REST is a major negative regulator of endocrine differentiation during pancreas organogenesis.

Multiple transcription factors have been shown to promote pancreatic ß-cell differentiation, yet much less is known about negative regulators. Earlier epigenomic studies suggested that the transcriptional repressor REST could be a suppressor of endocrinogenesis in the embryonic pancreas. However, pancreatic Rest knockout mice failed to show abnormal numbers of endocrine cells, suggesting that REST is not a major regulator of endocrine differentiation. Using a different conditional allele that enables profound REST inactivation, we observed a marked increase in pancreatic endocrine cell formation. REST inhibition also promoted endocrinogenesis in zebrafish and mouse early postnatal ducts and induced ß-cell-specific genes in human adult duct-derived organoids. We also defined genomic sites that are bound and repressed by REST in the embryonic pancreas. Our findings show that REST-dependent inhibition ensures a balanced production of endocrine cells from embryonic pancreatic progenitors.

Innervation of the pancreas in development and disease.

The autonomic nervous system innervates the pancreas by sympathetic, parasympathetic and sensory branches during early organogenesis, starting with neural crest cell invasion and formation of an intrinsic neuronal network. Several studies have demonstrated that signals from pancreatic neural crest cells direct pancreatic endocrinogenesis. Likewise, autonomic neurons have been shown to regulate pancreatic islet formation, and have also been implicated in type I diabetes. Here, we provide an overview of recent progress in mapping pancreatic innervation and understanding the interactions between pancreatic neurons, epithelial morphogenesis and cell differentiation. Finally, we discuss pancreas innervation as a factor in the development of diabetes.

Islet Neogenesis Associated Protein (INGAP) induces the differentiation of an adult human pancreatic ductal cell line into insulin-expressing cells through stepwise activation of key transcription factors for embryonic beta cell development.

Regeneration of ß-cells in diabetic patients is an important goal of diabetes research. Islet Neogenesis Associated Protein (INGAP) was discovered in the partially duct-obstructed hamster pancreas. Its bioactive fragment, pentadecapeptide 104-118 (INGAP-P), has been shown to reverse diabetes in animal models and to improve glucose homeostasis in patients with diabetes in clinical trials. Further development of INGAP as a therapy for diabetes requires identification of target cells in the pancreas and characterization of the mechanisms of action. We hypothesized that adult human pancreatic ductal cells retain morphogenetic plasticity and can be induced by INGAP to undergo endocrine differentiation. To test this hypothesis, we treated the normal human pancreatic ductal cell line (HPDE) with either INGAP-P or full-length recombinant protein (rINGAP) for short-term periods. Our data show that this single drug treatment induces both proliferation and transdifferentiation of HPDE cells, the latter being characterized by the rapid sequential activation of endocrine developmental transcription factors Pdx-1, Ngn3, NeuroD, IA-1, and MafA and subsequently the expression of insulin at both the mRNA and the protein levels. After 7 days, C-peptide was detected in the supernatant of INGAP-treated cells, reflecting their ability to secrete insulin. The magnitude of differentiation was enhanced by embedding the cells in Matrigel, which led to islet-like cluster formation. The islet-like clusters cells stained positive for nuclear Pdx-1 and Glut 2 proteins, and were expressing Insulin mRNA. These new data suggest that human adult pancreatic ductal cells retain morphogenetic plasticity and demonstrate that a short exposure to INGAP triggers their differentiation into insulin-expressing cells in vitro. In the context of the urgent search for a regenerative and/or cellular therapy for diabetes, these results make INGAP a promising therapeutic candidate.

Single-cell chromatin accessibility of developing murine pancreas identifies cell state-specific gene regulatory programs.

Numerous studies have characterized the existence of cell subtypes, along with their corresponding transcriptional profiles, within the developing mouse pancreas. The upstream mechanisms that initiate and maintain gene expression programs across cell states, however, remain largely unknown. Here, we generate single-nucleus ATAC-Sequencing data of developing murine pancreas and perform an integrated, multi-omic analysis of both chromatin accessibility and RNA expression to describe the chromatin landscape of the developing pancreas at both E14.5 and E17.5 at single-cell resolution. We identify candidate transcription factors regulating cell fate and construct gene regulatory networks of active transcription factor binding to regulatory regions of downstream target genes. This work serves as a valuable resource for the field of pancreatic biology in general and contributes to our understanding of lineage plasticity among endocrine cell types. In addition, these data identify which epigenetic states should be represented in the differentiation of stem cells to the pancreatic beta cell fate to best recapitulate in vitro the gene regulatory networks that are critical for progression along the beta cell lineage in vivo.

