Hyperosmotic Stress Induces a Specific Pattern for Stress Granule Formation in Human-Induced Pluripotent Stem Cells

Abstract

Stress granules (SGs) are assemblies of selective messenger RNAs (mRNAs), translation factors, and RNA-binding proteins in small untranslated messenger ribonucleoprotein (mRNP) complexes in the cytoplasm. Evidence indicates that different types of cells have shown different mechanisms to respond to stress and the formation of SGs. In the present work, we investigated how human-induced pluripotent stem cells (hiPSCs/IMR90-1) overcome hyperosmotic stress compared to a cell line that does not harbor pluripotent characteristics (SH-SY5Y cell line). Gradient concentrations of NaCl showed a different pattern of SG formation between hiPSCs/IMR90-1 and the nonpluripotent cell line SH-SY5Y. Other pluripotent stem cell lines (hiPSCs/CRTD5 and hESCs/H9 (human embryonic stem cell line)) as well as nonpluripotent cell lines (BHK-21 and MCF-7) were used to confirm this phenomenon. Moreover, the formation of hyperosmotic SGs in hiPSCs/IMR90-1 was independent of eIF2α phosphorylation and was associated with low apoptosis levels. In addition, a comprehensive proteomics analysis was performed to identify proteins involved in regulating this specific pattern of hyperosmotic SG formation in hiPSCs/IMR90-1. We found possible implications of microtubule organization on the response to hyperosmotic stress in hiPSCs/IMR90-1. We have also unveiled a reduced expression of tubulin that may protect cells against hyperosmolarity stress while inhibiting SG formation without affecting stem cell self-renewal and pluripotency. Our observations may provide a possible cellular mechanism to better understand SG dynamics in pluripotent stem cells.

Stress Factors as Possible Regulators of Pluripotent Stem Cell Survival and Differentiation

“…ESCs exposed to sorbitol, for instance, showed increased pSAPK expression and ultimately underwent apoptosis in a dose-dependent manner [ 11 , 16 ]. However, in hiPSCs exposed to sodium chloride or sorbitol, stress granule formation was observed, preserving cell morphology and pluripotency marker expression, and no apoptosis occurred [ 75 ]. Additionally, the exposure of ESCs to oxidative stress that occurred due to zearalenone treatment resulted in an increased expression of apoptosis markers [ 13 ], whereas hiPSCs exposed to sodium arsenite exhibited stress granule formation with decreased pluripotency marker expression [ 76 ].…”

“…In contrast, SH-SY5Y cells exhibited increased features of late apoptosis as the NaCl concentration increased. Additionally, caspase 3 activation (an indicator of active apoptosis) was not detected in hiPSCs/IMR90-1 at 400 mM NaCl, suggesting that the absence of SG formation in these cells is not attributed to apoptosis activation [ 75 ].…”

“…Conversely, the top-scoring IPA protein network in SH-SY5Y cells was associated with RNA damage, repair, post-transcriptional modifications, and protein synthesis. These findings highlight the distinct survival mechanisms activated in each cell group when subjected to hyperosmotic stress [ 75 ].…”

Digitalized organoids: integrated pipeline for 3D high-speed analysis of organoid structures using multilevel segmentation and cellular topology

“…After 1 week of HCT116 spheroids formation as described above, osmotic stress was applied by adding NaCl into the culture medium as previously described 30,31 . Complete medium supplemented with 100 mM NaCl (7647-14-5, Merck) was used as hypertonic medium.…”

Cellular Stress: Modulator of Regulated Cell Death

“…Cells are exposed to various external stressors such as temperature fluctuations, infections, nutrient deprivations, radiation, chemicals, and toxins. In addition, changes in redox states, ionic imbalances, accumulation of unfolded proteins, oncogene activation, etc., contribute to intrinsic cellular stress [ 2 , 3 , 4 , 5 , 6 , 7 ]. Various signaling pathways that are activated during stress, such as the heat shock response (HSR), unfolded protein response (UPR), DNA damage response (DDR), and response to oxidative stress, efficiently revert the stress-induced alterations to restore cellular homeostasis and prevent cell death.…”