Cell Loss during Pseudoislet Formation Hampers Profound Improvements in Islet Lentiviral Transduction Efficacy for Transplantation Purposes

Abstract

Islet transplantation is a promising treatment in type 1 diabetes, but the need for chronic immunosuppression is a major hurdle to broad applicability. Ex vivo introduction of agents by lentiviral vectors-improving beta-cell resistance against immune attack-is an attractive path to pursue. The aim of this study was to investigate whether dissociation of islets to single cells prior to viral infection and reaggregation before transplantation would improve viral transduction efficacy without cytotoxicity. This procedure improved transduction efficacy with a LV-pWPT-CMV-EGFP construct from 11.2 +/- 4.1% at MOI 50 in whole islets to 80.0 +/- 2.8% at MOI 5. Viability (as measured by Hoechst/PI) and functionality (as measured by glucose challenge) remained high. After transplantation, the transfected pseudoislet aggregates remained EGFP positive for more than 90 days and the expression of EGFP colocalized primarily with the insulin-positive beta-cells. No increased vulnerability to immune attack was observed in vitro or in vivo. These data demonstrate that dispersion of islets prior to lentiviral transfection and reaggregation prior to transplantation is a highly efficient way to introduce genes of interest into islets for transplantation purposes in vitro and in vivo, but the amount of beta-cells needed for normalization of glycemia was more than eightfold higher when using dispersed cell aggregates versus unmanipulated islets. The high price to pay to reach stable and strong transgene expression in islet cells is certainly an important cell loss.

Pseudoislets in stirred-suspension culture exhibit enhanced cell survival, propagation and insulin secretion

“…The enhanced insulin secretion of PIs comprised of ␤-cells or mixed islet cells (Cirulli et al, 1994;Halban et al, 1987;Hopcroft et al, 1985a;Serre-Beinier et al, 2000) make them ideal for use in basic diabetes research, platforms for high-throughput screening of drug candidates for diabetes, and bioartificial pancreas devices. Furthermore, cell loss during the formation of PIs intended for transplantation impedes improvements of their survival through ␤-cell engineering (Callewaert et al, 2007). The bioreactor method results in the generation of PIs with high cell viability, and can be scaled up for the generation of cellular material in sufficient quantities for research and clinical applications.…”

Improved physiological properties of gravity-enforced reassembled rat and human pancreatic pseudo-islets

“…50%, which is several‐fold higher than the 10% described by Callewaert et al . (). At first sight, a 50% cell loss may seem rather high; however, this number also includes the loss of damaged islet tissue that usually occurs simply as a consequence of the isolation procedure, leading to a disruption of the normal cell–matrix relationship (Thomas et al ., ).…”

RNA interference for improving the outcome of islet transplantation

“…In addition, lentiviral vectors induced immune system activation was also minimal [258]. Under optimized conditions, lentivirus could transduce around 10% to 30% of islet cells in an intact islet, which are mainly the cells at the periphery [253, 259, 260]. This is still enough to have significant effect on the viability, probably due to the “barrier” effect, that is shield the core of islets by protecting the periphery of islets [253].…”

“…Callewaert et al demonstrated increased transduction efficiency from around 11.2% to 80.0% by dissociation of islets to single cells prior to transduction and re-aggregation of islets before transplantation [260]. However, improvement in the transfection efficiency did not translate into the improved function of islets.…”