoptotic Foxo3a via its phosphorylation. Downregulation of Pten was further confirmed in the LCM islets of pancreatectomized mice compared to the sham-operated mice. Moreover, overexpression of miR-132 correlated with increased proliferation of EndoC-ßH1 cells. The regeneration of beta cells following partial pancreatectomy was lower in the miR-132/212-/- mice than the control littermates. CONCLUSIONS: This study provides compelling evidence about the critical role of miR-132 for the regeneration of mouse islet beta cells through the downregulation of its target Pten. Hence, the miR-132/Pten/Akt/Foxo3 signaling pathway may represent a suitable target to enhance beta cell mass.

Human pancreatic islet three-dimensional chromatin architecture provides insights into the genetics of type 2 diabetes.

Genetic studies promise to provide insight into the molecular mechanisms underlying type 2 diabetes (T2D). Variants associated with T2D are often located in tissue-specific enhancer clusters or super-enhancers. So far, such domains have been defined through clustering of enhancers in linear genome maps rather than in three-dimensional (3D) space. Furthermore, their target genes are often unknown. We have created promoter capture Hi-C maps in human pancreatic islets. This linked diabetes-associated enhancers to their target genes, often located hundreds of kilobases away. It also revealed >1,300 groups of islet enhancers, super-enhancers and active promoters that form 3D hubs, some of which show coordinated glucose-dependent activity. We demonstrate that genetic variation in hubs impacts insulin secretion heritability, and show that hub annotations can be used for polygenic scores that predict T2D risk driven by islet regulatory variants. Human islet 3D chromatin architecture, therefore, provides a framework for interpretation of T2D genome-wide association study (GWAS) signals.

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders.

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a routine procedure is widely utilized throughout the world and its main limitation resides in the poor efficacy of transgene integration, resulting in a low yield of founder animals. Only few percent of animals born after implantation of injected fertilized oocytes have integrated the transgene. In contrast, lentiviral vectors are powerful tools for integrative gene transfer and their use to transduce fertilized oocytes allows highly efficient production of founder transgenic mice with an average yield above 70%. Furthermore, any mouse strain can be used to produce transgenic animal and the penetrance of transgene expression is extremely high, above 80% with lentiviral mediated transgenesis compared to DNA microinjection. The size of the DNA fragment that can be cargo by the lentiviral vector is restricted to 10 kb and represents the major limitation of this method. Using a simple and easy to perform injection procedure beneath the zona pellucida of fertilized oocytes, more than 50 founder animals can be produced in a single session of microinjection. Such a method is highly adapted to perform, directly in founder animals, rapid gain and loss of function studies or to screen genomic DNA regions for their ability to control and regulate gene expression in vivo.

Differentiation of Sendai Virus-Reprogrammed iPSC into ß Cells, Compared with Human Pancreatic Islets and Immortalized ß Cell Line.

BACKGROUND: New sources of insulin-secreting cells are strongly in demand for treatment of diabetes. Induced pluripotent stem cells (iPSCs) have the potential to generate insulin-producing cells (iß). However, the gene expression profile and secretory function of iß still need to be validated in comparison with native ß cells. METHODS: Two clones of human iPSCs, reprogrammed from adult fibroblasts through integration-free Sendai virus, were differentiated into iß and compared with donor pancreatic islets and EndoC-ßH1, an immortalized human ß cell line. RESULTS: Both clones of iPSCs differentiated into insulin+ cells with high efficiency (up to 20%). iß were negative for pluripotency markers (Oct4, Sox2, Ssea4) and positive for Pdx1, Nkx6.1, Chromogranin A, PC1/3, insulin, glucagon and somatostatin. iß basally secreted C-peptide, glucagon and ghrelin and released insulin in response either to increasing concentration of glucose or a depolarizing stimulus. The comparison revealed that iß are remarkably similar to donor derived islets in terms of gene and protein expression profile and similar level of heterogeneity. The ability of iß to respond to glucose instead was more related to that of EndoC-ßH1. DISCUSSION: We demonstrated that insulin-producing cells generated from iPSCs recapitulate fundamental gene expression profiles and secretory function of native human ß cells.

Oligodendrocyte precursor survival and differentiation requires chromatin remodeling by Chd7 and Chd8.

