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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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Transplantation of hES cell–derived cells induces hyperglucagonemia and glucagon intolerance. A: Blood glucose levels after a 4-h morning fast. B: Blood glucose and plasma glucagon levels after an overnight fast and after 45-min refeeding period at 99 days after transplant (Tx). Glucagon was below the level of detection for control and human islet Tx groups (<40 pg/ml). C: Blood glucose and plasma mouse insulin levels (mINS; inset) at day 62 after transplant in response to intraperitoneal arginine injection (2 g/kg). D: Plasma glucagon levels for intraperitoneally arginine test shown in panel C. E: Blood glucose and plasma mouse insulin levels (mINS; inset) after a 4-h morning fast at day 77 after transplant in response to oral glucose challenge (2 g/kg). F: Blood glucose and plasma mouse insulin levels (mINS; inset) at day 92 after transplant in response to intraperitoneal glucagon injection (1 μg/kg). G: Whole body insulin sensitivity was assessed by injecting recombinant human insulin (0.4 units/kg) at day 114 after transplant. H: Plasma immunoreactive glucagon (left) and insulin (right) levels in response to insulin injection shown in panel G. n = 5–7 animals/group; *P < 0.05, **P < 0.01, ***P < 0.001 vs. control; #P < 0.05 vs. respective 0-min time point (Student t test). N.D., not detected. (A high-quality color representation of this figure is available in the online issue.)
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Figure 4: Transplantation of hES cell–derived cells induces hyperglucagonemia and glucagon intolerance. A: Blood glucose levels after a 4-h morning fast. B: Blood glucose and plasma glucagon levels after an overnight fast and after 45-min refeeding period at 99 days after transplant (Tx). Glucagon was below the level of detection for control and human islet Tx groups (<40 pg/ml). C: Blood glucose and plasma mouse insulin levels (mINS; inset) at day 62 after transplant in response to intraperitoneal arginine injection (2 g/kg). D: Plasma glucagon levels for intraperitoneally arginine test shown in panel C. E: Blood glucose and plasma mouse insulin levels (mINS; inset) after a 4-h morning fast at day 77 after transplant in response to oral glucose challenge (2 g/kg). F: Blood glucose and plasma mouse insulin levels (mINS; inset) at day 92 after transplant in response to intraperitoneal glucagon injection (1 μg/kg). G: Whole body insulin sensitivity was assessed by injecting recombinant human insulin (0.4 units/kg) at day 114 after transplant. H: Plasma immunoreactive glucagon (left) and insulin (right) levels in response to insulin injection shown in panel G. n = 5–7 animals/group; *P < 0.05, **P < 0.01, ***P < 0.001 vs. control; #P < 0.05 vs. respective 0-min time point (Student t test). N.D., not detected. (A high-quality color representation of this figure is available in the online issue.)

Mentions: To test whether stage 6 clusters were lineage-restricted to become α-cells and to assess the physiologic regulation of glucagon secretion from these cells, normoglycemic B6.129S7-RagTm1Mom/J mice were transplanted with ∼1.9 × 106 stage 6 clusters. Animals were followed with routine blood glucose tracking and additional metabolic tests for up to 5 months after transplantation. Cell recipients showed occasionally elevated 4-h fasted blood glucose levels compared with control animals (Fig. 4A). At 99 days after transplant, prolonged (∼16 h) fasting resulted in elevated plasma glucagon levels in cell recipients (399.2 ± 32.5 pg/ml) compared with control animals, where glucagon levels were largely undetectable (<40 pg/ml). Feeding significantly reduced plasma glucagon to 227.7 ± 46.3 pg/ml in cell transplant recipients (Fig. 4B). Importantly, postprandial blood glucose levels were not significantly different between groups (Fig. 4B).


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)

Transplantation of hES cell–derived cells induces hyperglucagonemia and glucagon intolerance. A: Blood glucose levels after a 4-h morning fast. B: Blood glucose and plasma glucagon levels after an overnight fast and after 45-min refeeding period at 99 days after transplant (Tx). Glucagon was below the level of detection for control and human islet Tx groups (<40 pg/ml). C: Blood glucose and plasma mouse insulin levels (mINS; inset) at day 62 after transplant in response to intraperitoneal arginine injection (2 g/kg). D: Plasma glucagon levels for intraperitoneally arginine test shown in panel C. E: Blood glucose and plasma mouse insulin levels (mINS; inset) after a 4-h morning fast at day 77 after transplant in response to oral glucose challenge (2 g/kg). F: Blood glucose and plasma mouse insulin levels (mINS; inset) at day 92 after transplant in response to intraperitoneal glucagon injection (1 μg/kg). G: Whole body insulin sensitivity was assessed by injecting recombinant human insulin (0.4 units/kg) at day 114 after transplant. H: Plasma immunoreactive glucagon (left) and insulin (right) levels in response to insulin injection shown in panel G. n = 5–7 animals/group; *P < 0.05, **P < 0.01, ***P < 0.001 vs. control; #P < 0.05 vs. respective 0-min time point (Student t test). N.D., not detected. (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 4: Transplantation of hES cell–derived cells induces hyperglucagonemia and glucagon intolerance. A: Blood glucose levels after a 4-h morning fast. B: Blood glucose and plasma glucagon levels after an overnight fast and after 45-min refeeding period at 99 days after transplant (Tx). Glucagon was below the level of detection for control and human islet Tx groups (<40 pg/ml). C: Blood glucose and plasma mouse insulin levels (mINS; inset) at day 62 after transplant in response to intraperitoneal arginine injection (2 g/kg). D: Plasma glucagon levels for intraperitoneally arginine test shown in panel C. E: Blood glucose and plasma mouse insulin levels (mINS; inset) after a 4-h morning fast at day 77 after transplant in response to oral glucose challenge (2 g/kg). F: Blood glucose and plasma mouse insulin levels (mINS; inset) at day 92 after transplant in response to intraperitoneal glucagon injection (1 μg/kg). G: Whole body insulin sensitivity was assessed by injecting recombinant human insulin (0.4 units/kg) at day 114 after transplant. H: Plasma immunoreactive glucagon (left) and insulin (right) levels in response to insulin injection shown in panel G. n = 5–7 animals/group; *P < 0.05, **P < 0.01, ***P < 0.001 vs. control; #P < 0.05 vs. respective 0-min time point (Student t test). N.D., not detected. (A high-quality color representation of this figure is available in the online issue.)
Mentions: To test whether stage 6 clusters were lineage-restricted to become α-cells and to assess the physiologic regulation of glucagon secretion from these cells, normoglycemic B6.129S7-RagTm1Mom/J mice were transplanted with ∼1.9 × 106 stage 6 clusters. Animals were followed with routine blood glucose tracking and additional metabolic tests for up to 5 months after transplantation. Cell recipients showed occasionally elevated 4-h fasted blood glucose levels compared with control animals (Fig. 4A). At 99 days after transplant, prolonged (∼16 h) fasting resulted in elevated plasma glucagon levels in cell recipients (399.2 ± 32.5 pg/ml) compared with control animals, where glucagon levels were largely undetectable (<40 pg/ml). Feeding significantly reduced plasma glucagon to 227.7 ± 46.3 pg/ml in cell transplant recipients (Fig. 4B). Importantly, postprandial blood glucose levels were not significantly different between groups (Fig. 4B).

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
Related in: MedlinePlus