Abstract
Modification of gene expression in pancreatic islets can be a powerful strategy for understanding the pathology of diabetes and developing novel therapeutic strategies against it. However, amenability of the isolated islets to genetic manipulation has been limited to only a subset of cells at the periphery due to poor penetration of transduction particles. To address this issue, we developed a standardized islet model, produced by optimized dissociation and controlled scaffold-free reaggregation of primary human islet cells. This process allowed for an ideal experimental window for accessing and manipulating the pancreatic endocrine cells at their single cell state, while enabling production of uniform islet microtissues displaying long-term (>28 days) and robust function. We used an adenovirus that allows tracking of transduced total cells, endocrine cells and beta cells by labeling them with three specific fluorescent reporters expressed from a single back-bone. To define the optimal transduction conditions, we introduced the virus at various titers during three different production stages; after islet dispersion, during and post reaggregation. We quantified transduction efficiency and viral penetration via 3D confocal microscopy followed by assessment of insulin secretory function, insulin content, and cell viability of transduced islet microtissues. Highly efficient (>75%) and uniform transduction was achieved when the virus was added after cell dispersion and during reaggregation. Approximately 80-95% of transduced cells were endocrine cells, of which 50-63% corresponded to β-cells. Although highly transduced islet microtissues displayed decreased chronic (35-50%), basal (55-62%) and stimulated (65-75%) insulin secretion, a significant fold induction of insulin secretion and unaltered insulin/ATP content was observed. Here we present efficient genetic manipulation of functional reaggregated islets by viral transduction as a novel tool for diabetes research.
Disclosure
B. Yesildag: None. J. Mir-Coll: Employee; Self; InSphero. Employee; Spouse/Partner; Roche Pharma. A. Neelakandhan: None. F. Forschler: Employee; Self; InSphero. A. Biernath: None. I.B. Leibiger: Consultant; Self; Biocrine AB. Consultant; Spouse/Partner; Biocrine AB. B. Leibiger: Consultant; Self; Biocrine AB. Consultant; Spouse/Partner; Biocrine AB. P. Berggren: None. T. Moede: None. C. Ammala: None.