Objective - 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 alpha cells.
Research Design and Methods - Using a combination of high-throughput small molecule screening and empirical testing, we developed a six-stage differentiation protocol for creating functional alpha cells. An extensive in vitro and in vivo characterization of the differentiated cells was performed.
Results - A high rate of synaptophysin expression (>75%) and robust expression of glucagon and the alpha cell-specific transcription factor ARX was achieved. Following a transient polyhormonal state in which cells co-express glucagon and insulin, maturation in vitro or in vivo resulted in depletion of insulin and other beta cell markers with concomitant enrichment of alpha cell markers. Following transplantation, these cells secreted fully processed, biologically active glucagon in response to physiological 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 alpha cell mass.
Conclusions - These results indicate that fully differentiated pancreatic endocrine cells can be created via step-wise 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 alpha cells to beta cells.
- Received April 22, 2010.
- Accepted September 20, 2010.
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