Summary The generation of insulin-producing pancreatic β cells from stem cells in vitro would provide an unprecedented cell source for drug discovery and cell transplantation therapy in diabetes. However, insulin-producing cells previously generated from human pluripotent stem cells (hPSC) lack many functional characteristics of bona fide β cells. Here we report a scalable differentiation protocol that can generate hundreds of millions of glucose-responsive β cells from hPSC in vitro. These stem cell derived β cells (SC-β) express markers found in mature β cells, flux Ca2+ in response to glucose, package insulin into secretory granules and secrete quantities of insulin comparable to adult β cells in response to multiple sequential glucose challenges in vitro. Furthermore, these cells secrete human insulin into the serum of mice shortly after transplantation in a glucose-regulated manner, and transplantation of these cells ameliorates hyperglycemia in diabetic mice.
In vitro differentiation of human stem cells can produce pancreatic β cells; the loss of this insulin-secreting cell type underlies type 1 diabetes. Here, as a step towards understanding this differentiation process, we report the transcriptional profiling of over 100,000 human cells undergoing in vitro β-cell differentiation, and describe the cells that emerged. We resolve populations that correspond to β cells, α-like poly-hormonal cells, non-endocrine cells that resemble pancreatic exocrine cells and a previously unreported population that resembles enterochromaffin cells. We show that endocrine cells maintain their identity in culture in the absence of exogenous growth factors, and that gene-expression changes associated with in vivo β-cell maturation are recapitulated in vitro. We implement a scalable re-aggregation technique to deplete non-endocrine cells and identify CD49a (also known as ITGA1) as a surface marker for the β-cell population, which allows magnetic sorting to a purity of 80%. Finally, we use a high-resolution sequencing time course to characterize gene-expression dynamics during human pancreatic endocrine induction, from which we develop a lineage model of in vitro β-cell differentiation. This study provides a deep perspective on human stem cell differentiation, and will guide future endeavours that focus on the differentiation of pancreatic islet cells, and applications in regenerative medicine.
SUMMARYThe direct induction of apoptosis has emerged as a powerful anti-cancer strategy, and small molecules that either inhibit or activate certain proteins in the apoptotic pathway have great potential as novel chemotherapeutic agents. Central to apoptosis is the activation of the zymogen procaspase-3 to caspase-3. Caspase-3 is the key "executioner" caspase, catalyzing the hydrolysis of a multitude of protein substrates within the cell. Interestingly, procaspase-3 levels are often elevated in cancer cells, suggesting a compound that directly stimulates the activation of procaspase-3 to caspase-3 could selectively induce apoptosis in cancer cells. We recently reported the discovery of a compound, PAC-1, which enhances procaspase-3 activity in vitro and induces apoptotic death in cancer cells in culture and in mouse xenograft models. Described herein is the mechanism by which PAC-1 activates procaspase-3 in vitro. We show that zinc inhibits the enzymatic activity of procaspase-3, and that PAC-1 strongly activates procaspase-3 in buffers that contain zinc. PAC-1 and zinc form a tight complex with one another, with a dissociation constant of approximately 42 nM. The combined data indicate that PAC-1 activates procaspase-3 in vitro by sequestering inhibitory zinc ions, thus allowing procaspase-3 to autoactivate itself to caspase-3. The small molecule mediated activation of procaspases has great therapeutic potential and thus this discovery of the in vitro mechanism of action of PAC-1 is critical to the development and optimization of other procaspase-activating compounds.
Angioblasts are multipotent progenitor cells that give rise to arteries or veins . Genetic disruption of the gridlock gene perturbs the artery/vein balance, resulting in generation of insufficient numbers of arterial cells . However, within angioblasts the precise biochemical signals that determine the artery/vein cell-fate decision are poorly understood. We have identified by chemical screening two classes of compounds that compensate for a mutation in the gridlock gene . Both target the VEGF signaling pathway and reveal two downstream branches emanating from the VEGF receptor with opposing effects on arterial specification. We show that activation of ERK (p42/44 MAP kinase) is a specific marker of early arterial progenitors and is among the earliest known determinants of arterial specification. In embryos, cells fated to contribute to arteries express high levels of activated ERK, whereas cells fated to contribute to veins do not. Inhibiting the phosphatidylinositol-3 kinase (PI3K) branch with GS4898 or known PI3K inhibitors, or by expression of a dominant-negative form of AKT promotes arterial specification. Conversely, inhibition of the ERK branch blocks arterial specification, and expression of constitutively active AKT promotes venous specification. In summary, chemical genetic analysis has uncovered unanticipated opposing roles of PI3K and ERK in artery/vein specification.
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