A Pro-Endocrine Pancreatic Islet Transcriptional Program Established During Development Is Retained in Human Gallbladder Epithelial Cells.

BACKGROUND & AIMS: Pancreatic islet ß-cells are factories for insulin production; however, ectopic expression of insulin also is well recognized. The gallbladder is a next-door neighbor to the developing pancreas. Here, we wanted to understand if gallbladders contain functional insulin-producing cells. METHODS: We compared developing and adult mouse as well as human gallbladder epithelial cells and islets using immunohistochemistry, flow cytometry, enzyme-linked immunosorbent assays, RNA sequencing, real-time polymerase chain reaction, chromatin immunoprecipitation, and functional studies. RESULTS: We show that the epithelial lining of developing, as well as adult, mouse and human gallbladders naturally contain interspersed cells that retain the capacity to actively transcribe, translate, package, and release insulin. We show that human gallbladders also contain functional insulin-secreting cells with the potential to naturally respond to glucose in vitro and in situ. Notably, in a non-obese diabetic (NOD) mouse model of type 1 diabetes, we observed that insulin-producing cells in the gallbladder are not targeted by autoimmune cells. Interestingly, in human gallbladders, insulin splice variants are absent, although insulin splice forms are observed in human islets. CONCLUSIONS: In summary, our biochemical, transcriptomic, and functional data in mouse and human gallbladder epithelial cells collectively show the evolutionary and developmental similarities between gallbladder and the pancreas that allow gallbladder epithelial cells to continue insulin production in adult life. Understanding the mechanisms regulating insulin transcription and translation in gallbladder epithelial cells would help guide future studies in type 1 diabetes therapy.

Human fetal pancreatic insulin-producing cells proliferate in vitro.

There have been considerable efforts towards understanding the potential of human pancreatic endocrine cells to proliferate and transition into mesenchymal cell populations. Since rodent studies have demonstrated that mouse insulin-producing cells do not proliferate in vitro, a similar possibility has been considered for human islet endocrine cells. Considering the inherent differences in mouse and human pancreatic islets, we decided to assess the potential of human fetal pancreatic insulin-producing cells to proliferate in vitro. We studied the proliferative potential of human fetal pancreatic islet-derived populations from second or third trimester fetal pancreas and characterized the cells that grow out during their expansion. We have used seven different approaches including in situ hybridization and immunostaining, quantitative estimation of multiple gene transcripts in populations as well as in single cells, clonal analysis of islet cells, assessment of heritable marks of active insulin promoter, and thymidine analog-based lineage tracing. Our studies demonstrate that human fetal pancreatic insulin-producing cells proliferate in vitro to generate mesenchymal cell populations. Interestingly, epigenetic modifications that mark open chromatin conformation of insulin promoter regions are retained even after a million fold expansion/proliferation in vitro. These findings demonstrate that hormone-producing cells in pancreatic islets proliferate in vitro and retain epigenetic marks that characterize an active insulin promoter. Such in vitro-derived mesenchymal cells may be of potential use in cell-replacement therapy for diabetes.

Expression of islet-specific microRNAs during human pancreatic development.

During pancreatic islet development, sequential changes in gene expression are known to be necessary for efficient differentiation and function of the endocrine pancreas. Several studies till now have demonstrated that microRNAs (miRNAs), which regulate translation of gene transcripts, influence gene expression cascades involved in pancreas development. Some of these miRNAs; miR-7 and miR-375 have been known to be expressed at high levels in pancreas and are also known to be involved in Zebrafish pancreas development as well as insulin secretion in mice. We demonstrate here that 4 different islet-specific microRNAs (miR-7, miR-9, miR-375 and miR-376) are expressed at high levels during human pancreatic islet development. Of these, miR-375, is seen to be differentially expressed in human islet beta- as well as non-beta-cells. Though no significant difference in abundance of miR-375 was noted in either cell type, analysis of islet-specific miRNA and mRNA in single cells show that non-beta cells contain higher levels of miR-375. Our data demonstrate that miRNAs that are known to be regulated during Zebrafish pancreatic development may play similar role in human pancreatic islet development.

The miR-30 family microRNAs confer epithelial phenotype to human pancreatic cells.

Epithelial-to-mesenchymal transition is a phenomenon necessary for embryonic development and also seen during certain pathological conditions.  We show here for the first time that reduction in miR-30 family microRNAs, is responsible for mesenchymal transition of primary cultures of human pancreatic epithelial cells.  We found that miR-30 family microRNAs target mesenchymal gene transcripts and maintain them in a translationally inactive state.  Forced depletion using miR-30 family specific anti-miRs leads to mesenchymal transition while ectopic overexpression maintains the epithelial phenotype.  We also show that miR-30 family microRNAs increase in abundance during differentiation of pancreatic islet-derived mesenchymal cells into hormone-producing islet-like cell aggregates.  Our studies in human adult diseased pancreas also demonstrate that miR-30 family microRNAs are expressed at lower abundance in fibrotic lesions during pancreatitis.  Together, our data confirm that miR-30 family microRNAs form a part of the regulatory signaling events involved in cellular response of pancreatic epithelial cells during mesenchymal transition.