Generation of Insulin-producing Islet-like Clusters from Human Embryonic Stem Cells

¹ Geron Corporation, Menlo Park, CA, USA
² University of Alberta, Edmonton, Alberta, Canada.

^* To whom correspondence should be addressed. E-mail: amajumdar{at}geron.com.

	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 self-renewing, pluripotent human embryonic stem cells (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 (Na-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 EGF, bFGF 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 IGFII 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 insulin/µg 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.

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