Stress Granules as a possible regulator of pluripotent stem cell self renewal and differentaition

Abstract

In cells subjected to environmental stresses, such as oxidative stress and heat shock (HS), dynamic ribonucleoprotein aggregates, known as Stress Granules (SGs)), are formed as a part of the cell response program. Different types of stresses control pluripotent stem cell (PSCs) ability to self renew and differentiate, indicating the possible role of SGs in regulating stem cell fate. In this study we compared the effects of oxidative (sodium arsenite (SA) and hydrogen peroxide (H2O2)) and thermal (Heat Shock) stresses on SG formation in human induced (hi) PSCs. Our aim was to examine whether these granules paly a role in regulating PSC self-renewal and differentiation. Our data showed that not all stressors induce SG formation in hiPSCs. Increasing SA concentartions, progressively increase the number of cells showing SGs, however, iPSCs treated with H2O2 exhibited no SG formation even with higher concentrations or longer periods of incubation. On the other hand, no granules were observed in cells kept at 37oC or exposed to mild HS (40oC) treatment, whereas at higher temperatures of 42oC, 100% of the cells formed SGs. Molecular analyses of the granules formed in iPSCs in response to stress conditions showed that they (i) contain the well-known SGs proteins markers (G3BP, TIAR, eIF4E, eIF4A, eIF3B, eIF4G, and PABP), (ii) are present in the cytoplasm in a physical attachment to processing bodies (PBs), and (iii) are disassembled after the removal of the stress. This data confirm that these iPSCs granules are per se SGs. In addition, the formation of SGs was associated with the stimulation of eIF2α phosphorylation in hiPSCs after SA and HS, but not H2O2, which confirm the stimulation of the stress response program. To test whether pluripotent marker proteins are recruited to SGs, we perform an initial screening for several pluripotent markers and confirmed that LIN28A and L1TD1 were SG markers and identified DPPA5 as a novel pluripotent marker that was weakly recruited to SGs. Altogether, our data introduce new aspects of how hiPSCs respond to adverse environmental conditions and identify SGs as a possible regulator of pluripotent stem cell self renewal and differentiation.