Dissecting the Transcriptional Landscape of the Proliferating Adults Human Beta Cells by single-cell RNA sequencing

Diabetes mellitus is a leading cause of morbidity and mortality worldwide, predicted to affect over 500 million people worldwide by 2030. Loss of beta-cell number and function underlies much of the pathology of diabetes. Therefore, in both T1D and T2D, ways to preserve or expand pancreatic beta-cell mass could be an effective therapeutic approach. Physiologically, beta-cell mass is maintained at optimal levels through slow turnover. In particular, in humans it has been shown that beta-cell mass expands several fold from birth and during about the first three years of childhood to stop gradually in adulthood. In the past few years, attempts have been made to develop high-throughput screens (HTS) to identify small-molecule inducers of pancreatic beta-cell expansion. Among the most recent results is the identification of the small molecule 5-iodotubercidin (5-IT). 5-IT has been shown to strongly and selectively increase human beta-cell proliferation in vitro and in vivo. Similar to harmine, it inhibits DYRK1A to promote proliferation. Interestingly, the percentage of proliferating beta cells never goes above 4-5%, even with compound treatment. Although physiologically relevant to maintain glucose homeostasis, the fact that only a subset of beta cells proliferates underlines the existence of an intrinsic heterogeneity among beta cells and opens the question of what is the transcriptional profile of these proliferating beta cells.

In the present proposal, I plan to use microfluidic approaches for single-cell transcriptome profiling to understand the complexity and heterogeneity of proliferating human beta cells after treatment with 5-IT. Microfluidics-based single-cell transcriptome profiling can discriminate with great detail the cell-cycle state of a specific cell by comparing the global pattern of gene expression with a set of genes known to be enriched in each of the phases of cell cycles. Pancreatic islets cells are particularly well suited for single-cell transcriptome analysis, because each of them express high levels of cell-specific hormones, allowing for fine association of a transcriptional profile to specific cell types. Since adult human beta cells are primarily locked in the G1 phase of the cell cycle, the availability of cell-specific markers, along with the possibility to identify the proliferating status of each cell, makes microfluidics-based single-cell transcriptome profiling a very powerful tool to 1) identify beta cells that respond to 5-IT by entering the cell cycle, and 2) understand transcriptional specificity of proliferating beta cells.