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.

Cyclic AMP is an important second messenger in the coordinated regulation of cellular metabolism. Its effects are mediated by cAMP-dependent protein kinase (PKA), which is assembled from two regulatory (R) and two catalytic (C) subunits. In mice there are four R genes (encoding RI alpha, RI beta, RII alpha, and RII beta) and two C gene (encoding C alpha and C beta), expressed in tissue-specific patterns. The RII beta isoform is abundant in brown and white adipose tissue and brain, with limited expression elsewhere. To elucidate its functions, we generated RII beta knockout mice. Here we report that mutants appear healthy but have markedly diminished white adipose tissue despite normal food intake. They are protected against developing diet-induced obesity and fatty livers. Mutant brown adipose tissue exhibits a compensatory increase in RI alpha, which almost entirely replaces lost RII beta, generating an isoform switch. The holoenzyme from mutant adipose tissue binds cAMP more avidly and is more easily activated than wild-type enzyme. This causes induction of uncoupling protein and elevations of metabolic rate and body temperature, contributing to the lean phenotype. Our results demonstrate a role for the RII beta holoenzyme in regulating energy balance and adiposity.

Synapse formation requires interactions between pre- and postsynaptic cells to establish the connection of a presynaptic nerve terminal with the neurotransmitter receptor-rich postsynaptic apparatus. At developing vertebrate neuromuscular junctions, acetylcholine receptor (AChR) clusters of nascent postsynaptic apparatus are not apposed by presynaptic nerve terminals. Two opposing activities subsequently promote the formation of synapses: positive signals stabilize the innervated AChR clusters, whereas negative signals disperse those that are not innervated. Although the nerve-derived protein agrin has been suggested to be a positive signal, the negative signals remain elusive. Here, we show that cyclin-dependent kinase 5 (Cdk5) is activated by ACh agonists and is required for the ACh agonist-induced dispersion of the AChR clusters that have not been stabilized by agrin. Genetic elimination of Cdk5 or blocking ACh production prevents the dispersion of AChR clusters in agrin mutants. Therefore, we propose that ACh negatively regulates neuromuscular synapse formation through a Cdk5-dependent mechanism.

The Cre recombinase (Cre) from bacteriophage P1 is an important tool for genetic engineering in mammalian cells. We constructed lentiviral vectors that efficiently deliver Cre in vitro and in vivo. Surprisingly, we found a significant reduction in proliferation and an accumulation in the G2͞M phase of Cre-expressing cells. To minimize the toxic effect of Cre, we designed a lentiviral vector that integrates into the host genome, expresses Cre in the target cell, and is subsequently deleted from the genome in a Credependent manner. Thus, the activity of Cre terminates its own expression (self-deleting). We showed efficient modification of target genes in vitro and in the brain after transduction with the self-deleting vectors. In contrast to sustained Cre expression, transient expression of Cre from the self-deleting vector induced significantly less cytotoxicity. Such a self-deleting Cre vector is a promising tool for the induction of conditional gene modifications with minimal Cre toxicity in vivo.gene transfer ͉ regulated gene expression ͉ Cre toxicity T he site-specific recombinase of the bacteriophage P1, Cre, catalyzes the recombination of two 34-bp sequences called loxP, which consist of two 13-bp palindromic sequences flanking an 8-bp core sequence (1, 2). Because of the high efficiency of the Cre͞loxP system in mammalian cells (3, 4), this system is now widely used for genetic engineering in vitro and in vivo (5, 6). Transfer of Cre in vivo can be achieved by the use of transgenic mice or by viral delivery to the target organ(s). Thus far, the transfer of Cre by using nonviral delivery vehicles such as liposomes has only been described for use in vitro (7).We have generated lentiviral vectors that carry the Cre recombinase and have analyzed their ability to mediate loxPspecific recombination. Lentiviruses are enveloped RNA viruses that belong to the family of complex retroviruses (for review see refs. 8 and 9). In contrast to prototypic retroviruses, such as murine leukemia virus, lentiviruses are able to transduce both dividing and nondividing cells. Lentiviral vectors pseudotyped with the G protein of the vesicular stomatitis virus can transduce a wide range of cells and have great potential for human gene therapy (10-13).The Cre lentiviral vectors efficiently transferred Cre to the target cells and induced Cre-mediated recombination of loxP sites in vitro and in vivo. The Cre-expressing cells unexpectedly exhibited a reduction in proliferation and accumulated in the G 2 ͞M phase of the cell cycle. To circumvent this toxic effect of Cre, we designed a Cre lentivirus vector that is itself subject to Cre-mediated excision (self-deleting or LV-Cre-SD). In this way, Cre expression is limited to only the time necessary for recombination. Our studies show that this approach works efficiently in vitro and in vivo. Materials and MethodsAnimals. Homozygous R26R mice (12-20 weeks of age) were anesthetized by i.p. injection of a ketamine (150 mg/kg)͞xylazine (15 mg/kg) mixture. Virus (3 l) was injected either ster...