Publication date: August 2017
Source:Biochimie, Volume 139
Author(s): Artur Gurgul, Jolanta Opiela, Klaudia Pawlina, Tomasz Szmatoła, Michał Bochenek, Monika Bugno-Poniewierska
The use of histone acetyl-transferase inhibitors such as trichostatin A (TSA) for epigenetic transformation of mesenchymal stem cells (MSCs), whose nuclei will be transferred into enucleated oocytes, is a novel approach in research involving somatic cell cloning of pigs and other mammalian species. Although the effectiveness of TSA in cloning applications was confirmed, processes and mechanisms underlying achieved effects are not yet fully understood, especially for pig MSCs. To contribute to this knowledge, in this study we performed a comprehensive transcriptome analysis using high-throughput sequencing of pig bone-marrow derived MSCs, both treated and untreated with TSA, and evaluated the effect of TSA administration on their transcription profile after 24 h of in vitro culture. The expression of selected positive and negative mesenchymal surface antigens was also evaluated in these cells by flow cytometry. Subsequently, the stability of induced expression changes was evaluated after another 55–72 h of culture without TSA. The results of this study showed that TSA does not affect the expression of the selected surface antigens related to MSC mesenchymal stemness origin, namely: CD90 (positive marker), CD31 and CD34 (negative markers) and has a wide stimulating effect on MSCs transcription, affecting genes across the whole genome with some minor signs of site-specific acting in regions on SSC2 and SSC6. TSA turned out to have a higher impact on already expressed genes with only minor abilities to induce expression of silenced genes. Genes with expression affected by TSA were related to a wide range of biological processes, however, we found some evidence for specific stimulation of genes associated with development, differentiation, neurogenesis or myogenesis. TSA also seemed to interfere with Wnt signaling pathways by upregulation of several engaged genes. The analysis of cell transcriptome after prolonged culture following the TSA removal, showed that the expression level of majority of genes affected by TSA is restored to the initial level. Nonetheless, the set of about six hundred genes responsible for e.g. adhesion, signal transduction and cell communication was altered even after 55–72 h of culture without TSA. TSA also enhanced expression of some of pluripotency marker genes (FGF2, LIF, TERT) but their expression was stabilized during further culture without TSA. The detailed analysis of factors connected with neuron-like differentiation allowed us to assume that TSA mostly stimulates neurogenic differentiation pathway in the pig MSCs possibly through interaction with Wnt-mediated signaling and thus triggers mechanisms conducive to epigenetic reprograming.
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