Differentiation of Human Pluripotent Stem Cells into Insulin-Producing Islet Clusters.

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and diabetes treatment. However, challenges remain in obtaining stem cell-derived beta cells that adequately mimic native human beta cells. Building upon previous studies, hPSC-derived islet cells have been generated to create a protocol with improved differentiation outcomes and consistency. The protocol described here utilizes a pancreatic progenitor kit during Stages 1-4, followed by a protocol modified from a paper previously published in 2014 (termed "R-protocol" hereafter) during Stages 5-7. Detailed procedures for using the pancreatic progenitor kit and 400 µm diameter microwell plates to generate pancreatic progenitor clusters, R-protocol for endocrine differentiation in a 96-well static suspension format, and in vitro characterization and functional evaluation of hPSC-derived islets, are included. The complete protocol takes 1 week for initial hPSC expansion followed by ~5 weeks to obtain insulin-producing hPSC islets. Personnel with basic stem cell culture techniques and training in biological assays can reproduce this protocol.

Protocol development to further differentiate and transition stem cell-derived pancreatic progenitors from a monolayer into endocrine cells in suspension culture.

The generation of functional ß-cells from human pluripotent stem cells (hPSCs) for cell replacement therapy and disease modeling of diabetes is being investigated by many groups. We have developed a protocol to harvest and aggregate hPSC-derived pancreatic progenitors generated using a commercially available kit into near uniform spheroids and to further differentiate the cells toward an endocrine cell fate in suspension culture. Using a static suspension culture platform, we could generate a high percentage of insulin-expressing, glucose-responsive cells. We identified FGF7 as a soluble factor promoting aggregate survival with no inhibitory effect on endocrine gene expression. Notch inhibition of pancreatic progenitor cells during aggregation improved endocrine cell induction in vitro and improved graft function following implantation and further differentiation in mice. Thus we provide an approach to promote endocrine formation from kit-derived pancreatic progenitors, either through extended culture or post implant.

Differentiation of human pluripotent stem cells into ß-cells: Potential and challenges.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) hold great potential as the basis for cell-based therapies of degenerative diseases, including diabetes. Current insulin-based therapies for diabetes do not prevent hyperglycaemia or the associated long-term organ damage. While transplantation of pancreatic islets can achieve insulin independence and improved glycemic control, it is limited by donor tissue scarcity, challenges of purifying islets from the pancreas, and the need for immunosuppression to prevent rejection of transplants. Large-scale production of ß-cells from stem cells is a promising alternative. Recent years have seen considerable progress in the optimization of in vitro differentiation protocols to direct hESCs/iPSCs into mature insulin-secreting ß-cells and clinical trials are now under way to test the safety and efficiency of hESC-derived pancreatic progenitor cells in patients with type 1 diabetes. Here, we discuss key milestones leading up to these trials in addition to recent developments and challenges for hESC/iPSC-based diabetes therapies and disease modeling.

Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells.

Transplantation of pancreatic progenitors or insulin-secreting cells derived from human embryonic stem cells (hESCs) has been proposed as a therapy for diabetes. We describe a seven-stage protocol that efficiently converts hESCs into insulin-producing cells. Stage (S) 7 cells expressed key markers of mature pancreatic beta cells, including MAFA, and displayed glucose-stimulated insulin secretion similar to that of human islets during static incubations in vitro. Additional characterization using single-cell imaging and dynamic glucose stimulation assays revealed similarities but also notable differences between S7 insulin-secreting cells and primary human beta cells. Nevertheless, S7 cells rapidly reversed diabetes in mice within 40 days, roughly four times faster than pancreatic progenitors. Therefore, although S7 cells are not fully equivalent to mature beta cells, their capacity for glucose-responsive insulin secretion and rapid reversal of diabetes in vivo makes them a promising alternative to pancreatic progenitor cells or cadaveric islets for the treatment of diabetes.

The tumor suppressor annexin A10 is a novel component of nuclear paraspeckles.

Annexin A10 is the latest identified member of the annexin family of Ca(2+)- and phospholipid-binding proteins. In previous studies, downregulation of annexin A10 was correlated with dedifferentiation, invasion, and tumor progression, pointing to a possible tumor suppressor role. However, the biochemical characteristics and functions of annexin A10 remain unknown. We show that annexin A10 displays biochemical characteristics atypical for an annexin, indicating a Ca(2+)- and membrane-binding-independent function. Annexin A10 co-localizes with the mRNA-binding proteins SFPQ and PSPC1 at paraspeckles, an only recently discovered nuclear body, and decreases paraspeckle numbers when overexpressed in HeLa cells. In addition, annexin A10 relocates to dark perinucleolar caps upon transcriptional inhibition of RNA polymerase II. We mapped the cap-binding function of annexin A10 to the proximal part of the core domain, which is missing in the short isoform of annexin A10, and show its independence from the remaining functional type II Ca(2+)-binding site. In contrast to this, paraspeckle recruitment required additional core regions and was negatively affected by the mutation of the last type II Ca(2+)-binding site. Additionally, we show that overexpression of annexin A10 in HeLa cells increases their sensitivity to apoptosis and reduces colony formation. The identification of unique nuclear and biochemical characteristics of annexin A10 points towards its membrane-independent role in paraspeckle-associated mRNA regulation or processing.