12331 Overcoming Limitations Of Identifying Proliferating β-cells In Dispersed Primary Islets Culture For Drug Discovery

Abstract Disclosure: H. Xu: None. S. Rebecca: None. S. Lee: None. J.P. Annes: None. Tremendous efforts have been made with screening platforms to identify islet β-cell mitogens that may be used to treat diabetes. However, promising compounds induce profound proliferation of islet non-β-cells in available screening platforms, calling for both β-cell targeted drug discovery and rigorous identification of β-cell identity to reduce false positivity. First, we used an established drug discovery dispersed islet cell-based platform(2D) to test the replication-promoting activity of GNF2133, a selective DYRK1A inhibitor that promotes β-cell proliferation, in isolated islets from human, rat and mouse. After 72 h in vitro culture, replication of non-β cells (PDX/Insulin negative) contributed to over 60% of the total replicating cells (Ki67+) in all species (human: 64%, rat: 71%, mouse: 67%). Mouse islets, which exhibit the greatest non-β-cell replication response upon GNF2133 treatment (4-fold above baseline, [50 nM]), were selected for subsequent studies. Next, we evaluated identification efficiency of β cell markers in this image-based platform. β cells, selected by the quantification value of insulin intensity, comprised 60-70% of total cell population at 48 h, 50-60% at 72 h and 20-30% at 96 h (p< 0.0001 for both comparisons: 48 h vs 72 h, 72 h vs 96 h). Basal replication of β-cells (Insulin+Ki67+) was 2.80%, 2.32%, 0.85% at 48 h, 72 h and 96 h, respectively. By comparison, the proportion of non-β cells showed an increasing trend as 2.48%, 6.49%, 8.68%, correspondingly. Identification with PDX1, another β cell marker, exhibited the same findings. Prolonged culture of dispersed islet cells, as these results indicate, favors growth of non-β-cells over β-cells. In addition, proliferating β-cells express lower levels of differentiation markers makes their discernment from replicating non-β cells more challenging. 窗体顶端 To minimize the duration of monolayer culture, replication induction was measured using intact islets with addition of BrdU during the last 24 h of culture. Then, islets were either directly fixed (3D) or dispersed and cultured for an additional 36 hour before being fixed (3D+2D). Immunostaining showed 70-80% PDX1 positive cells from both methods, which resembles in vivo β cell distribution within primary mouse islets. Basal β-cell replication in 3D+2D method was 0.30%, similar to in vivo BrdU labelling level. Upon mitogen treatment, PDX1+BrdU+ cells increased to 6.57% in 3D+2D method, slightly lower than 10.02% in 3D assay (p<0.05) yet significantly higher than 1.15% in 2D assay (<0.01). In summary, we developed a 3D+2D assay that enhances β-cell survival and the β-cell replication-induction response. This platform, while offering reduced throughput, provides a robust platform for discovery of β-cell replication-promoting compounds. Further validation with human islets is needed. Presentation: 6/2/2024