ABSTRACT: Our team has previously characterized a subpopulation of ductal progenitor cells with progenitor-like characteristics. These cells proliferate in response to stimulation of the BMP pathway by BMP-7 and, following withdrawal of this growth factor, differentiate into all three adult epithelial pancreatic lineages (ductal, acinar and endocrine), both in vitro and after transplantation. scRNAseq analysis of sorted human pancreatic ductal cells has confirmed the existence of progenitor-like clusters whose transcriptomic signature suggests a role for stress-mediated de-differentiation in the acquisition of their stemness. Importantly, the application of cell trajectory analyses upon integration of this ductal dataset with other published datasets that also include endocrine and acinar cells revealed the potential existence of ducto-acinar and ducto-endocrine differentiation axes. However, these conclusions were qualified by the fact that they came from the amalgamation of multiple datasets from many different donors, processed and analyzed in different ways, and also in the absence of a defined regenerative stimulus whose effect could be investigated in a truly continuous manner. We hypothesized that the sequential scRNAseq of same-donor HPSs would address the above constraints and allow us, for the first time, to dissect cell fate changes at the single cell level in response to BMP-7 in a “whole human pancreas” setting. To test this hypothesis, we conducted the longitudinal scRNAseq analysis of human pancreatic slices (HPSs) subjected to a 10-day regimen consisting of 5 days of exposure to BMP-7 followed by 5 days without. The most immediately apparent observation is the transcriptomic heterogeneity of the ductal and acinar compartments, which most earlier analyses (based on isolated islet samples that at best contained a small proportion of ductal and acinar cell types) have failed to detect. With the exception of a recent single nucleus RNAseq report, no other similar study thus far has presented the field with a true whole-pancreas analysis where the different cell types of the organ retain their natural proportion. Integration of single-cell datasets obtained from same-donor HPSs at different time points, or in the presence or absence of treatment, allowed us to conduct cell trajectory analyses using true temporal information, rather than by pseudotemporal inferences. This integration revealed population shifts consistent with BMP-7-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-producing cell populations. These findings contrast with the pattern observed in untreated controls, whose cluster composition and distribution remain largely unchanged with time –another observation that further confirms the robustness of the HPS model to faithfully mirror the cellular makeup of the human pancreas over a 10-day period. In summary, our study provides the first longitudinal scRNAseq analysis of whole human pancreatic tissue, mapping true cell fate trajectories in a manner that was simply not possible prior to the development of long-term HPS culture techniques. These experiments unequivocally confirm pancreatic plasticity in a dynamic fashion and validate the reliability of this novel human-based model for countless other research applications.
