Highly efficient ex vivo lentiviral transduction of primary human pancreatic exocrine cells

Jeetindra R A Balak,Natascha De Graaf,Eelco J P De Koning,Ton J Rabelink,Rob C Hoeben,Arnaud Zaldumbide,Françoise Carlotti

doi:10.1038/s41598-019-51763-z

Abstract

The lack of efficient gene transfer methods into primary human pancreatic exocrine cells hampers studies on the plasticity of these cells and their possible role in beta cell regeneration. Therefore, improved gene transfer protocols are needed. Lentiviral vectors are widely used to drive ectopic gene expression in mammalian cells, including primary human islet cells. Here we aimed to optimize gene transfer into primary human exocrine cells using modified lentiviral vectors or transduction conditions. We evaluated different promoters, viral envelopes, medium composition and transduction adjuvants. Transduction efficiency of a reporter vector was evaluated by fluorescence microscopy and flow cytometry. We show that protamine sulfate-assisted transduction of a VSV-G-pseudotyped vector expressing eGFP under the control of a CMV promoter in a serum-free environment resulted in the best transduction efficiency of exocrine cells, reaching up to 90% of GFP-positive cells 5 days after transduction. Our findings will enable further studies on pancreas (patho)physiology that require gene transfer such as gene overexpression, gene knockdown or lineage tracing studies.

Highlights

The lack of efficient gene transfer methods into primary human pancreatic exocrine cells hampers studies on the plasticity of these cells and their possible role in beta cell regeneration
Because it is hypothesized that the putative progenitor cell subpopulation involved in beta cell regeneration is located in the ductal compartment of the pancreas, initial experiments were performed on human pancreatic exocrine cultures that were enriched for ductal cells by monolayer culture on tissue-culture treated plastic, to which the pancreatic ductal cells selectively adhere[36]
Because these preliminary experiments indicated that transduction of human exocrine cells during monolayer expansion was very poor (

Summary

Introduction

The lack of efficient gene transfer methods into primary human pancreatic exocrine cells hampers studies on the plasticity of these cells and their possible role in beta cell regeneration. We optimized the transduction conditions of primary human adult exocrine cells with a HIV-1 (human immune deficiency virus 1) based, replication-deficient, lentiviral reporter vector. This novel protocol can be used for improved transgene expression in primary human exocrine tissue, which will enable further investigations on the putative role of these cells in beta cell regeneration

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