BMP signaling and regenerative plasticity: Correlating dynamic scRNAseq and real-time anatomical remodeling in T1D pancreatic slices - PROJECT SUMMARY The cellular plasticity of the human pancreas is implied from multiple scRNAseq studies. However, these have been invariably based on static datasets from which fate trajectories can only be inferred using pseudotemporal estimations. Furthermore, the reliance on isolated islet preparations for the conduct of these analyses has resulted in a drastic underrepresentation of other non-endocrine cell types, hindering our ability to accurately interrogate exocrine-endocrine interactions. The long-term culture of human pancreatic slices (HPSs) has presented the field with an opportunity to sidestep these limitations by longitudinally tracking tissue plasticity at the single- cell level. Combining single-cell transcriptomic datasets from same-donor HPSs at different time points, with or without a known regenerative stimulus (BMP signaling), has led to the integration of dynamic datasets that store true temporal or treatment-dependent information. This novel approach (Dynamic SliceSeq, or DSSeq for short) has revealed population shifts consistent with the BMP-mediated progenitor cell activation, the blurring of ductal/acinar boundaries, the formation of clear ducto-acinar-endocrine differentiation axes and, notably, the appearance of transitional insulin+ cell populations ‘caught in the act’ of adopting endocrine fates. Our research is the first to unveil human pancreatic plasticity at the single cell level as a function of treatment and time. In vitro lineage tracing indicates that BMP signaling also elicits the formation of functional glucose-responsive insulin+ cells within the exocrine compartment of slices from type 1 diabetic (t1D) donors. Our first hypothesis is that the path through which these cells arise in samples from non-diabetic donors will be largely preserved in those with autoimmune diabetes, and potentially even reinforced as a result of compensatory responses. We further hypothesize that the ductoacinar-endocrine differentiation axis identified by DSSeq (where progenitor populations of BMP-stimulated ductal cells differentiate into endocrine cells through an intermediate acinar-like stage) may mirror the process of embryonic ductal delamination. In particular, we expect BMP signaling to induce such progenitors to migrate into the acinar parenchyma prior to their coalescence into islets. To test these hypotheses, we will pursue the following specific aims: (1) DSSeq analysis of BMP-induced endocrine regeneration in HPSs from t1D donors; (2) Longitudinal resolution of ductal tissue remodeling and neogenesis of insulin+ cells within their native histological microenvironment; and (3) Spatial transcriptomics analysis of islet- duct interfaces in histological samples from nPOD’s t1D collection. The development of long-term HPS culture techniques, and especially DSSeq, has enabled the dissection of regenerative responses directly in live tissue from t1D donors with an unprecedented degree of resolution. The completion of our research objectives is expected to correlate the dynamic compartmental plasticity revealed by DSSeq with real-time histological changes induced by BMP signaling in HPSs from diabetic donors. Beyond the significance of these findings from a basic science perspective, our research will have a direct impact on the design of therapeutic approaches to regenerate β-cells in diabetic patients.
