Pluripotent embryonic stem (ES) cells are characterized by their capacity to self-renew indefinitely while maintaining the potential to differentiate into all cell types of an adult organism. Both the undifferentiated and differentiated states are defined by specific gene expression programs that are regulated at the chromatin level. By employing the KDM6-specific GSK-J4 inhibitor and by targeted gene knockout/knockdown (KO/KD), we have analyzed the contribution of the H3K27me2,3-specific demethylases KDM6A and KDM6B to murine ES cell differentiation. We observe that inhibition of the H3K27 demethylase activity induces DNA damage along with activation of the DNA damage response (DDR) and cell death in differentiating but not in undifferentiated ES cells. Laser microirradiation experiments revealed that the H3K27me3 mark but not the KDM6B protein co-localize with γH2AX+ sites of DNA damage in differentiating ES cells. Lack of H3K27me3 attenuates the GSK-J4-induced DDR in differentiating Eed KO ES cells. Collectively our findings indicate that differentiating ES cells depend on KDM6 and that the H3K27me3 demethylase activity is critically involved in DDR and survival of differentiating ES cells.

Repressor element 1-silencing transcription factor (REST) plays a crucial role in the differentiation of neural progenitor cells (NPCs). C-terminal domain small phosphatases (CTDSPs) are REST effector proteins that reduce RNA polymerase II activity on genes required for neurogenesis. miR-26b regulates neurogenesis in zebrafish by targeting ctdsp2 mRNA, but the molecular events triggered by this microRNA (miR) remain unknown. Here, we show in a murine embryonic stem cell differentiation paradigm that inactivation of miR-26 family members disrupts the formation of neurons and astroglia and arrests neurogenesis at the neural progenitor level. Furthermore, we show that miR-26 directly targets Rest, thereby inducing the expression of a large set of REST complex-repressed neuronal genes, including miRs required for induction of the neuronal gene expression program. Our data identify the miR-26 family as the trigger of a self-amplifying system required for neural differentiation that acts upstream of REST-controlled miRs.

Adipose-derived stromal/stem cells (ASCs) have been shown to exert regenerative functions, which are mainly attributed to the secretion of trophic factors. Upon transplantation, ASCs are facing an ischemic environment characterized by oxygen and nutrient deprivation. However, current knowledge on the secretion capacity of ASCs under such conditions is limited. Thus, the present study focused on the secretory function of ASCs under glucose and oxygen deprivation as major components of ischemia. After exposure to glucose/oxygen deprivation, ASCs maintained distinct viability, but the metabolic activity was greatly reduced by glucose limitation. ASCs were able to secrete a broad panel of factors under glucose/oxygen deprivation as revealed by a cytokine antibody array. Quantification of selected factors by ELISA demonstrated that glucose deprivation in combination with hypoxia led to markedly higher secretion levels of the angiogenic and anti-apoptotic factors IL-6, VEGF, and stanniocalcin-1 as compared to the hypoxic condition alone. A conditioned medium of glucose/oxygen-deprived ASCs promoted the viability and tube formation of endothelial cells, and the proliferation and migration of fibroblasts. These findings indicate that ASCs are stimulated by ischemia-like stress conditions to secrete trophic factors and would be able to exert their beneficial function in an ischemic environment.