Single-cell longitudinal analysis of regeneration in human pancreatic slices

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PROJECT SUMMARY We have described a subpopulation of human pancreatic ductal cells with progenitor-like characteristics. These cells can be sorted using antibodies against the BMP receptor 1A (ALK3bright+) and P2RY1, a surrogate surface marker for PDX1 identified by our team. ALK3bright+/P2RY1+ cells proliferate when exposed to BMP-7, and differentiate into all adult pancreatic cell types, including functional β-cells, upon BMP-7 withdrawal. scRNAseq of the ALK3bright+ fraction of human pancreatic donors confirmed the existence of clusters with progenitor-like features, with substantial evidence suggesting that such features may be acquired by de- differentiation. When transplanted into immune-deficient mice, sorted cell populations enriched in markers differentially expressed in these clusters self-organize into ‘micro-pancreata’ with native-like cytoarchitecture and functional endocrine cells. More recently, we have developed the means to study the dynamic processes of progenitor cell-dependent regeneration using human pancreatic slices (HPSs). We have achieved functional long-term (>10d) culture of HPSs, which allows for the longitudinal tracking of β-cell regeneration in a setting that is widely considered the closest approximation in vitro to a native pancreas. BMP signaling-dependent regeneration was established in slices from healthy and, more importantly, T1D and T2D donors. Numerous single-cell analyses of the pancreas, included our own, support the emerging view that specific lineages are in a state of flux between differentiation stages. However, all these analyses only offer a snapshot of the tissue at any given time point. Conclusions about potential developmental/regeneration paths are exclusively based on bioinformatics inferences. We hypothesize that the combination of long-term organotypic culture with lineage-tracing in vitro and sequential single-cell analyses will allow us, for the first time, to dynamically map cell fate changes –e.g., by conducting longitudinal scRNAseq of slices from the same donor across multiple time points after BMP-7 addition. Furthermore, we contend that this system will enable the real- time visualization and in-depth, single-cell resolution study of potential de-differentiation events, should they happen in response to different sources of stress in human slices. These approaches offer a wealth of new research possibilities that were downright unfeasible prior to the development of these methods. Our research design is expected to help us realize the full potential of single-cell transcriptomics to unveil dynamic biological processes, model human pancreatic disease, and, ultimately, enable the development of novel therapeutic approaches to induce regeneration. We will test the above hypotheses by pursuing the following specific aims: (1) Longitudinal scRNAseq of same-donor T1D/T2D HPSs following stimulation of the BMP pathway. (2) Study of the effect of stress, inflammation and de-differentiation on BMP-7-mediated induction of β-cell formation in HPSs; (3) Functional characterization of neogenic β-cells through “slice-on-a-chip” approaches; and (4) Determination of in vivo regeneration potential of progenitor cells sorted from healthy and T1D/T2D donors using a xenotransplantation model.