*Purpose: To monitor time-dependent cellular changes, with emphasis on beta cell regeneration, in pancreatic islets following treatment with Lacripep^TM-like peptide N-104 by mass cytometry. Background: Lacritin is a multifunctional tear protein with prosecretory, prosurvival and mitogenic properties. Islets are Lacritin responsive, and prominently express known elements of the Lacripep™ receptor complex and signaling pathways. N-104 peptide comprises the fifteen C-terminal amino acids of Lacritin. Mass cytometry was used to measure the expression of proliferative markers of all major endocrine cells as well exocrine cells.

*Methods: Mouse islets were cultured with 4 µM N-104, or negative control peptide C95, or left untreated for 24 hours, following which islets were dissociated into single cells by treatment with trypsin. Single cells were fixed and stained with 35 antibody markers. Mass cytometry data were obtained on CyTOF2 (Fluidigm) at the Flow Cytometry core at UVA.

*Results: At the single cell level, N-104 stimulated the proliferation of all major endocrine cells. Ki67 was measured as a marker for proliferation. Proliferation of alpha cells, identified by the expression of marker Proprotein convertase 2 was 0.90% for N-104 treated islets, 0.32% for untreated islets and 0.24% for negative control C-95. Proliferation of alpha cells using Glucagon as marker was 1.12%, 0.64% and 0.69% for N-104 treated, untreated and C-95 treated islets respectively. Proliferation of beta cells as identified by cytoplasmic insulin and proinsulin was the most significant – in keeping with the enhancement of glucose-stimulated insulin secretion that was observed in parallel. Proliferation of beta cells, identified by NKX6.1 was 2.88% for N-104 treated islets, 1.28% for untreated samples and 1.62% for C95 treated islets. Proinsulin showed increases of 3.87%, 1.00% and 1.44% and Insulin showed increases of 5.02%, 1.14% and 1.92% for N-104 treated, untreated and C-95 treated samples respectively. To a lesser degree, N-104 also enhanced delta and ductal cells proliferation – without substantially affecting acinar cells identified by markers PDX1, SST, and CD44.

*Conclusions: Our data indicate that systemic N-104 treatment promoted single cell proliferation in islets, especially that of beta cells, without affecting exocrine acinar cell proliferation.

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