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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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Related in: MedlinePlus

Cell type specificity of the rat insulin II promoter in human adult pancreas.Crude human islet preparations were transduced with lentiviruses expressing eGFP under the control of the rat insulin II promoter and analyzed 72 hours after infection. Cultures were photographed under a fluorescent inverted microscope (panels A and B), fixed and sectioned. C–D: staining for GFP (green) and insulin (red). E staining for GFP (green) and amylase (red). F: staining for GFP (green) and CK19 (red). Scale bars: 20 µm.
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pone-0003559-g004: Cell type specificity of the rat insulin II promoter in human adult pancreas.Crude human islet preparations were transduced with lentiviruses expressing eGFP under the control of the rat insulin II promoter and analyzed 72 hours after infection. Cultures were photographed under a fluorescent inverted microscope (panels A and B), fixed and sectioned. C–D: staining for GFP (green) and insulin (red). E staining for GFP (green) and amylase (red). F: staining for GFP (green) and CK19 (red). Scale bars: 20 µm.

Mentions: We next transduced human fetal pancreases with lentiviral vector expressing eGFP under the control of a 405 bp-fragment of the rat insulin II promoter (RIP405). This portion of the rat promoter is sufficient for beta cell restricted expression in rodents [15]. To determine whether this also applies for human cells, an islet cell-enriched fraction of pancreatic cells was transduced with pTRIP ΔU3.RIP405-eGFP. We observed intense staining in cell clusters (Fig. 4, panels A and B). We then sectioned clusters and co-stained for eGFP and endocrine, acinar or ductal markers. All eGFP-positive cells stained positive for insulin (Fig. 4, panel C, D) whereas neither amylase-expressing acinar cells, nor cytokeratin 19-expressing ductal cells (Fig. 4, panel E, F) stained positive for eGFP. Thus, this 405 bp fragment of the rat insulin II promoter is sufficient for beta cell-restricted expression of a transgene in the human adult pancreas.


Beta cells within single human islets originate from multiple progenitors.

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

Cell type specificity of the rat insulin II promoter in human adult pancreas.Crude human islet preparations were transduced with lentiviruses expressing eGFP under the control of the rat insulin II promoter and analyzed 72 hours after infection. Cultures were photographed under a fluorescent inverted microscope (panels A and B), fixed and sectioned. C–D: staining for GFP (green) and insulin (red). E staining for GFP (green) and amylase (red). F: staining for GFP (green) and CK19 (red). Scale bars: 20 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003559-g004: Cell type specificity of the rat insulin II promoter in human adult pancreas.Crude human islet preparations were transduced with lentiviruses expressing eGFP under the control of the rat insulin II promoter and analyzed 72 hours after infection. Cultures were photographed under a fluorescent inverted microscope (panels A and B), fixed and sectioned. C–D: staining for GFP (green) and insulin (red). E staining for GFP (green) and amylase (red). F: staining for GFP (green) and CK19 (red). Scale bars: 20 µm.
Mentions: We next transduced human fetal pancreases with lentiviral vector expressing eGFP under the control of a 405 bp-fragment of the rat insulin II promoter (RIP405). This portion of the rat promoter is sufficient for beta cell restricted expression in rodents [15]. To determine whether this also applies for human cells, an islet cell-enriched fraction of pancreatic cells was transduced with pTRIP ΔU3.RIP405-eGFP. We observed intense staining in cell clusters (Fig. 4, panels A and B). We then sectioned clusters and co-stained for eGFP and endocrine, acinar or ductal markers. All eGFP-positive cells stained positive for insulin (Fig. 4, panel C, D) whereas neither amylase-expressing acinar cells, nor cytokeratin 19-expressing ductal cells (Fig. 4, panel E, F) stained positive for eGFP. Thus, this 405 bp fragment of the rat insulin II promoter is sufficient for beta cell-restricted expression of a transgene in the human adult pancreas.

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