How beta cell mass homeostasis is achieved throughout human lifetime? Overall, the extreme paucity of in vivo observations in humans and the limited relevance of rodent models largely explain our poor current Everolimus knowledge about human beta cell proliferation and call for new models to study its control. In vitro studies on isolated human adult islets were also carried to provide insights into the control of the cell cycle. For instance, several cell cycle regulators or transcription factors, either alone or in combinations, appear to stimulate the proliferation of human adult beta cells. However, these studies provide questionable conclusions as being mostly based on vast overexpression. Moreover, standard proliferation markers i.e. BrdU incorporation and Ki67 expression were used in these studies. Yet, these markers may actually reveal an abortive cell cycle, presumably linked to DNA damages, in beta cells submitted to mitogenic stimulations, as in other terminally differentiated cell types. Accordingly, except in very few studies, whether induction of these proliferation markers is translated in an increased number of beta cells remains to be demonstrated. Again, new methods and tools to study human beta proliferation would be highly desirable to overcome these limits. Toward this goal, we set up here human beta cell lines stably expressing the Fucci cell cycle indicators. We provide here several lines of evidence indicating that these new Fucci cell lines are reliable and convenient tools to decipher the control of cell cycle and differentiation of human pancreatic beta cells. We evaluated whether this correlation holds true for human beta cells. We FACS sorted and immediately replated three G1 subpopulations from growing EndoC-bH2-OFP-GFZ according to their level of orange fluorescence . Twenty two hours later, the percentage of green cells was 4.3%, 23.4% and 37.2% in cells derived from the low, medium and high orange fractions, respectively. This indicates that cells with the highest level of the orange fluorescence are more likely nearby the G1/S boundary. Confirming this conclusion, parallel quantitative RT-PCR analyses revealed that the low, medium and high orange fraction contained increasing amounts of PCNA and Cyclin E1 mRNA, two markers of late G1 . Therefore, EndoC-bH2 Fucci cell lines provide a mean to purify populations of living human beta cells enriched in cells in different sub-steps of G1. Note however that the high orange population is possibly not entirely made of G1 cells but may rather contain some early S phase cells which have not yet accumulated enough Green Fucci marker to be detected as positive for green fluorescence. The above-described results demonstrated a nearly perfect correlation between the Fucci indicators and DNA content. However cell cycle analysis by Hoechst 33342 staining is relatively imprecise. In addition, a small, albeit consistent, fraction of green cells was detected within the «G1 peak», most of them being thus yellow . This could reveal some degree of leakiness of the green Fucci marker in human beta cells.