Although the function of the mammalian pancreas hinges on complex interactions of distinct cell types, gene expression profiles have primarily been described with bulk mixtures. Here we implemented a droplet-based, single-cell RNA-seq method to determine the transcriptomes of over 12,000 individual pancreatic cells from four human donors and two mouse strains. Cells could be divided into 15 clusters that matched previously characterized cell types: all endocrine cell types, including rare epsilon-cells; exocrine cell types; vascular cells; Schwann cells; quiescent and activated stellate cells; and four types of immune cells. We detected subpopulations of ductal cells with distinct expression profiles and validated their existence with immuno-histochemistry stains. Moreover, among human beta- cells, we detected heterogeneity in the regulation of genes relating to functional maturation and levels of ER stress. Finally, we deconvolved bulk gene expression samples using the single-cell data to detect disease-associated differential expression. Our dataset provides a resource for the discovery of novel cell type-specific transcription factors, signaling receptors, and medically relevant genes.

A Single-Cell Transcriptomic Map of the Human and Mouse Pancreas Reveals Inter- and Intra-cell Population Structure.

The gut-liver axis in liver disease: Pathophysiological basis for therapy.

Systems Metabolic Engineering Strategies: Integrating Systems and Synthetic Biology with Metabolic Engineering

Synthetic Biology Tools to Engineer Microbial Communities for Biotechnology

The intestine is a major source of systemic ammonia (NH3); thus, capturing part of gut NH3 may mitigate disease symptoms in conditions of hyperammonemia such as urea cycle disorders and hepatic encephalopathy. As an approach to the lowering of blood ammonia arising from the intestine, we engineered the orally delivered probiotic Escherichia coli Nissle 1917 to create strain SYNB1020 that converts NH3 to l-arginine (l-arg). We up-regulated arginine biosynthesis in SYNB1020 by deleting a negative regulator of l-arg biosynthesis and inserting a feedback-resistant l-arg biosynthetic enzyme. SYNB1020 produced l-arg and consumed NH3 in an in vitro system. SYNB1020 reduced systemic hyperammonemia, improved survival in ornithine transcarbamylase-deficient spfash mice, and decreased hyperammonemia in the thioacetamide-induced liver injury mouse model. A phase 1 clinical study was conducted including 52 male and female healthy adult volunteers. SYNB1020 was well tolerated at daily doses of up to 1.5 × 1012 colony-forming units administered for up to 14 days. A statistically significant dose-dependent increase in urinary nitrate, plasma 15N-nitrate (highest dose versus placebo, P = 0.0015), and urinary 15N-nitrate was demonstrated, indicating in vivo SYNB1020 activity. SYNB1020 concentrations reached steady state by the second day of dosing, and excreted cells were alive and metabolically active as evidenced by fecal arginine production in response to added ammonium chloride. SYNB1020 was no longer detectable in feces 2 weeks after the last dose. These results support further clinical development of SYNB1020 for hyperammonemia disorders including urea cycle disorders and hepatic encephalopathy.

An engineered E. coli Nissle improves hyperammonemia and survival in mice and shows dose-dependent exposure in healthy humans.

Phenylketonuria (PKU) is a genetic disease that is characterized by an inability to metabolize phenylalanine (Phe), which can result in neurotoxicity. To provide a potential alternative to a protein-restricted diet, we engineered Escherichia coli Nissle to express genes encoding Phe-metabolizing enzymes in response to anoxic conditions in the mammalian gut. Administration of our synthetic strain, SYNB1618, to the Pahenu2/enu2 PKU mouse model reduced blood Phe concentration by 38% compared with the control, independent of dietary protein intake. In healthy Cynomolgus monkeys, we found that SYNB1618 inhibited increases in serum Phe after an oral Phe dietary challenge. In mice and primates, Phe was converted to trans-cinnamate by SYNB1618, quantitatively metabolized by the host to hippurate and excreted in the urine, acting as a predictive biomarker for strain activity. SYNB1618 was detectable in murine or primate feces after a single oral dose, permitting the evaluation of pharmacodynamic properties. Our results define a strategy for translation of live bacterial therapeutics to treat metabolic disorders.

Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria

Although stem cell populations mediate regeneration of rapid turnover tissues, such as skin, blood, and gut, a stem cell reservoir has not been identified for some slower turnover tissues, such as the pancreatic islet. Despite lacking identifiable stem cells, murine pancreatic β cell number expands in response to an increase in insulin demand. Lineage tracing shows that new β cells are generated from proliferation of mature, differentiated β cells; however, the mechanism by which these mature cells sense systemic insulin demand and initiate a proliferative response remains unknown. Here, we identified the β cell unfolded protein response (UPR), which senses insulin production, as a regulator of β cell proliferation. Using genetic and physiologic models, we determined that among the population of β cells, those with an active UPR are more likely to proliferate. Moreover, subthreshold endoplasmic reticulum stress (ER stress) drove insulin demand-induced β cell proliferation, through activation of ATF6. We also confirmed that the UPR regulates proliferation of human β cells, suggesting that therapeutic UPR modulation has potential to expand β cell mass in people at risk for diabetes. Together, this work defines a stem cell-independent model of tissue homeostasis, in which differentiated secretory cells use the UPR sensor to adapt organ size to meet demand.

Insulin demand regulates β cell number via the unfolded protein response

240 - Genetically Engineered E. Coli Nissle Attenuates Hyperammonemia in a Mouse Model of Hepatic Encephalopathy by Metabolizing Gut Ammonia and Increasing Urea Production

Binh Ha

Papers

Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria

Insulin demand regulates β cell number via the unfolded protein response

240 - Genetically Engineered E. Coli Nissle Attenuates Hyperammonemia in a Mouse Model of Hepatic Encephalopathy by Metabolizing Gut Ammonia and Increasing Urea Production