Dataset Information

Human liver stem cell-derived extracellular vesicles reduce injury in a model of normothermic machine perfusion of rat livers previously exposed to a prolonged warm ischemia.

ABSTRACT: Livers from donors after circulatory death (DCD) are a promising option to increase the donor pool, but their use is associated with higher complication rate and inferior graft survival. Normothermic machine perfusion (NMP) keeps the graft at 37°C, providing nutrients and oxygen supply. Human liver stem cell-derived extracellular vesicles (HLSC-EVs) are able to reduce liver injury and promote regeneration. We investigated the efficacy of a reconditioning strategy with HLSC-EVs in an experimental model of NMP. Following total hepatectomy, rat livers were divided into 4 groups: (i) healthy livers, (ii) warm ischemic livers (60 min of warm ischemia), (iii) warm ischemic livers treated with 5 × 10⁸ HLSC-EVs/g-liver, and (iv) warm ischemic livers treated with a 25 × 10⁸ HLSC-EVs/g-liver. NMP lasted 6 h and HLSC-EVs (Unicyte AG, Germany) were administered within the first 15 min. Compared to controls, HLSC-EV treatment significantly reduced transaminases release. Moreover, HLSC-EVs enhanced liver metabolism by promoting phosphate utilization and pH self-regulation. As compared to controls, the higher dose of HLSC-EV was associated with significantly higher bile production and lower intrahepatic resistance. Histologically, this group showed reduced necrosis and enhanced proliferation. In conclusion, HLSC-EV treatment during NMP was feasible and effective in reducing injury in a DCD model with prolonged warm ischemia.

SUBMITTER: De Stefano N

PROVIDER: S-EPMC9291857 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Human liver stem cell-derived extracellular vesicles reduce injury in a model of normothermic machine perfusion of rat livers previously exposed to a prolonged warm ischemia.

De Stefano Nicola N Navarro-Tableros Victor V Roggio Dorotea D Calleri Alberto A Rigo Federica F David Ezio E Gambella Alessandro A Bassino Daniela D Amoroso Antonio A Patrono Damiano D Camussi Giovanni G Romagnoli Renato R

Transplant international : official journal of the European Society for Organ Transplantation 20210901 9

Livers from donors after circulatory death (DCD) are a promising option to increase the donor pool, but their use is associated with higher complication rate and inferior graft survival. Normothermic machine perfusion (NMP) keeps the graft at 37°C, providing nutrients and oxygen supply. Human liver stem cell-derived extracellular vesicles (HLSC-EVs) are able to reduce liver injury and promote regeneration. We investigated the efficacy of a reconditioning strategy with HLSC-EVs in an experimental ...[more]

PMID: 34448268

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Project description:There is a ever widening gap between the availability of donor livers suitable for transplantation and liver transplant demand of a growing waiting list. To expand the pool of liver grafts, donation after circulatory death is increasingly being used. Livers procured after circulatory death are exposed to a harmful period of warm Ischemia (WI) during the donation process, reducing post-transplant results significantly. Additionally, the conventional technique of static cold storage does not protect grafts that suffered WI. New dynamic preservation strategies by means of Normothermic Machine Perfusion (NMP) showed enhanced preservation of these type of grafts, but it is unknown how NMP influences liver cell biology during perfusion. Hepatocytes and cholnagiocytes have been shown to release Extracellular Vesicles (EVs) in the systemic circulation and bile, respectively. We hypothesized that EVs are released during liver NMP as well, in perfusate and bile. EVs have been separated from perfusate and bile during NMP of porcine livers, and the RNA cargo of the EVs has been sequenced to gain insights on liver cell biology during machine perfusion. Pigs were randomly allocated to undergo liver procurement either immediately after the start of surgery (no-WI, n=5) or after a period of WI of 60-minutes (clamping of thoracic aorta; WI-60, n=5). All livers (n=10) were cooled down and flushed out with ice-cold preservation solution, according to standard clinical practice. Porcine blood was collected and washed with a cell saver to obtain packed red blood cells for NMP. The hepatic artery, portal vein, and inferior vena cava were cannulated and after a period of about 2-hours of cold storage, NMP was started. All liver underwent NMP for 6-hours. Packed red blood-based perfusate samples were taken at 1, 3, and 6h of NMP; whereas, the bile produced the first 3-hours and the last 3-hours was pooled in 2 samples. Perfusate and bile samples were processed immediately for EVs separation with charge-based precipitation in a mixture of protamine and polyethylene glycol. The total RNA was extracted from EVs. Libraries of mRNA and of small RNA were prepared and analyzed for quality and size range, and sequenced on an Illumina HiSeq4000 instrument. Count-based differential expression analysis was done with R-based Bioconductor package DESeq2, and the following comparisons were performed: - Unpaired (between groups) comparison - perfusate samples WI-60 vs. no-WI - bile samples WI-60 vs. no-WI - Paired (withing group) comparison - no-WI - perfusate sample 1h vs. perfusate sample 3h - perfusate sample 1-3h vs. perfusate sample 6h - bile sample 1-3h vs. bile sample 3-6h - perfusate samples vs. bile samples - WI-60 - bile sample 1-3h vs. bile sample 3-6h - perfusate samples vs. bile samples Results were adjusted for multiple testing with the Benjamini-Hochberg procedure, to control for false discovery rate. Additionally, the function of the transcripts identified in the EVs of both groups and of the differentially expressed transcripts was investigated with gene ontology enrichment analysis for the domains \\"biological process\\" and \\"pathways\\".

Dataset Information

Human liver stem cell-derived extracellular vesicles reduce injury in a model of normothermic machine perfusion of rat livers previously exposed to a prolonged warm ischemia.

Publications

Human liver stem cell-derived extracellular vesicles reduce injury in a model of normothermic machine perfusion of rat livers previously exposed to a prolonged warm ischemia.

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