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Production of functional glucagon-secreting α-cells from human embryonic stem cells.

Rezania A, Riedel MJ, Wideman RD, Karanu F, Ao Z, Warnock GL, Kieffer TJ - Diabetes (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: BetaLogics Venture, Centocor Research and Development, Skillman, New Jersey, USA.

ABSTRACT

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 α- cells.

Research design and methods: 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.

Results: 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.

Conclusions: 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.

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Hormone content and secretion kinetics of hES cell–derived cells. A: Glucagon and insulin content of stage 6 clusters, normalized for total DNA content and expressed as fold difference over human islets (n = 3 samples for ES-derived cells and n = 8 for human islets). Error bars indicate SE. B: Twenty-four hour bioactive glucagon release from hES cells at indicated differentiation stages (n = 2). N.D., not detected. C and D: Glucagon (C) and insulin (D) secretion from perifused human islets and stage 6 clusters (n = 4 chambers for each) in response to 15 mmol/l glucose (Glu), 30 mmol/l KCl and 15 mmol/l arginine (Arg). E: Glucagon secretion from perifused stage 6 clusters (n = 4 chambers) in response to 3 mmol/l glucose (3Glu) or 16.7 mmol/l (16.7Glu) with or without 1 μmol/l of the somatostatin analog octreotide (Sst), 100 μmol/l carbachol (Cch), or 15 mmol/l arginine (Arg) as indicated. (A high-quality color representation of this figure is available in the online issue.)
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Figure 3: Hormone content and secretion kinetics of hES cell–derived cells. A: Glucagon and insulin content of stage 6 clusters, normalized for total DNA content and expressed as fold difference over human islets (n = 3 samples for ES-derived cells and n = 8 for human islets). Error bars indicate SE. B: Twenty-four hour bioactive glucagon release from hES cells at indicated differentiation stages (n = 2). N.D., not detected. C and D: Glucagon (C) and insulin (D) secretion from perifused human islets and stage 6 clusters (n = 4 chambers for each) in response to 15 mmol/l glucose (Glu), 30 mmol/l KCl and 15 mmol/l arginine (Arg). E: Glucagon secretion from perifused stage 6 clusters (n = 4 chambers) in response to 3 mmol/l glucose (3Glu) or 16.7 mmol/l (16.7Glu) with or without 1 μmol/l of the somatostatin analog octreotide (Sst), 100 μmol/l carbachol (Cch), or 15 mmol/l arginine (Arg) as indicated. (A high-quality color representation of this figure is available in the online issue.)

Mentions: Glucagon and insulin protein content were ∼10-fold higher and 10-fold lower in the stage 6 clusters than in human islets, respectively (Fig. 3A). Biologically active glucagon secretion was first detected at stage 5 and increased in stage 6 (Fig. 3B). Stage 6 clusters exhibited insulin secretion in response to KCl, and arginine, albeit at quite low levels (Fig. 3D), with no significant response to glucose (Fig. 3D). In some cases, basal secretion of glucagon in low glucose was significantly greater than that observed in human islets (Fig. 3C), although in some cases, basal glucagon secretion was similar (Fig. 3E). Both KCl and arginine stimulated glucagon secretion (Fig. 3C and E) as did the acetylcholine analog carbachol (Fig. 3E). Glucagon release was diminished in the presence of the somatostatin analog octreotide or high glucose, suggesting that glucagon secretion can be physiologically regulated in these cells (Fig. 3E). After extended culture of stage 6 clusters in vitro, the percentage of insulin-positive and insulin/glucagon copositive cells decreased markedly. Conversely, the percentage of glucagon-positive cells was increased (Fig. 2D), whereas the proportion of cells expressing the endocrine marker synaptophysin remained high at ∼75% (Fig. 2E).


Production of functional glucagon-secreting α-cells from human embryonic stem cells.

Rezania A, Riedel MJ, Wideman RD, Karanu F, Ao Z, Warnock GL, Kieffer TJ - Diabetes (2010)

Hormone content and secretion kinetics of hES cell–derived cells. A: Glucagon and insulin content of stage 6 clusters, normalized for total DNA content and expressed as fold difference over human islets (n = 3 samples for ES-derived cells and n = 8 for human islets). Error bars indicate SE. B: Twenty-four hour bioactive glucagon release from hES cells at indicated differentiation stages (n = 2). N.D., not detected. C and D: Glucagon (C) and insulin (D) secretion from perifused human islets and stage 6 clusters (n = 4 chambers for each) in response to 15 mmol/l glucose (Glu), 30 mmol/l KCl and 15 mmol/l arginine (Arg). E: Glucagon secretion from perifused stage 6 clusters (n = 4 chambers) in response to 3 mmol/l glucose (3Glu) or 16.7 mmol/l (16.7Glu) with or without 1 μmol/l of the somatostatin analog octreotide (Sst), 100 μmol/l carbachol (Cch), or 15 mmol/l arginine (Arg) as indicated. (A high-quality color representation of this figure is available in the online issue.)
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Related In: Results  -  Collection

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Figure 3: Hormone content and secretion kinetics of hES cell–derived cells. A: Glucagon and insulin content of stage 6 clusters, normalized for total DNA content and expressed as fold difference over human islets (n = 3 samples for ES-derived cells and n = 8 for human islets). Error bars indicate SE. B: Twenty-four hour bioactive glucagon release from hES cells at indicated differentiation stages (n = 2). N.D., not detected. C and D: Glucagon (C) and insulin (D) secretion from perifused human islets and stage 6 clusters (n = 4 chambers for each) in response to 15 mmol/l glucose (Glu), 30 mmol/l KCl and 15 mmol/l arginine (Arg). E: Glucagon secretion from perifused stage 6 clusters (n = 4 chambers) in response to 3 mmol/l glucose (3Glu) or 16.7 mmol/l (16.7Glu) with or without 1 μmol/l of the somatostatin analog octreotide (Sst), 100 μmol/l carbachol (Cch), or 15 mmol/l arginine (Arg) as indicated. (A high-quality color representation of this figure is available in the online issue.)
Mentions: Glucagon and insulin protein content were ∼10-fold higher and 10-fold lower in the stage 6 clusters than in human islets, respectively (Fig. 3A). Biologically active glucagon secretion was first detected at stage 5 and increased in stage 6 (Fig. 3B). Stage 6 clusters exhibited insulin secretion in response to KCl, and arginine, albeit at quite low levels (Fig. 3D), with no significant response to glucose (Fig. 3D). In some cases, basal secretion of glucagon in low glucose was significantly greater than that observed in human islets (Fig. 3C), although in some cases, basal glucagon secretion was similar (Fig. 3E). Both KCl and arginine stimulated glucagon secretion (Fig. 3C and E) as did the acetylcholine analog carbachol (Fig. 3E). Glucagon release was diminished in the presence of the somatostatin analog octreotide or high glucose, suggesting that glucagon secretion can be physiologically regulated in these cells (Fig. 3E). After extended culture of stage 6 clusters in vitro, the percentage of insulin-positive and insulin/glucagon copositive cells decreased markedly. Conversely, the percentage of glucagon-positive cells was increased (Fig. 2D), whereas the proportion of cells expressing the endocrine marker synaptophysin remained high at ∼75% (Fig. 2E).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: BetaLogics Venture, Centocor Research and Development, Skillman, New Jersey, USA.

ABSTRACT

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 α- cells.

Research design and methods: 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.

Results: 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.

Conclusions: 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.

Show MeSH