Generation of functional human pancreatic ? cells in vitro.
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ABSTRACT:
SUBMITTER: Pagliuca FW
PROVIDER: S-EPMC4617632 | BioStudies | 2014-01-01
REPOSITORIES: biostudies
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Project description:The generation of insulin-producing pancreatic 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 cells (SC) 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. Differentiated cells were sorted and processed for RNA isolation using the MARIS protocol published previously (PMID: 24516164.) Human embryonic stem cell (hESC) line HUES8 was differentiated into SC-beta cells. Two biological replicates were analyzed. Those data were normalized together with and compared to existing, previously published data from Hrvatin et al. ( (PMID: 24516164) from human islet -derived insulin+ cells, undifferentiated HUES8 hES cells, and insulin+ cells derived from HUES8 cells according to previously published protocols.
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Project description:Recent advances in human pluripotent stem cell (hPSC) differentiation protocols have generated insulin-producing cells resembling pancreatic β cells. While these stem cell-derived β (SC-β) cells are capable of undergoing glucose-stimulated insulin secretion (GSIS), insulin secretion per cell remains low compared with islets and cells lack dynamic insulin release. Herein, we report a differentiation strategy focused on modulating transforming growth factor β (TGF-β) signaling, controlling cellular cluster size, and using an enriched serum-free media to generate SC-β cells that express β cell markers and undergo GSIS with first- and second-phase dynamic insulin secretion. Transplantation of these cells into mice greatly improves glucose tolerance. These results reveal that specific time frames for inhibiting and permitting TGF-β signaling are required during SC-β cell differentiation to achieve dynamic function. The capacity of these cells to undergo GSIS with dynamic insulin release makes them a promising cell source for diabetes cellular therapy.
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Project description:Transplantation of human pluripotent stem cells (hPSC) differentiated into insulin-producing ? cells is a regenerative medicine approach being investigated for diabetes cell replacement therapy. This report presents a multifaceted transplantation strategy that combines differentiation into stem cell-derived ? (SC-?) cells with 3D printing. By modulating the parameters of a low-cost 3D printer, we created a macroporous device composed of polylactic acid (PLA) that houses SC-? cell clusters within a degradable fibrin gel. Using finite element modeling of cellular oxygen diffusion-consumption and an in vitro culture system that allows for culture of devices at physiological oxygen levels, we identified cluster sizes that avoid severe hypoxia within 3D-printed devices and developed a microwell-based technique for resizing clusters within this range. Upon transplantation into mice, SC-? cell-embedded 3D-printed devices function for 12 weeks, are retrievable, and maintain structural integrity. Here, we demonstrate a novel 3D-printing approach that advances the use of differentiated hPSC for regenerative medicine applications and serves as a platform for future transplantation strategies.
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