Spatial and transcriptional heterogeneity of pancreatic beta cell neogenesis revealed by a time-resolved reporter system

While pancreatic beta cells have been shown to originate from endocrine progenitors in ductal regions, it remains unclear precisely where beta cells emerge from and which transcripts define newborn beta cells. We therefore investigated characteristics of newborn beta cells extracted by a time-resolved reporter system.We established a mouse model, 'Ins1-GFP; Timer', which provides spatial information during beta cell neogenesis with high temporal resolution. Single-cell RNA-sequencing (scRNA-seq) was performed on mouse beta cells sorted by fluorescent reporter to uncover transcriptomic profiles of newborn beta cells. scRNA-seq of human embryonic stem cell (hESC)-derived beta-like cells was also performed to compare newborn beta cell features between mouse and human.Fluorescence imaging of Ins1-GFP; Timer mouse pancreas successfully dissected newly generated beta cells as green fluorescence-dominant cells. This reporter system revealed that, as expected, some newborn beta cells arise close to the ducts (βduct); unexpectedly, the others arise away from the ducts and adjacent to blood vessels (βvessel). Single-cell transcriptomic analyses demonstrated five distinct populations among newborn beta cells, confirming spatial heterogeneity of beta cell neogenesis such as high probability of glucagon-positive βduct, musculoaponeurotic fibrosarcoma oncogene family B (MafB)-positive βduct and musculoaponeurotic fibrosarcoma oncogene family A (MafA)-positive βvessel cells. Comparative analysis with scRNA-seq data of mouse newborn beta cells and hESC-derived beta-like cells uncovered transcriptional similarity between mouse and human beta cell neogenesis including microsomal glutathione S-transferase 1 (MGST1)- and synaptotagmin 13 (SYT13)-highly-expressing state.The combination of time-resolved histological imaging with single-cell transcriptional mapping demonstrated novel features of spatial and transcriptional heterogeneity in beta cell neogenesis, which will lead to a better understanding of beta cell differentiation for future cell therapy.Raw and processed single-cell RNA-sequencing data for this study has been deposited in the Gene Expression Omnibus under accession number GSE155742.