Oligodendrocyte precursor cells (OPCs) constitute the main proliferative cells in the adult brain, and deregulation of OPC proliferation-differentiation balance results in either glioma formation or defective adaptive (re)myelination. OPC differentiation requires significant genetic reprogramming, implicating chromatin remodeling. Mounting evidence indicates that chromatin remodelers play important roles during normal development and their mutations are associated with neurodevelopmental defects, with CHD7 haploinsuficiency being the cause of CHARGE syndrome and CHD8 being one of the strongest autism spectrum disorder (ASD) high-risk-associated genes. Herein, we report on uncharacterized functions of the chromatin remodelers Chd7 and Chd8 in OPCs. Their OPC-chromatin binding profile, combined with transcriptome and chromatin accessibility analyses of Chd7-deleted OPCs, demonstrates that Chd7 protects nonproliferative OPCs from apoptosis by chromatin closing and transcriptional repression of p53 Furthermore, Chd7 controls OPC differentiation through chromatin opening and transcriptional activation of key regulators, including Sox10, Nkx2.2, and Gpr17 However, Chd7 is dispensable for oligodendrocyte stage progression, consistent with Chd8 compensatory function, as suggested by their common chromatin-binding profiles and genetic interaction. Finally, CHD7 and CHD8 bind in OPCs to a majority of ASD risk-associated genes, suggesting an implication of oligodendrocyte lineage cells in ASD neurological defects. Our results thus offer new avenues to understand and modulate the CHD7 and CHD8 functions in normal development and disease.

The Wnt/ß-catenin pathway determines the predisposition and efficiency of liver-to-pancreas reprogramming.

Transdifferentiation (TD) is the direct reprogramming of adult cells into cells of alternate fate and function. We have previously shown that liver cells can be transdifferentiated into beta-like, insulin-producing cells through ectopic expression of pancreatic transcription factors (pTFs). However, the efficiency of the process was consistently limited to <15% of the human liver cells treated in culture. The data in the current study suggest that liver-to-pancreas TD is restricted to a specific population of liver cells that is predisposed to undergo reprogramming. We isolated TD-predisposed subpopulation of liver cells from >15 human donors using a lineage tracing system based on the Wnt response element, part of the pericentral-specific promoter of glutamine synthetase. The cells, that were propagated separately, consistently exhibited efficient fate switch and insulin production and secretion in >60% of the cells upon pTF expression. The rest of the cells, which originated from 85% of the culture, resisted TD. Both populations expressed the ectopic pTFs with similar efficiencies, followed by similar repression of hepatic genes. Our data suggest that the TD-predisposed cells originate from a distinct population of liver cells that are enriched for Wnt signaling, which is obligatory for efficient TD. In TD-resistant populations, Wnt induction is insufficient to induce TD. An additional step of chromatin opening enables TD of these cells. CONCLUSION: Liver-to-pancreas TD occurs in defined predisposed cells. These cells' predisposition is maintained by Wnt signaling that endows the cells with the plasticity needed to alter their transcriptional program and developmental fate when triggered by ectopic pTFs. These results may have clinical implications by drastically increasing the efficacy of TD in future clinical uses. (Hepatology 2018).

The EndoC-ßH1 cell line is a valid model of human beta cells and applicable for screenings to identify novel drug target candidates.

OBJECTIVE: To characterize the EndoC-ßH1 cell line as a model for human beta cells and evaluate its beta cell functionality, focusing on insulin secretion, proliferation, apoptosis and ER stress, with the objective to assess its potential as a screening platform for identification of novel anti-diabetic drug candidates. METHODS: EndoC-ßH1 was transplanted into mice for validation of in vivo functionality. Insulin secretion was evaluated in cells cultured as monolayer and as pseudoislets, as well as in diabetic mice. Cytokine induced apoptosis, glucolipotoxicity, and ER stress responses were assessed. Beta cell relevant mRNA and protein expression were investigated by qPCR and antibody staining. Hundreds of proteins or peptides were tested for their effect on insulin secretion and proliferation. RESULTS: Transplantation of EndoC-ßH1 cells restored normoglycemia in streptozotocin induced diabetic mice. Both in vitro and in vivo, we observed a clear insulin response to glucose, and, in vitro, we found a significant increase in insulin secretion from EndoC-ßH1 pseudoislets compared to monolayer cultures for both glucose and incretins. Apoptosis and ER stress were inducible in the cells and caspase 3/7 activity was elevated in response to cytokines, but not affected by the saturated fatty acid palmitate. By screening of various proteins and peptides, we found Bombesin (BB) receptor agonists and Pituitary Adenylate Cyclase-Activating Polypeptides (PACAP) to significantly induce insulin secretion and the proteins SerpinA6, STC1, and APOH to significantly stimulate proliferation. ER stress was readily induced by Tunicamycin and resulted in a reduction of insulin mRNA. Somatostatin (SST) was found to be expressed by 1% of the cells and manipulation of the SST receptors was found to significantly affect insulin secretion. CONCLUSIONS: Overall, the EndoC-ßH1 cells strongly resemble human islet beta cells in terms of glucose and incretin stimulated insulin secretion capabilities. The cell line has an active cytokine induced caspase 3/7 apoptotic pathway and is responsive to ER stress initiation factors. The cells' ability to proliferate can be further increased by already known compounds as well as by novel peptides and proteins. Based on its robust performance during the functionality assessment assays, the EndoC-ßH1 cell line was successfully used as a screening platform for identification of novel anti-diabetic drug candidates.