Dataset Information

Comparative Effect of MSC Secretome to MSC Co-culture on Cardiomyocyte Gene Expression Under Hypoxic Conditions in vitro.

ABSTRACT: Introduction:Despite major leaps in regenerative medicine, the regeneration of cardiomyocytes after ischemic conditions remains to elucidate. It is crucial to understand hypoxia induced cellular mechanisms to provide advanced treatment options, including the use of stem cell paracrine factors for myocardial regeneration. Materials and Methods:In this study, the regenerative potential of hypoxic human cardiomyocytes (group Hyp-CMC) in vitro was evaluated when co-cultured with human bone-marrow derived MSC (group Hyp-CMC-MSC) or stimulated with the secretome of MSC (group Hyp-CMC-SMSC). The secretome of normoxic MSC and CMC, and the hypoxic CMC was analyzed with a cytokine panel. Gene expression changes of HIF-1?, proliferation marker Ki-67 and cytokinesis marker RhoA over different reoxygenation time periods of 4, 8, 24, 48, and 72 h were analyzed in comparison to normoxic CMC and MSC. Further, the proinflammatory cytokine IL-18 protein expression change, metabolic activity and proliferation was assessed in all experimental setups. Results and Conclusion:HIF-1? was persistently overexpressed in Hyp-CMC-SMSC as compared to Hyp-CMC (except at 72 h). Hyp-CMC-MSC showed a weaker HIF-1? expression than Hyp-CMC-SMSC in most tested time points, except after 8 h. The Ki-67 expression showed the strongest upregulation in Hyp-CMC after 24 and 48 h incubation, then returned to baseline level, while a temporary increase in Ki-67 expression in Hyp-CMC-MSC at 4 and 8 h and at 48 h in Hyp-CMC-SMSC could be observed. RhoA was increased in normoxic MSCs and in Hyp-CMC-SMSC over time, but not in Hyp-CMC-MSC. A temporary increase in IL-18 protein expression was detected in Hyp-CMC-SMSC and Hyp-CMC. Our study demonstrates timely dynamic changes in expression of different ischemia and regeneration-related genes of CMCs, depending from the culture condition, with stronger expression of HIF-1?, RhoA and IL-18 if the hypoxic CMC were subjected to the secretome of MSCs.

SUBMITTER: Kastner N

PROVIDER: S-EPMC7571272 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Comparative Effect of MSC Secretome to MSC Co-culture on Cardiomyocyte Gene Expression Under Hypoxic Conditions <i>in vitro</i>.

Kastner Nina N Mester-Tonczar Julia J Winkler Johannes J Traxler Denise D Spannbauer Andreas A Rüger Beate M BM Goliasch Georg G Pavo Noemi N Gyöngyösi Mariann M Zlabinger Katrin K

Frontiers in bioengineering and biotechnology 20201005

<h4>Introduction</h4>Despite major leaps in regenerative medicine, the regeneration of cardiomyocytes after ischemic conditions remains to elucidate. It is crucial to understand hypoxia induced cellular mechanisms to provide advanced treatment options, including the use of stem cell paracrine factors for myocardial regeneration.<h4>Materials and methods</h4>In this study, the regenerative potential of hypoxic human cardiomyocytes (group Hyp-CMC) <i>in vitro</i> was evaluated when co-cultured wit ...[more]

PMID: 33123511

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Project description:The unicellular microalga Euglena gracilis produces wax esters for ATP acquisition under low-oxygen conditions. The regulatory mechanism of wax ester production is not yet understood. Indeed, our previous transcriptomic analysis showed that transcript levels of genes involved in the wax ester synthesis hardly changed under hypoxic conditions, suggesting contribution of post-transcriptional regulation. In this study, we conducted a proteome analysis of E. gracilis mitochondria, as this organelle employs the fatty-acid synthesis pathway under hypoxic conditions. Mitochondria were isolated from E. gracilis SM-ZK strain treated with both aerobic and hypoxic conditions and used for shotgun proteomic analysis. Three independent proteomic analyses succeeded in identifying a total of 714 non-redundant proteins. Of these, 229 were detected in common to all experiments, and 116 were significantly recognized as differentially expressed proteins. GO enrichment analysis suggested dynamic changes in mitochondrial metabolic pathways and redox reactions under aerobic and hypoxic conditions. Protein levels of bifunctional enzymes isocitrate lyase and malate synthase in glyoxylate cycle were 1.35-fold higher under hypoxic conditions. Abundances of the propionyl-CoA synthetic enzymes, succinyl-CoA synthetase and propionyl-CoA carboxylase, were also 1.35- and 1.47-fold higher, respectively, under hypoxic conditions. Protein levels of pyruvate:NADP+ oxidoreductase, a key enzyme for anaerobic synthesis of acetyl-CoA, which serves as a C2 donor for fatty acids, showed a 1.68-fold increase under hypoxic conditions, whereas those of pyruvate dehydrogenase subunits showed a 0.77-0.81-fold decrease. Protein levels of the fatty-acid synthesis enzymes, 3-ketoacyl-CoA thiolase isoforms (KAT1 and KAT2), 3-hydroxyacyl-CoA dehydrogenases, and acyl-CoA dehydrogenase were up-regulated by 1.20- to 1.42-fold in response to hypoxic treatment. Overall, our proteomic analysis revealed that wax ester synthesis-related enzymes are up-regulated at the protein level post-transcriptionally to promote wax ester production in E. gracilis under low-oxygen conditions.