Pancreas agenesis mutations disrupt a lead enhancer controlling a developmental enhancer cluster.

Sequence variants in cis-acting enhancers are important for polygenic disease, but their role in Mendelian disease is poorly understood. Redundancy between enhancers that regulate the same gene is thought to mitigate the pathogenic impact of enhancer mutations. Recent findings, however, have shown that loss-of-function mutations in a single enhancer near PTF1A cause pancreas agenesis and neonatal diabetes. Using mouse and human genetic models, we show that this enhancer activates an entire PTF1A enhancer cluster in early pancreatic multipotent progenitors. This leading role, therefore, precludes functional redundancy. We further demonstrate that transient expression of PTF1A in multipotent progenitors sets in motion an epigenetic cascade that is required for duct and endocrine differentiation. These findings shed insights into the genome regulatory mechanisms that drive pancreas differentiation. Furthermore, they reveal an enhancer that acts as a regulatory master key and is thus vulnerable to pathogenic loss-of-function mutations.

Dynamic Click Hydrogels for Xeno-Free Culture of Induced Pluripotent Stem Cells.

Xeno-free, chemically defined poly(ethylene glycol) (PEG)-based hydrogels are being increasingly used for in vitro culture and differentiation of human induced pluripotent stem cells (hiPSCs). These synthetic matrices provide tunable gelation and adaptable material properties crucial for guiding stem cell fate. Here, sequential norbornene-click chemistries are integrated to form synthetic, dynamically tunable PEG-peptide hydrogels for hiPSCs culture and differentiation. Specifically, hiPSCs are photoencapsulated in thiol-norbornene hydrogels crosslinked by multiarm PEG-norbornene (PEG-NB) and proteaselabile crosslinkers. These matrices are used to evaluate hiPSC growth under the influence of extracellular matrix properties. Tetrazine-norbornene (Tz-NB) click reaction is then employed to dynamically stiffen the cell-laden hydrogels. Fast reactive Tz and its stable derivative methyltetrazine (mTz) are tethered to multiarm PEG, yielding mono-functionalized PEG-Tz, PEG-mTz, and dualfunctionalized PEG-Tz/mTz that react with PEG-NB to form additional crosslinks in the cell-laden hydrogels. The versatility of Tz-NB stiffening is demonstrated with different Tz-modified macromers or by intermittent incubation of PEG-Tz for temporal stiffening. Finally, the Tz-NB-mediated dynamic stiffening is explored for 4D culture and definitive endoderm differentiation of hiPSCs. Overall, this dynamic hydrogel platform affords exquisite controls of hydrogel crosslinking for serving as a xeno-free and dynamic stem cell niche.

A Specialized Niche in the Pancreatic Microenvironment Promotes Endocrine Differentiation.

The interplay between pancreatic epithelium and the surrounding microenvironment is pivotal for pancreas formation and differentiation as well as adult organ homeostasis. The mesenchyme is the main component of the embryonic pancreatic microenvironment, yet its cellular identity is broadly defined, and whether it comprises functionally distinct cell subsets is not known. Using genetic lineage tracing, transcriptome, and functional studies, we identified mesenchymal populations with different roles during pancreatic development. Moreover, we showed that Pbx transcription factors act within the mouse pancreatic mesenchyme to define a pro-endocrine specialized niche. Pbx directs differentiation of endocrine progenitors into insulin- and glucagon-positive cells through non-cell-autonomous regulation of ECM-integrin interactions and soluble molecules. Next, we measured functional conservation between mouse and human pancreatic mesenchyme by testing identified mesenchymal factors in an iPSC-based differentiation model. Our findings provide insights into how lineage-specific crosstalk between epithelium and neighboring mesenchymal cells underpin the generation of different pancreatic cell types.

