Project description:Exosomes transport biologically active cargo (e.g., proteins and microRNA) between cells, including many of the paracrine factors that mediate the beneficial effects associated with stem-cell therapy. Stem cell derived exosomes, in particular mesenchymal stem cells (MSCs), have been shown previously to largely replicate the therapeutic activity associated with the cells themselves, which suggests that exosomes may be a useful cell-free alternative for the treatment of cardiovascular disorders. However, the mechanisms that govern how exosomes home to damaged cells and tissues or the uptake and distribution of exosomal cargo are poorly characterized, because techniques for distinguishing between exosomal proteins and proteins in the targeted tissues are lacking. Here, we report the development of an in-vivo model that enabled the visualization, tracking, and quantification of proteins from systemically administered MSC exosomes. The model uses bioorthogonal chemistry and cell-selective metabolic labeling to incorporate the noncanonical amino acid azidonorleucine (ANL) into the MSC proteome. ANL incorporation is facilitated via expression of a mutant (L274G) methionyl-tRNA-synthetase (MetRS*) and subsequent incubation with ANL-supplemented media; after which ANL can be covalently linked to alkyne-conjugated reagents (e.g., dyes, resins) via click chemistry. Our results demonstrate that when the exosomes produced by ANL-treated, MetRS*-expressing MSCs were systemically administered to mice, the ANL-labeled exosomal proteins could be accurately and reliably identified, isolated, and quantified from a variety of mouse organs, and that myocardial infarction (MI) both increased the abundance of exosomal proteins and redistributed a number of them from the membrane fraction of intact hearts to the cytosol of cells in infarcted hearts. Additionally, we found that Desmoglein-1c is enriched in MSC exosomes and taken up by ischemic myocardium. Collectively, our results indicate that this newly developed bioorthogonal system can provide crucial insights into exosome homing, as well as the uptake and biodistribution of exosomal proteins.

Project description:Yorkshire pigs were divergently selected for residual feed intake (RFI) for 8 generations. Pigs with low RFI have reduced feed intake. Based on the resource allocation theory, we hypothesize that these low RFI pigs might have compromised immune response to immune stimulation. To test this hypothesis, twenty eight gilts of initial body weight (BW) of 63 4 kg from pigs selected for low RFI and high RFI were randomly selected for the ISU RFI selection project and utilized in two replicates (14 pigs/replicate) for this study. All pigs were housed in individual metabolism pens, had free access to water and were fed a corn-soy-based diet twice daily, and feed restricted (1.5 kg/day) as previously described. After a 9-day adaptation period pigs were randomly assigned within line to either a control (n = 6, three pigs per line) or lipopolysaccharide (LPS) challenge (n = 8, 4 pigs per line) group. Pigs in the challenge group were then repeatedly challenged with an intramuscular injection of 30, 36, 39, and 42 g/kg BW of LPS from E. coli O5:B5 (sigma-Aldrich, St. Louis, MO) dissolved in saline solution at time points 0, 48, 96, and 144 hours post initial injection (hpi) to compensate for LPS tolerance after initial injection, respectively, while animals in the control group were injected with an equivalent volume of saline solution at the equivalent time points. Rectal temperatures of individual pigs were measured immediately before the initial injection (serving as baseline, called 0 hpi for convenience), and 2, 4, 6, and 24 hpi, and 0, 4, 24 hours after each subsequent injection for all pigs. Blood samples were collected from the jugular vein into TempusTM Blood RNA tubes (Life Technologies, Grand Island, NY) for long-term storage at -80 C, EDTA tubes (BD, Franklin Lakes, NJ) for CBC tests and serum tubes for cytokine assays at 0, 2, 6, 24 and 168 hpi. At 168 hpi, all pigs were euthanized via barbiturate overdose, exsanguinated and tissue samples including the longissimus dorsi muscle, liver, spleen, and ileum were isolated, cleaned an frozen for later use. This study only focused on samples and data collected for the first 24 hours. Total RNA was extracted from the 32 blood samples of the treatment group in Replicate 2, collected at 0, 2, 6, and 24 hpi, by using preserved blood RNA purification kit I (Norgen Biotek Corp, Thorold, Ontario) per manufacturers instruction. On-column DNA digestion was performed as described using DNase I (Qiagen, Valencia, CA). Globin transcripts (HBB and HBA) were depleted by following an RNase H-mediated method. The quantity and integrity of the RNA were monitored by using Nanodrop 2000 (Thermo Scientific, Waltham, MA) and Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) before and after globin depletion. The efficiency of globin depletion of each sample was checked by conventional RT-qPCR with -actin and GAPDH as the internal controls. Globin depletion efficiencies for all RNA samples were above 85%.

			Action	DRS
	20220725_sampleDescription.xlsx	Xlsx
	AcquisitionMethodComparison_Fig1_DIA_raw.zip	Other
	AcquisitionMethodComparison_Fig1_DIA_results.zip	Other
	AcquisitionMethodComparison_Fig1_offlineFractionation_raw.zip	Other
	AcquisitionMethodComparison_Fig1_offlineFractionation_results.zip	Other

Dataset Information

Cell-selective proteomics segregates pancreatic cancer subtypes by extracellular proteins in tumors and circulation

Dataset's files

Publications

Proteomics reveals distinct mechanisms regulating the release of cytokines and alarmins during pyroptosis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets