ABSTRACT
Mesenchymal stem/stromal cells (MSCs) are self-renewing multipotent cells with regenerative, secretory and immunomodulatory capabilities that are beneficial for the treatment of various diseases. To avoid the issues that come with using tissue-derived MSCs in therapy, MSCs may be generated by the differentiation of human embryonic stems cells (hESCs) in culture. However, the changes that occur during the differentiation process have not been comprehensively characterized. Here, we combined transcriptome, proteome and phosphoproteome profiling to perform an in-depth, multi-omics study of the hESCs-to-MSCs differentiation process. Based on RNA-to-protein correlation, we determined a set of high confidence genes that are important to differentiation. Among the earliest and strongest induced proteins with extensive differential phosphorylation was AHNAK, which we hypothesized to be a defining factor in MSC biology. We observed two distinct expression waves of developmental HOX genes and an AGO2-to-AGO3 switch in gene silencing. Exploring the kinetic of noncoding ORFs during differentiation, we mapped new functions to well annotated long noncoding RNAs (CARMN, MALAT, NEAT1, LINC00152) as well as new candidates which we identified to be important to the differentiation process. Phosphoproteome analysis revealed ESC and MSC-specific phosphorylation motifs with PAK2 and RAF1 as top predicted upstream kinases in MSCs. Our data represent a rich systems-level resource on ESC-to-MSC differentiation that will be useful for the study of stem cell biology.
Subject(s)
Gene Expression Profiling/methods , Gene Regulatory Networks , Human Embryonic Stem Cells/cytology , Mesenchymal Stem Cells/cytology , Proteomics/methods , Cell Differentiation , Cells, Cultured , Chromatography, Liquid , Gene Expression Regulation , Human Embryonic Stem Cells/metabolism , Humans , Mass Spectrometry , Mesenchymal Stem Cells/metabolism , Phosphorylation , Protein Interaction Maps , Sequence Analysis, RNAABSTRACT
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.
Subject(s)
Cell Culture Techniques/methods , Embryonic Stem Cells/metabolism , Isotope Labeling , Proteomics/methods , Amino Acids/chemistry , Animals , Cell Differentiation , Culture Media/chemistry , Embryonic Stem Cells/cytology , Humans , MiceABSTRACT
Cancer metastasis causes approximately 90% of all cancer-related death and independent of the advancement of cancer therapy, a majority of late stage patients suffers from metastatic cancer. Metastasis implies cancer cell migration and invasion throughout the body. Migration requires the formation of pseudopodia in the direction of movement, but a detailed understanding of this process and accordingly strategies of prevention remain elusive. Here, we use quantitative proteomic profiling of human cancer pseudopodia to examine this mechanisms essential to metastasis formation, and identify potential candidates for pharmacological interference with the process. We demonstrate that Prohibitins (PHBs) are significantly enriched in the pseudopodia fraction derived from cancer cells, and knockdown of PHBs, as well as their chemical inhibition through Rocaglamide (Roc-A), efficiently reduces cancer cell migration.
Subject(s)
Antineoplastic Agents/therapeutic use , Neoplasm Metastasis/prevention & control , Pseudopodia/metabolism , Repressor Proteins/metabolism , Antineoplastic Agents/pharmacology , Benzofurans/pharmacology , Cell Line, Tumor , Cell Movement/drug effects , Gene Knockdown Techniques , Humans , Prohibitins , Proteomics/methods , Pseudopodia/drug effects , Repressor Proteins/antagonists & inhibitors , Repressor Proteins/geneticsABSTRACT
We show that HTLV-1 negative leukemia cells are more sensitive to TQ due to higher levels of drug-induced reactive oxygen species (ROS). PreG1 population in HTLV-1 negative Jurkat and CEM was higher than HTLV-1 transformed HuT-102 and MT-2 cells. Peripheral blood mononuclear cells were more resistant. Hoechst staining indicated more features of apoptosis, namely nuclear blebs and shrunken nuclei in HuT-102 than Jurkat. A greater depletion of the antioxidant enzyme glutathione occurred in Jurkat, which consequently led to an increase in ROS, loss of mitochondrial membrane potential, cytochrome c release, activation of caspases 3 and 9, and cleavage of PARP. Treatment with z-VAD-fmk partially reversed TQ-induced apoptosis, suggesting a caspase-dependent mechanism. N-acetyl cysteine prevented apoptosis providing evidence that cell death is ROS-dependent. Catalase prevented apoptosis to a lesser extent than NAC. In summary, TQ induces apoptosis in adult T cell leukemia/lymphoma by decreasing glutathione and increasing ROS, and levels of ROS underlie the differential cellular response to TQ. Our data suggest a potential therapeutic role for TQ in sensitizing HTLV-I-negative T-cell lymphomas.