Figure 8. Fucci indicators reveal growth-arrest and terminal differentiation of human beta cells.

(A) Exponentially growing EndoC-βH2-PGF2AOF Fucci cells were transduced with a retrovector encoding either a selectable marker (zeocin resistance) or both Zeocin resistance and the Cre recombinase, then selected in a zeocin containing medium and analyzed by flow cytometry for their green and orange fluorescences 13 days later. Doublets were excluded from the analysis. EndoC-βH2 cells depends upon two floxed transgenes for their proliferation, SV40LT and hTERT. After 13 days (time of transduction taken as day 0), green EndoC-βH2-PGF2AOF cells become much rarer while the proportion of orange cells inflates in cells transduced with a Cre-encoding vector (right) compared to control cells (left). (B) Control and Cre-transduced cells were analyzed for expression of Cre, SV40LT, Ki67 and INSULIN mRNA by quantitative RT-PCR. The maximal value is arbitrarily taken as 100 for each transcript (C) The same experiment as in A was carried out and control and Cre-transduced cells were FACS sorted after 12 days (time of transduction taken as day 0) according to the intensity of the orange fluorescence: the bulk of orange control cells (fraction 0), the exactly corresponding fraction among CRE-transduced orange cells (fraction 1) and the brightest Cre-transduced orange cells (fraction 2). (D) Each fraction harvested in (C) was then submitted to quantitative RT-PCR analyses for the expression of Cre, SV40LT, Ki67, INSULIN and IAPP mRNA. Two PCR experiments (except three for Insulin) done with the same cDNA samples, were performed (two duplicates per sample), and the mean of the values as well as standard deviations are shown. The maximal value is arbitrarily taken as 100 for each transcript (bottom panels).