Human pancreatic exocrine cells were cultured in 3D suspension and formed pancreatospheres composed of acinar-derived and duct-like cells. We investigated, up to 6 days, the fate of human pancreatic acinar cells using fluorescein-conjugated Ulex Europaeus Agglutinin 1 lectin, a previously published acinar-specific non-genetic lineage tracing strategy. At day 4, fluorescence-activated cell sort for the intracellularly incorporated FITC-conjugated UEA1 lectin and the duct-specific CA19.9 surface marker, distinguished acinar-derived cells (UEA1+CA19.9−) from duct-like cells (UEA1−CA19.9+) and acinar-to-duct-like transdifferentiated cells (UEA1+CA19.9+). mRNA expression analysis of the acinar-derived (UEA1+CA19.9−) and duct-like (UEA1-CA19.9+) cell fractions with concomitant immunocytochemical analysis of the pancreatospheres revealed acquisition of an embryonic signature in the UEA1+CA19.9− acinar-derived cells characterized by de novo expression of SOX9 and CD142, robust expression of PDX1 and surface expression of GP2. The colocalisation of CD142, a multipotent pancreatic progenitor surface marker, PDX1, SOX9 and GP2 is reminiscent of a cellular state present during human embryonic development. Addition of TGF-beta signalling inhibitor Alk5iII, induced a 28-fold increased KI67-labeling in pancreatospheres, more pronounced in the CD142+GP2+ acinar-derived cells. These findings with human cells underscore the remarkable plasticity of pancreatic exocrine acinar cells, previously described in rodents, and could find applications in the field of regenerative medicine.
Maintenance of the pancreatic acinar cell phenotype suppresses tumor formation. Hence, repetitive acute or chronic pancreatitis, stress conditions in which the acinar cells dedifferentiate, predispose for cancer formation in the pancreas. Dedifferentiated acinar cells acquire a large panel of duct cell-specific markers. However, it remains unclear to what extent dedifferentiated acini differ from native duct cells and which genes are uniquely regulating acinar cell dedifferentiation. Moreover, most studies have been performed on mice since the availability of human cells is scarce. Here, we applied a non-genetic lineage tracing method of human pancreatic exocrine acinar and duct cells that allowed cell-type-specific gene expression profiling by RNA sequencing. Subsequent to this discovery analysis, one transcription factor that was unique for dedifferentiated acinar cells was functionally characterized. RNA sequencing analysis showed that human dedifferentiated acinar cells expressed genes in “Pathways of cancer” with a prominence of MECOM (EVI-1), a transcription factor that was not expressed by duct cells. During mouse embryonic development, pre-acinar cells also transiently expressed MECOM and in the adult mouse pancreas, MECOM was re-expressed when mice were subjected to acute and chronic pancreatitis, conditions in which acinar cells dedifferentiate. In human cells and in mice, MECOM expression correlated with and was directly regulated by SOX9. Mouse acinar cells that, by genetic manipulation, lose the ability to upregulate MECOM showed impaired cell adhesion, more prominent acinar cell death, and suppressed acinar cell dedifferentiation by limited ERK signaling. In conclusion, we transcriptionally profiled the two major human pancreatic exocrine cell types, acinar and duct cells, during experimental stress conditions. We provide insights that in dedifferentiated acinar cells, cancer pathways are upregulated in which MECOM is a critical regulator that suppresses acinar cell death by permitting cellular dedifferentiation.
Pancreatic acinar cells are a cell type of origin for pancreatic cancer that become progressively less sensitive to tumorigenesis induced by oncogenic Kras mutations after birth. This sensitivity is increased when Kras mutations are combined with pancreatitis. Molecular mechanisms underlying these observations are still largely unknown. To identify these mechanisms, we generated the first CRISPR-edited mouse models that enable detection of wild-type and mutant KRAS proteins in vivo. Analysis of these mouse models revealed that more than 75% of adult acinar cells are devoid of detectable KRAS protein. In the 25% of acinar cells expressing KRAS protein, transcriptomic analysis highlighted a slight upregulation of the RAS and MAPK pathways. However, at the protein level, only marginal pancreatic expression of essential KRAS effectors, including C-RAF, was observed. The expression of KRAS and its effectors gradually decreased after birth. The low sensitivity of adult acinar cells to Kras mutations resulted from low expression of KRAS and its effectors and the subsequent lack of activation of RAS/MAPK pathways. Pancreatitis triggered expression of KRAS and its effectors as well as subsequent activation of downstream signaling; this induction required the activity of EGFR. Finally, expression of C-RAF in adult pancreas was required for pancreatic tumorigenesis. In conclusion, our study reveals that control of the expression of KRAS and its effectors regulates the sensitivity of acinar cells to transformation by oncogenic Kras mutations. Significance: This study generates new mouse models to study regulation of KRAS during pancreatic tumorigenesis and highlights a novel mechanism through which pancreatitis sensitizes acinar cells to Kras mutations.
In vitro cultured pancreatic acinar cells rapidly differentiate. Low temperature exposure prevents this process and improves the efficiency of acinar cell labelling with adenovirus vectors. This may help in tracing β-cell neogenesis from human pancreatic acinar cells.
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