1998-LB: Previously Frozen Beta-Cell Transcriptional Response to Cytokine Treatment Mirrors That of Fresh Beta Cells

Introduction & Objective: Proinflammatory cytokines contribute to pancreatic beta cell death and dysfunction underlying Type I Diabetes (T1D) pathogenesis. We previously used 10X Genomics single-nucleus RNA-seq (snRNA-seq) and multiome sequencing (snRNA-seq and snATAC-seq from the same nucleus) to profile the transcriptional response of pancreatic islet cell types to cytokine treatment (IL-1B and TNF-a; N=6 treated and 6 untreated). Fresh islets were used; however, it would be convenient to use frozen islets to examine beta cell response to cytokines. In the current study, we sought to determine whether beta cells of frozen islets showed a similar transcriptional response to cytokines as those from fresh islets.

Methods: We selected frozen islet samples from three donors. After thawing, one sample from each donor was treated with cytokines as in our previous study and one sample from each donor was left untreated; samples were then re-frozen and underwent single-nucleus multiome profiling using a pooled design to minimize batch effects. We used Seurat to cluster nuclei and identify beta cells, and performed a differential gene expression analysis (DESeq2) on pseudobulk expression between the treated and untreated beta cells from each donor.

Results: 3,758 of 5,228 pass-QC nuclei were beta cell nuclei. 3,693 of 18,436 tested genes were differentially expressed (DE) between the conditions (5% FDR). There was significant overlap between DE genes in the current study and in the fresh islet study (two-sided Fisher’s Exact test p = 2.6x10^-250, odds ratio = 14.5). Of 827 DE genes in the fresh islet study that were also tested in the current study, 794 (96.0%) showed the same direction-of-effect in the current study (regardless of significance; two-sided Fisher’s Exact test p = 3.4x10^-173, odds ratio = 647.8).

Conclusion: Frozen islets show a similar transcriptional response to cytokines as fresh islets, suggesting they are appropriate to use in future research studies on this topic.