Temporal single-cell regeneration studies: the greatest thing since sliced pancreas?

Abstract

The application of single-cell analytic techniques to the study of stem/progenitor cell niches supports the emerging view that pancreatic cell lineages are in a state of flux between differentiation stages. For all their value, however, such analyses merely offer a snapshot of the cellular palette of the tissue at any given time point. Conclusions about potential developmental/regeneration paths are solely based on bioinformatics inferences. In this context, the advent of new techniques for the long-term culture and lineage tracing of human pancreatic slices offers a virtual window into the native organ and presents the field with a unique opportunity to serially resolve pancreatic regeneration dynamics at the single-cell level.

Keywords: human pancreatic slices; pancreatic regeneration; pseudotime; single-cell RNA sequencing; β-cell neogenesis.

Figure 1.. Described applications of pancreatic slices.

Recent refinements on pancreatic slicing techniques have enabled the execution of live studies on each of the main cell types found in the native pancreas (acinar, ductal, endocrine, endothelial, immune, neuronal) in their proper anatomical context. Representative references are shown next to each approach.

Key figure.. Prospective applications of long-term cultured slices for the temporal study of pancreatic regeneration.

(A) Same-donor serial HPSs can be exposed to a regeneration stimulus (e.g., BMP-7). Conducting scRNAseq of dissociated slices at different time points is expected to yield true temporal information about population dynamics, including, potentially, fate trajectories leading to β-cell neogenesis. (B) Lineage-tracing approach to dissect regeneration at the single-cell level. Co-transduction of HPSs with adenoviruses carrying a loxP-Red-loxP-Green reporter and a Human Insulin Promoter (HIP)-driven Cre results in green labeling of pre-existing β-cells and red tagging of all other transduced cells. Dissociation, fluorescent sorting and scRNAseq of each fraction would resolve the various sub-populations of transduced non-endocrine cells (red) and insulin-producing cells (green) (shown only for red cells in the panel). Upon addition of a regenerative stimulus, some non-endocrine cells (red) will progressively become green through an intermediate red + green (yellow) stage, as described in [64]. A second sampling, dissociation, fluorescence sorting and scRNAseq of slices treated in this manner would allow for the single-cell transcriptomic profiling of the red + green fraction, consisting of cells at different stages of fate-switching. Neogenic β-cells (green) would appear after the resolution of the above transitional events, days after the original labeling of pre-existing β-cells. Dissociation/sorting/scRNAseq of green cells at this stage would identify the transcriptomic signature of neogenic β-cells, and reveal potential differences with pre-existing ones. Charting regeneration pathways at the single-cell level is an expected outcome of the temporal integration of datasets obtained at each time point. Additional multi-omics approaches and other confirmatory assays should be used for confirmation of these findings.