Generation of islet-like cell aggregates from human non-pancreatic cancer cell lines

To explore a novel source for the derivation of islets, we examined the differentiation potential of human non-pancreatic cancer cell lines, HeLa (cervical carcinoma cell line) and MCF-7 (breast cancer cell line). These cells were subjected to a serum-free, three-step sequential differentiation protocol which gave two distinct cell populations: single cells and cellular aggregates. Subsequent analysis confirmed their identity as pancreatic acinar cells and islet-like cell aggregates (ICAs), as evidenced by amylase secretion and diphenylthiocarbazone staining respectively. Reverse transcriptase-PCR and immunocytochemistry assessment of the ICAs revealed the expression of pancreatic specific markers Ngn-3, Glut-2, Pax-6 and Isl-1. These ICAs secreted insulin in response to glucose challenge, confirming their functionality. We propose that ICAs generated from HeLa and MCF-7 cell lines could form a promising in vitro platform of human islet equivalents (hIEQs) for diabetes research

Mouse Leydig cells express multiple P2X receptor subunits

ATP acts on cellular membranes by interacting with P2X (ionotropic) and P2Y (metabotropic) receptors. Seven homomeric P2X receptors (P2X1–P2X7) and seven heteromeric receptors (P2X1/2, P2X1/4, P2X1/5, P2X2/3, P2X2/6, P2X4/6, P2X4/7) have been described. ATP treatment of Leydig cells leads to an increase in [Ca2+]i and testosterone secretion, supporting the hypothesis that Ca2+ signaling through purinergic receptors contributes to the process of testosterone secretion in these cells. Mouse Leydig cells have P2X receptors with a pharmacological and biophysical profile resembling P2X2. In this work, we describe the presence of several P2X receptor subunits in mouse Leydig cells. Western blot experiments showed the presence of P2X2, P2X4, P2X6, and P2X7 subunits. These results were confirmed by immunofluorescence. Functional results support the hypothesis that heteromeric receptors are present in these cells since 0.5 μM ivermectin induced an increase (131.2 ± 5.9%) and 3 μM ivermectin a decrease (64.2 ± 4.8%) in the whole-cell currents evoked by ATP. These results indicate the presence of functional P2X4 subunits. P2X7 receptors were also present, but they were non-functional under the present conditions because dye uptake experiments with Lucifer yellow and ethidium bromide were negative. We conclude that a heteromeric channel, possibly P2X2/4/6, is present in Leydig cells, but with an electrophysiological and pharmacological phenotype characteristic of the P2X2 subunit