However, BrdU positive cells were detected in the RPE at 24 h PR only in the presence of FGF2, when the RPE became p27Kip1 negative, suggesting that RPE cells had entered the cell cycle. We did not selleckchem Sorafenib observed BrdU positive cells in the RPE at 24 h PR in the absence of FGF2 when the RPE was still p27Kip1 positive, there fore, FGF2 is necessary for the cell cycle Inhibitors,Modulators,Libraries entry, and even tually for RPE transdifferentiation. To analyze the process of dedifferentiation, we won dered if injury was sufficient to initiate changes in gene expression of pluripotency inducing fac tors, genes associated with the RPE specification, and genes Inhibitors,Modulators,Libraries associated with retina progenitors, as well as eye field transcriptional factors. We used the following time points of analysis as a reference, 6 h PR, 24 h PR and 72 h PR.
mRNA levels for all different genes were evaluated by quantitative RT PCR using Inhibitors,Modulators,Libraries RPE samples collected by laser capture microdissection. Surprisingly, at 6 h PR, we observed activation of gene expression of sox2, c myc and klf4 and over the basal levels detected in unin jured eyes. However, the expression of sox2 decreased by 72 h PR to the basal levels. Although the injury was sufficient to up regulate sox2, c myc and klf4, which are present in ret ina progenitors, the absence of the tran scripts for oct4 and nanog that are present in embryonic stem cells suggest that the RPE cells do not become pluri potent, but do acquire some plasticity. In agreement with Inhibitors,Modulators,Libraries our results, in vitro culture of RPE cells, isolated from adult human donor eyes, showed high levels of c myc and klf4 compared to human embryonic stem cells, however, oct4 and nanog were not detected by immunostaining or RT qPCR.
Among all the pluripotency inducing factors, c Myc, Klf4 Inhibitors,Modulators,Libraries and Sox2 are the common factors expressed in regenerating tissues. It is of note that we did not detect Diabete expression of oct4 in the RPE before or after injury. Interestingly, in zebrafish, klf4 and oct4 are expressed in the uninjured retina and transiently increase during the process of M��ller glia dedifferentiation. Also in zebrafish, the knockdown of morpholino against pou5f1 impairs fin regeneration, sug gesting that Oct4 might be crucial for regeneration in this organism. The process of RPE dedifferentiation was evidenced by the down regulation of RPE specification genes mitf and tyr con comitantly with an up regulation of neural retina progeni tors ascl1 and chx10. We also decided to analyze if the dedifferentiated RPE cells go back into the lineage of eye formation. Different factors are crucial for eye formation, the most important are the eye field transcriptional factors that are expressed in the anterior neural plate in the region specified to be come the eyes.