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Beta cells within single human islets originate from multiple progenitors.

Scharfmann R, Xiao X, Heimberg H, Mallet J, Ravassard P - PLoS ONE (2008)

Bottom Line: By performing gene transfer at low multiplicity of infection, we created a chimeric graft with a subpopulation of human beta cells expressing GFP and found both GFP-positive and GFP-negative beta cells within single islets.The detection of both labeled and unlabeled beta cells in single islets demonstrates that beta cells present in a human islet are derived from multiple progenitors thus providing the first dynamic analysis of human islet formation during development.This human transgenic-like tool can be widely used to elucidate dynamic genetic processes in human tissue formation.

View Article: PubMed Central - PubMed

Affiliation: University Paris-Descartes, Faculty of Medicine, INSERM, Necker Hospital, U845, Paris, France.

ABSTRACT

Background: In both humans and rodents, glucose homeostasis is controlled by micro-organs called islets of Langerhans composed of beta cells, associated with other endocrine cell types. Most of our understanding of islet cell differentiation and morphogenesis is derived from rodent developmental studies. However, little is known about human islet formation. The lack of adequate experimental models has restricted the study of human pancreatic development to the histological analysis of different stages of pancreatic development. Our objective was to develop a new experimental model to (i) transfer genes into developing human pancreatic cells and (ii) validate gene transfer by defining the clonality of developing human islets.

Methods and findings: In this study, a unique model was developed combining ex vivo organogenesis from human fetal pancreatic tissue and cell type-specific lentivirus-mediated gene transfer. Human pancreatic progenitors were transduced with lentiviruses expressing GFP under the control of an insulin promoter and grafted to severe combined immunodeficient mice, allowing human beta cell differentiation and islet morphogenesis. By performing gene transfer at low multiplicity of infection, we created a chimeric graft with a subpopulation of human beta cells expressing GFP and found both GFP-positive and GFP-negative beta cells within single islets.

Conclusion: The detection of both labeled and unlabeled beta cells in single islets demonstrates that beta cells present in a human islet are derived from multiple progenitors thus providing the first dynamic analysis of human islet formation during development. This human transgenic-like tool can be widely used to elucidate dynamic genetic processes in human tissue formation.

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Human beta cells within a single islet are derived from more than one progenitor.Human fetal pancreases were dissociated, transduced with lentiviruses expressing GFP under the control of the insulin promoter, grafted and analyzed 4.5 months later. A: double staining for insulin (red) and GFP (green) showing that GFP is only found in islets; B: double staining for glucagon (red) and GFP (green) showing that GFP is not found in alpha cells; C: double staining for insulin (red) and GFP (green) showing that GFP is only found in a subpopulation of beta cells. D is an enlargement of panel C (dotted square). Scale bars: A: 50 µm; B–D: 25 µm.
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pone-0003559-g005: Human beta cells within a single islet are derived from more than one progenitor.Human fetal pancreases were dissociated, transduced with lentiviruses expressing GFP under the control of the insulin promoter, grafted and analyzed 4.5 months later. A: double staining for insulin (red) and GFP (green) showing that GFP is only found in islets; B: double staining for glucagon (red) and GFP (green) showing that GFP is not found in alpha cells; C: double staining for insulin (red) and GFP (green) showing that GFP is only found in a subpopulation of beta cells. D is an enlargement of panel C (dotted square). Scale bars: A: 50 µm; B–D: 25 µm.

Mentions: We finally transduced human fetal pancreases with pTRIP ΔU3.RIP405-eGFP at the multiplicity of infection shown to target a limited proportion of progenitors. Pancreases were then grafted into scid mice and removed 4.5 months later. Islets of Langerhans had developed during the transplantation period (Fig. 5A). eGFP expression was specific to beta cells within the islets (Fig. 5, panels A, B and C), consistent with the specificity of the rat insulin II promoter. If all beta cells within an islet are derived from a single progenitor, they should all be positive for eGFP. However, if they are derived from more than one progenitor, a mixed pattern should be found. We counted 2730 islets and found that 23 contained eGFP-positive cells. In all labeled islets, only a subpopulation of insulin-positive cells was positive for eGFP (Fig. 5). These findings demonstrate that beta cells within a single human islet are derived from more than one progenitor.


