Mesenchymal Stromal Cell-Derived Factors Promote Tissue Repair in a Small-for-Size Ischemic Liver Model but Do Not Protect against Early Effects of Ischemia and Reperfusion Injury

Abstract

Loss of liver mass and ischemia/reperfusion injury (IRI) are major contributors to postresectional liver failure and small-for-size syndrome. Mesenchymal stromal cell- (MSC-) secreted factors are described to stimulate regeneration after partial hepatectomy. This study investigates if liver-derived MSC-secreted factors also promote liver regeneration after resection in the presence of IRI. C57BL/6 mice underwent IRI of 70% of their liver mass, alone or combined with 50% partial hepatectomy (PH). Mice were treated with MSC-conditioned medium (MSC-CM) or unconditioned medium (UM) and sacrificed after 6 or 24 hours (IRI group) or after 48 hours (IRI + PH group). Blood and liver tissue were analyzed for tissue injury, hepatocyte proliferation, and gene expression. In the IRI alone model, serum ALT and AST levels, hepatic tissue damage, and inflammatory cytokine gene expression showed no significant differences between both treatment groups. In the IRI + PH model, significant reduction in hepatic tissue damage as well as a significant increase in hepatocyte proliferation was observed after MSC-CM treatment.

Conclusion: Mesenchymal stromal cell-derived factors promote tissue regeneration of small-for-size livers exposed to ischemic conditions but do not protect against early ischemia and reperfusion injury itself. MSC-derived factors therefore represent a promising treatment strategy for small-for-size syndrome and postresectional liver failure.

Figure 1

Effects of MSC-CM on body and liver weight after IRI + PH. (a) Body weight change from surgery to harvest; (b) harvest liver weight versus initial liver weight; (c) harvest liver weight to body weight ratio.

Figure 2

Effects of MSC-CM on hepatic tissue injury. H&E stained liver tissue sections were classified based on the percentage of damaged tissue: no injury, 0–25%, 25–50%, 50–75%, or >75% of liver tissue affected. This figure shows the percentage of animals with a certain injury score (a) 6 hours after IRI, (b) 24 hours after IRI, and (c) 48 hours after IRI + PH with representative pictures showing approximately 25% necrotic liver tissue in the UM group compared to no clear necrotic tissue in the MSC-CM group.

Figure 3

Effects of MSC-CM on serum injury markers. Serum ALT levels at (a) 6 hours after IRI, (b) 24 hours after IRI, and (c) 48 hours after IRI + PH. Serum AST levels at (d) 6 hours after IRI, (e) 24 hours after IRI, and (f) 48 hours after IRI + PH.

Figure 4

Effects of MSC-CM on hepatocyte proliferation. Livers were processed for immunohistochemistry on BrdU to quantify hepatocyte proliferation. This figure shows the percentage of BrdU positive hepatocytes (a) 6 hours after IRI, (b) 24 hours after IRI, and (c) 48 hours after IRI + PH; ^∗ p ≤ 0.05.

Figure 5

Effects of MSC-CM on hepatic gene expression. Hepatic gene expression levels were determined by quantitative RT-PCR and normalized against TBP. Expression levels of inflammation related genes at (a) 6 hours after IRI, (b) 24 hours after IRI, and (c) 48 hours after IRI + PH; (d) expression levels of cell cycle related genes at 48 hours after IRI + PH.

Figure 6

Functional analysis of cellular processes altered by L-MSC-conditioned medium. Proteins present in serum-free MSC-CM were analyzed using Mass Spectrometry. Out of 2060 proteins identified, 861 related to functional networks in Ingenuity Pathway Analysis. Top cellular processes involved proliferation of hepatocytes and nonparenchymal cells.