Analysis of Differentiation Protocols Defines a Common Pancreatic Progenitor Molecular Signature and Guides Refinement of Endocrine Differentiation.

Several distinct differentiation protocols for deriving pancreatic progenitors (PPs) from human pluripotent stem cells have been described, but it remains to be shown how similar the PPs are across protocols and how well they resemble their in vivo counterparts. Here, we evaluated three differentiation protocols, performed RNA and assay for transposase-accessible chromatin using sequencing on isolated PPs derived with these, and compared them with fetal human pancreas populations. This enabled us to define a shared transcriptional and epigenomic signature of the PPs, including several genes not previously implicated in pancreas development. Furthermore, we identified a significant and previously unappreciated cross-protocol variation of the PPs through multi-omics analysis and demonstrate how such information can be applied to refine differentiation protocols for derivation of insulin-producing beta-like cells. Together, our study highlights the importance of a detailed characterization of defined cell populations derived from distinct differentiation protocols and provides a valuable resource for exploring human pancreatic development.

Expansion of Adult Human Pancreatic Tissue Yields Organoids Harboring Progenitor Cells with Endocrine Differentiation Potential.

Generating an unlimited source of human insulin-producing cells is a prerequisite to advance ß cell replacement therapy for diabetes. Here, we describe a 3D culture system that supports the expansion of adult human pancreatic tissue and the generation of a cell subpopulation with progenitor characteristics. These cells display high aldehyde dehydrogenase activity (ALDHhi), express pancreatic progenitors markers (PDX1, PTF1A, CPA1, and MYC), and can form new organoids in contrast to ALDHlo cells. Interestingly, gene expression profiling revealed that ALDHhi cells are closer to human fetal pancreatic tissue compared with adult pancreatic tissue. Endocrine lineage markers were detected upon in vitro differentiation. Engrafted organoids differentiated toward insulin-positive (INS+) cells, and circulating human C-peptide was detected upon glucose challenge 1 month after transplantation. Engrafted ALDHhi cells formed INS+ cells. We conclude that adult human pancreatic tissue has potential for expansion into 3D structures harboring progenitor cells with endocrine differentiation potential.

Establishment of a rapid and footprint-free protocol for differentiation of human embryonic stem cells into pancreatic endocrine cells with synthetic mRNAs encoding transcription factors.

BACKGROUND: Transplantation of pancreatic ß cells generated in vitro from pluripotent stem cells (hPSCs) such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) has been proposed as an alternative therapy for diabetes. Though many differentiation protocols have been developed for this purpose, lentivirus-mediated forced expression of transcription factors (TF)-PDX1 and NKX6.1-has been at the forefront for its relatively fast and straightforward approach. However, considering that such cells will be used for therapeutic purposes in the future, it is desirable to develop a procedure that does not leave any footprint on the genome, as any changes of DNAs could potentially be a source of unintended, concerning effects such as tumorigenicity. In this study, we attempted to establish a novel protocol for rapid and footprint-free hESC differentiation into a pancreatic endocrine lineage by using synthetic mRNAs (synRNAs) encoding PDX1 and NKX6.1. We also tested whether siPOU5F1, which reduces the expression of pluripotency gene POU5F1 (also known as OCT4), can enhance differentiation as reported previously for mesoderm and endoderm lineages. METHODS: synRNA-PDX1 and synRNA-NKX6.1 were synthesized in vitro and were transfected five times to hESCs with a lipofection reagent in a modified differentiation culture condition. siPOU5F1 was included only in the first transfection. Subsequently, cells were seeded onto a low attachment plate and aggregated by an orbital shaker. At day 13, the degree of differentiation was assessed by quantitative RT-PCR (qRT-PCR) and immunohistochemistry for endocrine hormones such as insulin, glucagon, and somatostatin. RESULTS: Both PDX1 and NKX6.1 expression were detected in cells co-transfected with synRNA-PDX1 and synRNA-NKX6.1 at day 3. Expression levels of insulin in the transfected cells at day 13 were 450 times and 14 times higher by qRT-PCR compared to the levels at day 0 and in cells cultured without synRNA transfection, respectively. Immunohistochemically, pancreatic endocrine hormones were not detected in cells cultured without synRNA transfection but were highly expressed in cells transfected with synRNA-PDX1, synRNA-NKX6.1, and siPOU5F1 at as early as day 13. CONCLUSIONS: In this study, we report a novel protocol for rapid and footprint-free differentiation of hESCs to endocrine cells.