Beta cells within single human islets originate from multiple progenitors.

Scharfmann R, Xiao X, Heimberg H, Mallet J, Ravassard P - PLoS ONE (2008)

Human beta cells within a single islet are derived from more than one progenitor.Human fetal pancreases were dissociated, transduced with lentiviruses expressing GFP under the control of the insulin promoter, grafted and analyzed 4.5 months later. A: double staining for insulin (red) and GFP (green) showing that GFP is only found in islets; B: double staining for glucagon (red) and GFP (green) showing that GFP is not found in alpha cells; C: double staining for insulin (red) and GFP (green) showing that GFP is only found in a subpopulation of beta cells. D is an enlargement of panel C (dotted square). Scale bars: A: 50 µm; B–D: 25 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2571119&req=5

pone-0003559-g005: Human beta cells within a single islet are derived from more than one progenitor.Human fetal pancreases were dissociated, transduced with lentiviruses expressing GFP under the control of the insulin promoter, grafted and analyzed 4.5 months later. A: double staining for insulin (red) and GFP (green) showing that GFP is only found in islets; B: double staining for glucagon (red) and GFP (green) showing that GFP is not found in alpha cells; C: double staining for insulin (red) and GFP (green) showing that GFP is only found in a subpopulation of beta cells. D is an enlargement of panel C (dotted square). Scale bars: A: 50 µm; B–D: 25 µm.
Mentions: We finally transduced human fetal pancreases with pTRIP ΔU3.RIP405-eGFP at the multiplicity of infection shown to target a limited proportion of progenitors. Pancreases were then grafted into scid mice and removed 4.5 months later. Islets of Langerhans had developed during the transplantation period (Fig. 5A). eGFP expression was specific to beta cells within the islets (Fig. 5, panels A, B and C), consistent with the specificity of the rat insulin II promoter. If all beta cells within an islet are derived from a single progenitor, they should all be positive for eGFP. However, if they are derived from more than one progenitor, a mixed pattern should be found. We counted 2730 islets and found that 23 contained eGFP-positive cells. In all labeled islets, only a subpopulation of insulin-positive cells was positive for eGFP (Fig. 5). These findings demonstrate that beta cells within a single human islet are derived from more than one progenitor.

Bottom Line: By performing gene transfer at low multiplicity of infection, we created a chimeric graft with a subpopulation of human beta cells expressing GFP and found both GFP-positive and GFP-negative beta cells within single islets.The detection of both labeled and unlabeled beta cells in single islets demonstrates that beta cells present in a human islet are derived from multiple progenitors thus providing the first dynamic analysis of human islet formation during development.This human transgenic-like tool can be widely used to elucidate dynamic genetic processes in human tissue formation.

View Article: PubMed Central - PubMed

Affiliation: University Paris-Descartes, Faculty of Medicine, INSERM, Necker Hospital, U845, Paris, France.

ABSTRACT

Background: In both humans and rodents, glucose homeostasis is controlled by micro-organs called islets of Langerhans composed of beta cells, associated with other endocrine cell types. Most of our understanding of islet cell differentiation and morphogenesis is derived from rodent developmental studies. However, little is known about human islet formation. The lack of adequate experimental models has restricted the study of human pancreatic development to the histological analysis of different stages of pancreatic development. Our objective was to develop a new experimental model to (i) transfer genes into developing human pancreatic cells and (ii) validate gene transfer by defining the clonality of developing human islets.

Methods and findings: In this study, a unique model was developed combining ex vivo organogenesis from human fetal pancreatic tissue and cell type-specific lentivirus-mediated gene transfer. Human pancreatic progenitors were transduced with lentiviruses expressing GFP under the control of an insulin promoter and grafted to severe combined immunodeficient mice, allowing human beta cell differentiation and islet morphogenesis. By performing gene transfer at low multiplicity of infection, we created a chimeric graft with a subpopulation of human beta cells expressing GFP and found both GFP-positive and GFP-negative beta cells within single islets.

Conclusion: The detection of both labeled and unlabeled beta cells in single islets demonstrates that beta cells present in a human islet are derived from multiple progenitors thus providing the first dynamic analysis of human islet formation during development. This human transgenic-like tool can be widely used to elucidate dynamic genetic processes in human tissue formation.

Show MeSH
Related in: MedlinePlus