Production of Functional Glucagon-Secreting α-Cells From Human Embryonic Stem Cells

• Published in: Diabetes (New York, N.Y.) Vol. 60; no. 1; pp. 239 - 247
• Main Authors: REZANIA, Alireza, RIEDEL, Michael J, WIDEMAN, Rhonda D, KARANU, Francis, ZILIANG AO, WARNOCK, Garth L, KIEFFER, Timothy J
• Format: Journal Article
• Language: English
• Published: Alexandria, VA American Diabetes Association 01.01.2011
• Subjects: Animals; Biological and medical sciences; Blood Glucose - metabolism; Cell Differentiation - physiology; Cell Line; Cytogenetics; Diabetes. Impaired glucose tolerance; DNA - analysis; DNA - genetics; Embryonic stem cells; Embryonic Stem Cells - cytology; Embryonic Stem Cells - secretion; Embryonic Stem Cells - transplantation; Endocrine pancreas. Apud cells (diseases); Endocrinopathies; Etiopathogenesis. Screening. Investigations. Target tissue resistance; Genetic aspects; Glucagon; Glucagon - analysis; Glucagon - secretion; Glucagon-Secreting Cells - cytology; Glucagon-Secreting Cells - secretion; Growth; Humans; Immunohistochemistry; Immunology and Transplantation; Insulin - analysis; Islands of Langerhans; Islets of Langerhans - cytology; Islets of Langerhans - secretion; Medical sciences; Mice; Proglucagon - genetics; Properties; Reverse Transcriptase Polymerase Chain Reaction; Canada; Endocrinopathy; Human; Pancreatic hormone; Embryo; Glucagon; Stem cell; Diabetes mellitus; Peptide hormone
• ISSN: 0012-1797; 1939-327X
• DOI: 10.2337/db10-0573

Differentiation of human embryonic stem (hES) cells to fully developed cell types holds great therapeutic promise. Despite significant progress, the conversion of hES cells to stable, fully differentiated endocrine cells that exhibit physiologically regulated hormone secretion has not yet been achieved. Here we describe an efficient differentiation protocol for the in vitro conversion of hES cells to functional glucagon-producing α- cells. Using a combination of small molecule screening and empirical testing, we developed a six-stage differentiation protocol for creating functional α-cells. An extensive in vitro and in vivo characterization of the differentiated cells was performed. A high rate of synaptophysin expression (>75%) and robust expression of glucagon and the α-cell transcription factor ARX was achieved. After a transient polyhormonal state in which cells coexpress glucagon and insulin, maturation in vitro or in vivo resulted in depletion of insulin and other β-cell markers with concomitant enrichment of α-cell markers. After transplantation, these cells secreted fully processed, biologically active glucagon in response to physiologic stimuli including prolonged fasting and amino acid challenge. Moreover, glucagon release from transplanted cells was sufficient to reduce demand for pancreatic glucagon, resulting in a significant decrease in pancreatic α-cell mass. These results indicate that fully differentiated pancreatic endocrine cells can be created via stepwise differentiation of hES cells. These cells may serve as a useful screening tool for the identification of compounds that modulate glucagon secretion as well as those that promote the transdifferentiation of α-cells to β-cells.