Cell culture and transfection
Islets were isolated from C57BL/6JRj mice (Janvier, Saint Berthevin, France), cultured overnight, and the peri-islet capsule was digested immediately before mRNA transfection. EndoC-bH3 cells (SARL Endocells, Paris, France) were cultured and immortalising transgenes were excised before transfection as described in Benazra et al. . Human islets were obtained from the Leiden University Medical Center. Human islets isolated at the Leiden University Medical Center that could not be used for clinical transplantation were used in the studies according to national laws and if research consent was available. See electronic supplementary material (ESM) Table 1 for donor characteristics, and ESM Methods.
Mus musculus Vegf-A (also known as Vegfa) was cloned in the vector pEtheRNA. In vitro transcription (IVT) was performed as previously reported . Modified Vegf-A mRNA was produced by incorporating pseudouridine and 5-methyl-cytidine during IVT [21, 22]. The production of mRNA encoding green fluorescent protein (Gfp) was as previously reported . See ESM Methods.
Measurement of insulin secretion
Isolated mouse islets were cultured overnight and transfected the following day as described above. Islets were incubated for 2 h in Ham’s F-10 (Gibco, Thermo Fischer Scientific, Waltham, MA, USA) with either 2 mmol/l or 20 mmol/l glucose. Insulin concentration was measured by ELISA (Mercodia, Uppsala, Sweden). See ESM Methods.
The Ethical Committee for Animal Use of Vrije Universiteit Brussel approved the experiments. 8–12-week-old male C57BL/6JRj mice (Janvier) were used for syngeneic transplantation and 8–12-week-old male severe combined immunodeficiency (SCID)-beige mice (CB17.Cg-PrkdcscidLystbg-J/Crl) (Charles River, Laboratories, L’Arbresle, France) for xenotransplantation of human islets or EndoC-bH3 cells. Mice were housed under standardised conditions (12 h dark/12 h light cycle) and fed a standard diet ad libitum. To evaluate graft vascularisation and size, mice were randomised to a non-transfected, or a Gfp or Vegf-A mRNA transfected group (henceforth referred to as ‘no RNA’, ‘GFP’, and ‘VEGF’) and received either 100 mouse islets, or 1×105 human islet cells or EndoC-bH3 cells under the kidney capsule. To evaluate the metabolic effect of mRNA transfection of the grafts, alloxan-induced diabetic C57BL/6JRj mice were randomised to no RNA, VEGF, or a modified Vegf-A mRNA-transfected group (henceforth referred to as modVEGF), received a marginal mass of 300 syngeneic mouse islets under the kidney capsule , and underwent metabolic measurements. Experimenters were blind to group assignment and outcome assessment. See ESM Methods.
Cells were fixed, embedded and stained for GFP for analysis of transfection efficiency. Islet grafts were fixed and processed as previously reported . Beta cells were stained for insulin and NKX6.1 and alpha cells for glucagon. Biotinylated tomato lectin was used for analysis of in vivo vascularization. See ESM Methods.
The relative mRNA expression levels of Ppia, Rig1, Ifna1 and Mx1 were analysed via qPCR as previously reported . Data are presented as average dCq compared to the reference gene Ppia. See ESM Methods.
One-way ANOVA, two-way ANOVA, Kruskal–Wallis or logrank tests were used as indicated. Data are presented as mean ± SEM. A value of p < 0.05 was considered statistically significant. See ESM Methods.