Figure 2.

The human pseudoislet genetics platform allows assessment of islet-specific gene functionalities in vitro and in vivo. A and B: Dispersed human islet cells were transduced with lentiviruses coding for GFP under a constitutive promoter to show that all islet cell types can be efficiently transduced; immunostaining of human pseudoislets showing β (INS⁺, orange arrows) (A and B), α (GCG⁺, white arrows) (A), and δ (STT⁺, orange arrows) (B) efficiently transduced (GFP⁺) islet cells. Scale bar: 20 μm. C: Functionality of transduced pseudoislets can be assessed through GSIS in vitro. The data are normalized to 2.8 mmol/L glucose insulin level. D: Transduced pseudoislets (500/mouse) can be transplanted under the kidney capsule of immunocompromised mice and subjected to an intraperitoneal glucose tolerance test (IPGTT) in vivo, allowing assessment of longer-term effects of genetic manipulations. E: Grafts can be recovered and transduced cells (in this case, GFP⁺ PDX⁺ [white arrows]) further evaluated by immunostaining. Scale bar: 40 μm. F and G: Molecular analysis can also be performed following redispersion of pseudoislets transduced with lentiviruses coding for GFP under a constitutive promoter and FACS sorting of infected, GFP⁺ (F) specific cell types (α: GCG⁺; β: INS⁺) (G). H: Following redispersion, single cells can also be used for patch-clamp or patch-seq. ATAC-seq, assay for transposase-accessible chromatin sequencing; RNA-seq, RNA sequencing.