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GNPS - C.acnes and S.aureus BHI coculture Pos_XAD

ABSTRACT: Monoculture and coculture of Cutibacterium acnes KPA171202 and Staphylococcus aureus JE2 on BHI liquid medium. Anaerobic culture conditions for 3-5 days. Cultures were centrifuged and supernatant removed. Extraction performed by addition of 100uL XAD16 slurry to the supernatant and incubation under shaking (2000 rpm, 4 h). XAD16 slurry removed and extracted with 100% MeOH. Extract was freeze-dried and resuspended for LC-MS/MS performed in an UltiMate 3000 UPLC system (Thermo Scientific) using a Kinetex 1.7 mm C18 reversed phase UHPLC column (50 X 2.1 mm) and Maxis Q-TOF mass spectrometer (Bruker Daltonics) equipped with ESI source.

INSTRUMENT(S): maXis

ORGANISM(S): Staphylococcus Aureus Je2 Cutibacterium Acnes Kpa171202

SUBMITTER: Michael A. Fischbach Katherine P. Lemon

PROVIDER: MSV000084701 | GNPS | Mon Dec 16 16:45:00 GMT 2019

REPOSITORIES: GNPS

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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.

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GNPS - C.acnes and S.aureus BHI coculture Pos_XAD

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