Project description:BACKGROUND: Gene expression studies related to cancer diagnosis and treatment are important. In order to conduct such experiment accurately, absolutely reliable housekeeping genes are essential to normalize cancer related gene expression. The most important characteristics of such genes are their presence in all cells and their expression levels remain relatively constant under different experimental conditions. However, no single gene of this group of genes manifests always stable expression levels under all experimental conditions. Incorrect choice of housekeeping genes leads to interpretation errors of experimental results including evaluation and quantification of pathological gene expression. Here, we examined (a) the degree of GAPDH expression regulation in Hep-1-6 mouse hepatoma and Hep-3-B and HepG2 human hepatocellular carcinoma cell lines as well as in human lung adenocarcinoma epithelial cell line (A-549) in addition to both HT-29, and HCT-116 colon cancer cell lines, under hypoxic conditions in vitro in comparison to other housekeeping genes like beta-actin, serving as experimental loading controls, (b) the potential use of GAPDH as a target for tumor therapeutic approaches was comparatively examined in vitro on both protein and mRNA level, by western blot and semi quantitative RT-PCR, respectively. FINDINGS: No hypoxia-induced regulatory effect on GAPDH expression was observed in the cell lines studied in vitro that were; Hep-1-6 mouse hepatoma and Hep-3-B and HepG2 human hepatocellular carcinoma cell lines, Human lung adenocarcinoma epithelial cell line (A-549), both colon cancer cell lines HT-29, and HCT-116. CONCLUSION: As it is the case for human hepatocellular carcinoma, mouse hepatoma, human colon cancer, and human lung adenocarcinoma, GAPDH represents an optimal choice of a housekeeping gene and/(or) loading control to determine the expression of hypoxia induced genes in tumors of different origin. The results confirm our previous findings in human glioblastoma that this gene is not an attractive target for tumor therapeutic approaches because of the lack of GAPDH regulation under hypoxia.

Project description:BackgroundTesticular tissue cryopreservation before gonadotoxic treatments allows fertility preservation in children suffering from cancer. Fertility restoration strategies, in particular in vitro maturation of prepubertal testicular tissue, are being developed mainly in animal models. The rat, widely used in biomedical research, including in reproductive biology, is a relevant model.ObjectivesTo determine whether sequential two-step culture protocols can improve the efficiency of rat in vitro spermatogenesis.Materials and methodsRat prepubertal testicular tissues were cultured on agarose gels with either a one-step or two-step protocol with or without polydimethylsiloxane (PDMS) ceiling chips. The progression of spermatogenesis, germ/Sertoli cell ratio, cell proliferation, seminiferous tubule area, and intratubular cell density were assessed by histological and immunohistochemical analyses. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays and Peanut Agglutinin (PNA) lectin labeling were performed to analyze the Deoxyribonucleic Acid (DNA) integrity and differentiation step of in vitro-produced spermatids.ResultsSequential two-step protocols allowed the production of spermatids with a higher efficiency compared with the one-step culture protocol. However, the efficiency was low, as less than 1.5% of tubules contained spermatids. Most of the in vitro-produced spermatids contained unfragmented DNA and were at an early step of differentiation. Rare elongating spermatids could be detected in the cultured explants. Although complete in vitro spermatogenesis could not be obtained with PDMS ceiling chips, entry into meiosis was promoted in one-step organotypic cultures.Discussion and conclusionComplete in vitro meiosis and the beginning of the elongation phase of spermiogenesis were obtained in a rat model using sequential culture methods. Because of their low efficiency, further work will be necessary to identify the culture conditions allowing the completion of spermiogenesis. These optimizations could pave the way for future applications, including the development of an in vitro fertility restoration procedure for childhood cancer survivors, which is still far from being clinically available.

Dataset Information

Comparative Effect of MSC Secretome to MSC Co-culture on Cardiomyocyte Gene Expression Under Hypoxic Conditions in vitro.

Publications

Comparative Effect of MSC Secretome to MSC Co-culture on Cardiomyocyte Gene Expression Under Hypoxic Conditions <i>in vitro</i>.

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