Project description:Background: Experimental studies have suggested that stem cells (SC) may exert their beneficial effects on the ischemic heart by paracrine activation of antiapoptotic pathways. In order to identify potential cardioprotective mediators, we performed a systematic analysis of the differential gene expression of lin-/c-kit+ SC after coculture with cardiomyocytes (CM). Methods: SC were separated from murine bone marrow and labeled with the green fluorescent CFDA. SC were then cocultivated with neonatal rat ventricular CM (NRVCM). After 48 h, we performed two cell sorting steps to generate a highly purified population of conditioned SC (>99%) and isolated the RNA. Next, we performed a genome-wide microarray analysis of cocultured vs. monocultured SC with Illumina Chips (Coculture n=3; Monoculture n=3). Our systematic analysis of differentially expressed genes focused on products that are secretable, membrane-bound and potentially involved in antiapoptotic signal transduction. Results: Our analysis resulted in 3 genes (out of more than 1000 differentially regulated genes) that met the criteria mentioned above and which could also be confirmed by RT-PCR. We found CCL-12 (12x, p<0.05), MIF (2x, p<0.05) and connexin 40 (4.5x; p<0.05) upregulated in our coculture model. An ELISA of cell culture supernatants was performed to proof secretability of candidate genes and showed that supernatants of coculture experiments have higher CCL-12 concentrations than monoculture experiments (20 pg/ml vs. 4 pg/ml; p<0.01). Next, we stimulated NRVCM with concentrated coculture supernatants which resulted in a significantly higher phosphorylation of AKT (p<0.01). Finally, NRVCM were labeled with Annexin-V and apoptosis of NRVCM in a co- and monoculture was measured by FACS. Thereby, we observed a reduction in apoptotic NRVCM in the coculture model (12% vs. 25%; p<0.05). Conclusion: Our results provide evidence that coculture of SC with NRVCM leads to an upregulation of antiapoptotic genes and a paracrine-mediated increase in the phosphorylation of AKT. This results in less apoptotis in cocultured NRVCM. We therefore conclude, that cell-cell interactions lead to a modified gene expression in SC that may in part explain the cardioprotective effects of stem cell therapy. We analysed six RNA samples extracted from three cocultures and three monocultures. Every sample represents an independant biological experiment, derived from individual preparations of cardiac myocytes and bone marrow stem cells.

Project description:Background: Experimental studies have suggested that stem cells (SC) may exert their beneficial effects on the ischemic heart by paracrine activation of antiapoptotic pathways. In order to identify potential cardioprotective mediators, we performed a systematic analysis of the differential gene expression of lin-/c-kit+ SC after coculture with cardiomyocytes (CM). Methods: SC were separated from murine bone marrow and labeled with the green fluorescent CFDA. SC were then cocultivated with neonatal rat ventricular CM (NRVCM). After 48 h, we performed two cell sorting steps to generate a highly purified population of conditioned SC (>99%) and isolated the RNA. Next, we performed a genome-wide microarray analysis of cocultured vs. monocultured SC with Illumina Chips (Coculture n=3; Monoculture n=3). Our systematic analysis of differentially expressed genes focused on products that are secretable, membrane-bound and potentially involved in antiapoptotic signal transduction. Results: Our analysis resulted in 3 genes (out of more than 1000 differentially regulated genes) that met the criteria mentioned above and which could also be confirmed by RT-PCR. We found CCL-12 (12x, p<0.05), MIF (2x, p<0.05) and connexin 40 (4.5x; p<0.05) upregulated in our coculture model. An ELISA of cell culture supernatants was performed to proof secretability of candidate genes and showed that supernatants of coculture experiments have higher CCL-12 concentrations than monoculture experiments (20 pg/ml vs. 4 pg/ml; p<0.01). Next, we stimulated NRVCM with concentrated coculture supernatants which resulted in a significantly higher phosphorylation of AKT (p<0.01). Finally, NRVCM were labeled with Annexin-V and apoptosis of NRVCM in a co- and monoculture was measured by FACS. Thereby, we observed a reduction in apoptotic NRVCM in the coculture model (12% vs. 25%; p<0.05). Conclusion: Our results provide evidence that coculture of SC with NRVCM leads to an upregulation of antiapoptotic genes and a paracrine-mediated increase in the phosphorylation of AKT. This results in less apoptotis in cocultured NRVCM. We therefore conclude, that cell-cell interactions lead to a modified gene expression in SC that may in part explain the cardioprotective effects of stem cell therapy.

Project description:Drug-induced liver injury (DILI) is an important clinical problem. Here we used a genomics approach to establish the critical drug-induced toxicity pathways that act in synergy with the pro-inflammatory cytokine tumor necrosis factor (TNF) to cause cell death of liver HepG2 cells. Transcriptomics of the cell injury stress response pathways initiated by two hepatoxicants, diclofenac and carbamazepine, revealed the endoplasmic reticulum (ER) stress/translational initiation signaling and Nrf2 antioxidant signaling as two major affected pathways, which was similar to that observed for the majority of ~80 DILI compounds in primary human hepatocytes. The ER stress was primarily related to PERK and ATF4 activation and subsequent expression of CHOP, which was all independent of TNFÎ± signaling. Identical ATF4 dependent transcriptional programs were observed in primary human hepatocytes as well as primary precision cut human liver slices. Targeted RNA interference studies revealed that while ER stress signaling through IRE1Î± and ATF6 acted cytoprotective, activation of the ER stress protein kinase PERK and subsequent expression of CHOP was pivotal for the onset of drug/TNF-induced apoptosis. While inhibition of the Nrf2-dependent adaptive oxidative stress response enhanced the drug/TNF cytotoxicity, Nrf2 signaling did not affect CHOP expression. Both hepatotoxic drugs enhanced expression of the translational initiation factor EIF4A1, which was essential for CHOP expression and drug/TNF-mediated cell killing. Our data support a model in which enhanced drug-induced translation initiates PERK-mediated CHOP signaling in an EIF4A1 dependent manner, thereby sensitizing towards caspase-8-dependent TNF induced apoptosis. We sought to determine the effect of co-exposure of certain drugs associated with idiosyncratic DILI with the cytokine TNFalpha to mimic drug exposure with inflammation signaling in HepG2 cells Human hepatoma HepG2 cells were obtained from American Type Culture Collection (ATCC, Wesel, Germany), cultured in DMEM supplemented with 10% (v/v) FBS, 25 U/mL penicillin and 25 Âµg/mL streptomycin and used for experiments between passage 5 and 20.

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An in vitro coculture system of human peripheral blood mononuclear cells with hepatocellular carcinoma-derived cells for predicting drug-induced liver injury

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