Generation of Insulin-Producing Islet-Like Clusters from Human Embryonic Stem Cells

Abstract

Recent success in pancreatic islet transplantation has energized the field to discover an alternative source of stem cells with differentiation potential to ␤ cells. Generation of glucose-responsive, insulin-producing ␤ cells from selfrenewing, pluripotent human ESCs (hESCs) has immense potential for diabetes treatment. We report here the development of a novel serum-free protocol to generate insulin-producing islet-like clusters (ILCs) from hESCs grown under feeder-free conditions. In this 36-day protocol, hESCs were treated with sodium butyrate and activin A to generate definitive endoderm coexpressing CXCR4 and Sox17, and CXCR4 and Foxa2. The endoderm population was then converted into cellular aggregates and further differentiated to Pdx1-expressing pancreatic endoderm in the presence of epidermal growth factor, basic fibroblast growth factor, and noggin. Soon thereafter, expression of Ptf1a and Ngn3 was detected, indicative of further pancreatic differentiation. The aggregates were finally matured in the presence of insulin-like growth factor II and nicotinamide. The temporal pattern of pancreas-specific gene expression in the hESC-derived ILCs showed considerable similarity to in vivo pancreas development, and the final population contained representatives of the ductal, exocrine, and endocrine pancreas. The hESC-derived ILCs contained 2%-8% human C-peptide-positive cells, as well as glucagon-and somatostatin-positive cells. Insulin content as high as 70 ng of insulin/g of DNA was measured in the ILCs, representing levels higher than that of human fetal islets. In addition, the hESC-derived ILCs contained numerous secretory granules, as determined by electron microscopy, and secreted human C-peptide in a glucose-dependent manner.

Sodium butyrate and dexamethasone promote exocrine pancreatic gene expression in mouse embryonic stem cells

“…Acinar cells constitute 90% of the pancreatic epithelium and play a central role in pancreatic diseases, such as pancreatitis and pancreatic cancer. Although recent studies have indicated that ES cells can be induced to differentiate into insulin-secreting cells [7,8] , until now there have been only limited methods for studying acinar-cell differentiation in vitro. Because both the liver and the pancreas develop from the common embryonic endoderm and because these two lineages can transdifferentiate into each other [18,19] , exogenous factors that induce hepatic differentiation may also affect pancreatic differentiation.…”

“…Certain differentiation factors involved in embryonic development can differentiate ES cells into cell types from all three germ layers in vitro [3][4][5] . More specifically, recent studies have demonstrated the capacity of ES cells to differentiate into endocrine pancreatic or insulin-producing cells in vitro [6][7][8] . However, the pancreas is composed of both exocrine and endocrine compartments, and although ES-derived pancreatic β-cell differentiation has been characterized, little is known regarding exocrine pancreatic differentiation.…”

“…Recent research demonstrated that sodium butyrate can activate early pancreatic development genes in ES cells [12] and induce ES cells to differentiate into islet-like clusters [8] . Our previous work has also suggested that sodium butyrate can induce endoderm differentiation.…”

CD24: A Novel Surface Marker for PDX1-Positive Pancreatic Progenitors Derived from Human Embryonic Stem Cells

“…Significant progresses have been made in directing human ESCs to differentiate into pancreatic progenitors and insulin-producing beta cells by stepwise induction [2][3][4][5][6][7][8]. However, although pancreatic progenitors can be efficiently generated from human ESCs, efficient in vitro differentiation into mature functional beta cells that are able to reverse diabetes in animal models has not been achieved.…”

Differentiation of Stem Cells into Insulin-Producing Cells: Current Status and Challenges

“…They indicated that iPS cells and ESCs exhibit similar morphological and genetic changes during ILCs differentiation. Differentiation of ILCs was performed according to four-stage protocol described previously by Jiang et al (2007b). The iPS-derived ILCs contained C-peptide-positive and glucagon-positive cells and released C-peptide upon glucose stimulation.…”