Physical contact between mesenchymal stromal cells and hepatocytes via tunneling nanotubes favor the utilization of hepatocyte lipids

Background:

Human mesenchymal stromal cells (MSC) ameliorated hepatic lipid accumulation in a mouse model of methionine-choline-deficient diet-induced NASH. So far, the underlying mechanism is unclear. In vitro co-culture of MSC with isolated mouse hepatocytes reduced the lipid content in the hepatocytes demonstrating a direct impact of MSC on hepatocyte lipid metabolism. It was the goal to identify, whether this impact is direct or indirect via MSC-born paracrine mechanisms.

Methods:

Mono- and co-cultures of mouse hepatocytes and human bone marrow-derived MSC were grown in hepatocyte growth medium (HGM, control group), or in either steatosis-inducing methionine-choline-deficient (MCD) medium, or in HGM containing 0.5 mM palmitic acid (C16:0) (treatment groups). Changes in expression of genes involved in lipid metabolism were examined by semi-quantitative RT-PCR. To detect potential paracrine effects mediated by the MSC, conditioned media were collected from hepatocytes, MSC and co-cultures of both, and hepatocytes under control and treatment conditions were cultured for an additional 1 and 2 days in these conditioned media. Lipid droplets were stained with oil red O and quantified by image analysis. To identify, whether there was direct cell-cell contact via tunneling nanotubes (TNT), which might transfer organelles involved in lipid oxidation like mitochondria, human mitochondria in the MSC were pre-labeled with MitoTracker Red CMXRos prior to co-culture. After 1 to 3 days, cells were fixed and stained with phalloidin and DAPI to visualize F-actin and nuclei. Images were captured with an Axio Observer.Z1- inverted fluorescent microscope with ApoTome.2 (Zeiss).

Results:

In co-cultures of hepatocytes and MSC under control and treatment conditions, the expression of mRNAs involved in hepatocyte lipid utilization (peroxisome proliferator-activated receptor-α [PPARα]) and peroxisomal β- (acetyl-Coenzyme A acyltransferase 1 [Acaa1]) as well as microsomal ω-oxidation (cytochrome P450 2E1 [Cyp2E1]) were upregulated as compared to hepatocytes cultured without MSC. This might indicate that the presence of MSC enhanced the capacity of fatty acid oxidation in hepatocytes. Neither conditioned medium collected from co-cultures of MSC and hepatocytes, nor from MSC mono-cultures, diminished lipid accumulation in hepatocytes under both control and treatment conditions as compared with medium collected from hepatocyte monocultures. This indicated that probably no paracrine effects of MSC were involved in the attenuation of hepatocyte lipid storage. A known feature of MSC is their capability to transfer organelles to donor cells via TNT. Here, we observed long filopodium-like, actin-containing protrusions originating from the MSC and touching the hepatocytes or other MSC probably representing TNT. MSC-derived pre-labeled mitochondria were detected in the hepatocytes suggesting the donation of mitochondria from MSC to the hepatocytes. Currently, experiments are in progress to show, whether other organelles such as peroxisomes might also be transferred from the MSC to hepatocytes via TNT.

Conclusion:

MSC increased degradation of hepatocyte lipids, which may be achieved by the delivery of organelles involved in lipid degradation through TNT via physical cell-cell contacts.