4 research outputs found
Hyperosmotic Stress Induces a Specific Pattern for Stress Granule Formation in Human-Induced Pluripotent Stem Cells
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
Adaptation of a Commonly Used, Chemically Defined Medium for Human Embryonic Stem Cells to Stable Isotope Labeling with Amino Acids in Cell Culture
Metabolic
labeling with stable isotopes is a prominent technique
for comparative quantitative proteomics, and stable isotope labeling
with amino acids in cell culture (SILAC) is the most commonly used
approach. SILAC is, however, traditionally limited to simple tissue
culture regimens and only rarely employed in the context of complex
culturing conditions as those required for human embryonic stem cells
(hESCs). Classic hESC culture is based on the use of mouse embryonic
fibroblasts (MEFs) as a feeder layer, and as a result, possible xenogeneic
contamination, contribution of unlabeled amino acids by the feeders,
interlaboratory variability of MEF preparation, and the overall complexity
of the culture system are all of concern in conjunction with SILAC.
We demonstrate a feeder-free SILAC culture system based on a customized
version of a commonly used, chemically defined hESC medium developed
by Ludwig et al. and commercially available as mTeSR1 [mTeSR1 is a
trade mark of WiCell (Madison, WI) licensed to STEMCELL Technologies
(Vancouver, Canada)]. This medium, together with adjustments to the
culturing protocol, facilitates reproducible labeling that is easily
scalable to the protein amounts required by proteomic work flows.
It greatly enhances the usability of quantitative proteomics as a
tool for the study of mechanisms underlying hESCs differentiation
and self-renewal. Associated data have been deposited to the ProteomeXchange
with the identifier PXD000151
Adaptation of a Commonly Used, Chemically Defined Medium for Human Embryonic Stem Cells to Stable Isotope Labeling with Amino Acids in Cell Culture
Metabolic
labeling with stable isotopes is a prominent technique
for comparative quantitative proteomics, and stable isotope labeling
with amino acids in cell culture (SILAC) is the most commonly used
approach. SILAC is, however, traditionally limited to simple tissue
culture regimens and only rarely employed in the context of complex
culturing conditions as those required for human embryonic stem cells
(hESCs). Classic hESC culture is based on the use of mouse embryonic
fibroblasts (MEFs) as a feeder layer, and as a result, possible xenogeneic
contamination, contribution of unlabeled amino acids by the feeders,
interlaboratory variability of MEF preparation, and the overall complexity
of the culture system are all of concern in conjunction with SILAC.
We demonstrate a feeder-free SILAC culture system based on a customized
version of a commonly used, chemically defined hESC medium developed
by Ludwig et al. and commercially available as mTeSR1 [mTeSR1 is a
trade mark of WiCell (Madison, WI) licensed to STEMCELL Technologies
(Vancouver, Canada)]. This medium, together with adjustments to the
culturing protocol, facilitates reproducible labeling that is easily
scalable to the protein amounts required by proteomic work flows.
It greatly enhances the usability of quantitative proteomics as a
tool for the study of mechanisms underlying hESCs differentiation
and self-renewal. Associated data have been deposited to the ProteomeXchange
with the identifier PXD000151