Autoimmunity

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66083/1/j.1365-4362.1981.tb00393.x.pd

A Scalable System for Production of Functional Pancreatic Progenitors from Human Embryonic Stem Cells

Development of a human embryonic stem cell (hESC)-based therapy for type 1 diabetes will require the translation of proof-of-principle concepts into a scalable, controlled, and regulated cell manufacturing process. We have previously demonstrated that hESC can be directed to differentiate into pancreatic progenitors that mature into functional glucose-responsive, insulin-secreting cells in vivo. In this study we describe hESC expansion and banking methods and a suspension-based differentiation system, which together underpin an integrated scalable manufacturing process for producing pancreatic progenitors. This system has been optimized for the CyT49 cell line. Accordingly, qualified large-scale single-cell master and working cGMP cell banks of CyT49 have been generated to provide a virtually unlimited starting resource for manufacturing. Upon thaw from these banks, we expanded CyT49 for two weeks in an adherent culture format that achieves 50–100 fold expansion per week. Undifferentiated CyT49 were then aggregated into clusters in dynamic rotational suspension culture, followed by differentiation en masse for two weeks with a four-stage protocol. Numerous scaled differentiation runs generated reproducible and defined population compositions highly enriched for pancreatic cell lineages, as shown by examining mRNA expression at each stage of differentiation and flow cytometry of the final population. Islet-like tissue containing glucose-responsive, insulin-secreting cells was generated upon implantation into mice. By four- to five-months post-engraftment, mature neo-pancreatic tissue was sufficient to protect against streptozotocin (STZ)-induced hyperglycemia. In summary, we have developed a tractable manufacturing process for the generation of functional pancreatic progenitors from hESC on a scale amenable to clinical entry

Impaired cholecystokinin-induced gallbladder emptying incriminated in spontaneous “black” pigment gallstone formation in germfree Swiss Webster mice

“Black” pigment gallstones form in sterile gallbladder bile in the presence of excess bilirubin conjugates (“hyperbilirubinbilia”) from ineffective erythropoiesis, hemolysis, or induced enterohepatic cycling (EHC) of unconjugated bilirubin. Impaired gallbladder motility is a less well-studied risk factor. We evaluated the spontaneous occurrence of gallstones in adult germfree (GF) and conventionally housed specific pathogen-free (SPF) Swiss Webster (SW) mice. GF SW mice were more likely to have gallstones than SPF SW mice, with 75% and 23% prevalence, respectively. In GF SW mice, gallstones were observed predominately in heavier, older females. Gallbladders of GF SW mice were markedly enlarged, contained sterile black gallstones composed of calcium bilirubinate and <1% cholesterol, and had low-grade inflammation, edema, and epithelial hyperplasia. Hemograms were normal, but serum cholesterol was elevated in GF compared with SPF SW mice, and serum glucose levels were positively related to increasing age. Aged GF and SPF SW mice had deficits in gallbladder smooth muscle activity. In response to cholecystokinin (CCK), gallbladders of fasted GF SW mice showed impaired emptying (females: 29%; males: 1% emptying), whereas SPF SW females and males emptied 89% and 53% of volume, respectively. Bilirubin secretion rates of GF SW mice were not greater than SPF SW mice, repudiating an induced EHC. Gallstones likely developed in GF SW mice because of gallbladder hypomotility, enabled by features of GF physiology, including decreased intestinal CCK concentration and delayed intestinal transit, as well as an apparent genetic predisposition of the SW stock. GF SW mice may provide a valuable model to study gallbladder stasis as a cause of black pigment gallstones.National Institutes of Health (U.S.) (Training Grant T32-OD10978-26)National Institutes of Health (U.S.) (Training Grant P30-ES002109)Kinship Foundation. Searle Scholars Progra