Efficient restricted gene expression in beta cells by lentivirus-mediated gene transfer into pancreatic stem/progenitor cells.
OBJECTIVE
Gene transfer into pancreatic beta cells, which produce and secrete insulin, is a promising strategy to protect such cells against autoimmune destruction and also to generate beta cells in mass, thereby providing a novel therapeutic approach to treat diabetic patients. Until recently, exogenous DNA has been directly transferred into mature beta cells with various levels of success. We investigated whether exogenous DNA could be stably transferred into pancreatic stem/progenitor cells, which would subsequently differentiate into mature beta cells expressing the transgene.


METHODS
We designed transplantation and tissue culture procedures to obtain ex vivo models of pancreatic development. We next constructed recombinant lentiviruses expressing enhanced green fluorescent protein (eGFP) under the control of either the rat insulin promoter or a ubiquitous promoter, and performed viral infection of rat embryonic pancreatic tissue.


RESULTS
Embryonic pancreas infected with recombinant lentiviruses resulted in endocrine cell differentiation and restricted cell type expression of the transgene according to the specificity of the promoter used in the viral construct. We next demonstrated that the efficiency of infection could be further improved upon infection of embryonic pancreatic epithelia, followed by their in vitro culture, using conditions that favour endocrine cell differentiation. Under these conditions, endocrine stem/progenitor cells expressing neurogenin 3 are efficiently transduced by recombinant lentiviral vectors. Moreover, when eGFP was placed under the control of the insulin promoter, 70.4% of the developed beta cells were eGFP-expressing cells. All of the eGFP-positive cells were insulin-producing cells.


CONCLUSIONS
We have demonstrated that mature rat pancreatic beta cells can be stably modified by infecting pancreatic stem/progenitor cells that undergo endocrine differentiation.
Castaing
M
M
INSERM EMI 0363, Faculté Necker, 156 rue Vaugirard, 75015 Paris, France.
Guerci
A
A
Mallet
J
J
Czernichow
P
P
Ravassard
P
P
Scharfmann
R
R
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins; D043422:Carboxypeptidases A / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology; D003587:Cytomegalovirus / Q000235:genetics; D015870:Gene Expression / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D005934:Glucagon / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D017404:In Situ Hybridization, Fluorescence; D007328:Insulin / Q000235:genetics / Q000378:metabolism; D007515:Islets of Langerhans / Q000378:metabolism*; D016086:Lentivirus / Q000235:genetics; D008297:Male; D051379:Mice; D016513:Mice, SCID; D009419:Nerve Tissue Proteins / Q000235:genetics; D010179:Pancreas / Q000166:cytology / Q000196:embryology; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D014162:Transfection / Q000379:methods; D014183:Transplantation, Heterologous; D019737:Transplants
D051792:Basic Helix-Loop-Helix Proteins; D043422:Carboxypeptidases A; D005934:Glucagon; D049452:Green Fluorescent Proteins; D007328:Insulin; D009419:Nerve Tissue Proteins; C407737:Neurog3 protein, mouse; C498696:Neurog3 protein, rat