Multi-site Neurogenin3 Phosphorylation Controls Pancreatic Endocrine Differentiation.

The proneural transcription factor Neurogenin3 (Ngn3) plays a critical role in pancreatic endocrine cell differentiation, although regulation of Ngn3 protein is largely unexplored. Here we demonstrate that Ngn3 protein undergoes cyclin-dependent kinase (Cdk)-mediated phosphorylation on multiple serine-proline sites. Replacing wild-type protein with a phosphomutant form of Ngn3 increases α cell generation, the earliest endocrine cell type to be formed in the developing pancreas. Moreover, un(der)phosphorylated Ngn3 maintains insulin expression in adult ß cells in the presence of elevated c-Myc and enhances endocrine specification during ductal reprogramming. Mechanistically, preventing multi-site phosphorylation enhances both Ngn3 stability and DNA binding, promoting the increased expression of target genes that drive differentiation. Therefore, multi-site phosphorylation of Ngn3 controls its ability to promote pancreatic endocrine differentiation and to maintain ß cell function in the presence of pro-proliferation cues and could be manipulated to promote and maintain endocrine differentiation in vitro and in vivo.

Neuroendocrine adenoma of the middle ear presented as paragaglioma: a case report.

BACKGROUND: Neuroendocrine adenoma of the middle ear (NAME) is a rare benign glandular tumor with neuroendocrine differentiation arising from the middle ear mucosa. The common symptoms and clinical findings of this tumor are usually non-specific. When the tumor appears as a reddish retrotympanic mass, it can be misdiagnosed to be a paraganglioma. CASE DESCRIPTION: A 38-year-old male presented with a 12-month history of left ear ​​fullness and tinnitus. Otoscopy revealed a reddish retrotympanic mass with an intact tympanic membrane. The imaging demonstrated a soft tissue mass adjacent to the eardrum and the ossicles, compatible with a paraganglioma. The mass was surgically removed through a posterior tympanotomy. The histological and immunohistochemical examination showed the specimen to be a neuroendocrine adenoma. Four years after the operation, the patient remains without any sign of recurrence. CONCLUSION: NAME is a rare tumor, mimicking tympanic paraganglioma, while its final diagnosis is only provided by histological and immunohistochemical analysis. HIPPOKRATIA 2017, 21(4): 201-203.

Notch1 signaling controls cell proliferation, apoptosis and differentiation in lung carcinoma.

OBJECTIVES: The role of Notch signaling in human lung cancer still remains unclear, and there has been and stills a debate, on the extent to which Notch ligands and receptors are involved in lung cancer development. This study was carried out to investigate the role of Notch1 signaling in the proliferation and differentiation of human lung cancer cells. METHODS: We used small interfering RNA (siRNA) technology to down-regulate the expression of Notch1 in small cell lung carcinoma (SCLC) cells; H69AR and SBC-3, as well as in non-small cell lung carcinoma (NSCLC) cells; A549 adenocarcinoma (ADC) and H2170 squamous cell carcinoma (SCC). Also, we transfected venus Notch1 intracellular domain (v.NICD) plasmid into the human SCLC line H69 and H1688. In addition, H1688 cells with activated Notch1 were injected into immune-compromised Rag2(-/-) Jak3(-/-) mice for analysis of ex vivo tumor growth and differentiation phenotype. RESULTS: Notch1 controls cell proliferation and apoptosis in both SCLC and A549; but not in H2170 cell line. Overexpression of Notch1 in SCLC markedly decreased cell proliferation via apoptosis. The subcutaneous tumors arising from xenotransplaned SCLC cells transfected with Notch1 showed "epithelial-like glandular" arrangement, with positive Alcian blue staining and reduction in neuroendocrine markers. CONCLUSION: Notch1 up regulation has an inhibitory effect on cell growth and NE differentiation in SCLC, with induction of an epithelial-like morphology of cells in tissue samples. In NSCLC, Notch1 expression has a tumor inhibitory effect on ADC cells, but not SCC cells.