Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer.
Cell-based therapies for treating insulin-dependent diabetes (IDD) can provide a more physiologic regulation of blood glucose levels in a less invasive fashion than daily insulin injections. Promising cells include intestinal enteroendocrine cells genetically engineered to secrete insulin in response to physiologic stimuli; responsiveness occurs at the exocytosis level to regulate the acute release of recombinant insulin. In this work, we established a human cellular model to demonstrate that meat hydrolysate can simultaneously stimulate glucagon-like peptide-1 (GLP-1, an enteroendocrine cell-derived incretin hormone) and recombinant insulin secretion from the engineered human NCI-H716 intestinal cell line. Cells were genetically modified using the recombinant adeno-associated virus (rAAV)-mediated insulin gene transfer. Recombinant cells were then differentiated to display endocrine features, in particular the formation of granule-like compartments. A fusion protein of insulin and enhanced green fluorescence protein (EGFP) was designed to reveal the compartments of localization of the fusion protein and assess its co-localization with endogenous GLP-1. Our work provides a unique human cellular model for regulated insulin release through genetic engineering of GLP-1-secreting intestinal cells, which is expected to be useful for cell-based therapies of IDD.
Tang
Shiue-Cheng
SC
School of Chemical Engineering, Georgia Tech-Emory Center for the Engineering of Living Tissues, and P.H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332, USA.
Sambanis
Athanassios
A
D002460:Cell Line; D000229:Dependovirus / Q000235:genetics*; D003922:Diabetes Mellitus, Type 1 / Q000628:therapy; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods; D049452:Green Fluorescent Proteins; D006801:Humans; D007328:Insulin / Q000235:genetics / Q000378:metabolism*; D000078790:Insulin Secretion; D007413:Intestinal Mucosa / Q000378:metabolism / Q000502:physiology*; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics; D018613:Microscopy, Confocal; D011994:Recombinant Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured
D007328:Insulin; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins