Endocrine Abstracts

NES2Y cells were derived several years ago from a patient following surgery for Congenital Hyperinsulinism (CHI). These cells have the inductive capacity to form insulin-secreting cells, but they are largely uncharacterized. The purpose of this study was to examine the expression and function of the ATP-binding cassette protein ABCG2 and to characterize the cells for their expression of markers of Side Population (SP) progenitor cells and Stellate Cells (SC). Cells were maintained under standard conditions with or without the addition of mitoxantrone (MTX, 20-50 nM for 1, 3 or 6 days) to the cell culture medium. PCR, immunohistochemistry, high content analysis and flow cytometry techniques were used to examine the molecular and functional properties of these cells. Based upon the exclusion of fluorescent dyes such Hoechst 33342 and DCV by ABCG2, we estimated that approximately 30% of native cells expressed a defined SP phenotype which was increased to approximately 60% following exposure to MTX. MTX-induction led to enhanced ABCG2 detection at the plasma membrane and the nucleus, and to a marked decrease in the rate of cell proliferation. Pancreatic endocrine progenitor cell selective markers were found (by RT-PCR) to be expressed over several passages including NKX2.2, PAX6, Nestin, PDX1, NKX6.1 and SOX9, but the cells did not express NEUROD1 and Neurogenin3. NES2Y cells were also found to express a number of SC markers including vinculin, vimentin, synaptophysin, α-smooth muscle actin, glial fibrillary acidic protein, neural cell adhesion molecule, desmin and CRP2. Immunofluorescence confirmed the presence of vimentin protein. Collectively these data suggest that NES2Y cells share common features of SC and SP stem cells and that they express progenitor cell markers consistent with their inductive capacity to form insulin-secreting cells.