Institute of Liver Studies- Dhawan Lab
King´s College London
Investigation of the mitochondrial transfer from human mesenchymal stem/stromal cells to human primary hepatocytes through tunnelling nanotubes as a potential mechanism for cell survival and function enhancement during hepatocyte transplantation
Raquel Fernandez Dacosta1, Charlotte Lee1, Simon Walker1, Sharon Lehec1, Emer Fitzpatrick1, Anil Dhawan1, Celine Filippi1.
1Dhawan Lab, Hepatocyte Biology and Transplantation Group, Institute of Liver Studies, King’s College London, London, United Kingdom
Background: Microencapsulation of primary human hepatocytes followed by intraperitoneal transplantation has been clinically proven as a safe and successful therapy to bridge patients with acute liver failure to recovery or liver transplantation. Nevertheless, the cell isolation process together with subsequent cryopreservation and thawing, damages hepatocytes, resulting in lower viability, adhesion capacity, and function, thus reducing the efficacy of this therapy. We and others have shown that mesenchymal stem/stromal cells (MSC) enhance hepatocyte viability and metabolism, mainly through direct cell-cell interactions. However, the precise intercellular events leading to hepatocyte function improvements are not yet known. As hepatocyte viability and metabolism is controlled by mitochondrial function, we are focusing our studies on the potential effects of MSC on hepatocyte mitochondria. MSC have been reported to rescue different types of injured cells by transfer of functional mitochondria through actin-based nanostructures called tunneling nanotubes (TNT). Therefore, we hypothesize that MSC enhance hepatocyte survival and restore some of their functions through a TNT-based mitochondria transfer that replaces the mitochondrial activity lost during the isolation process.
Aims: (i) To assess if a TNT-based transfer of mitochondria occurs between MSC and human primary hepatocytes and (ii) to determine the ideal TNT-inhibition conditions, and allow the investigation of the role that TNT-based mitochondrial transfer have on hepatocyte viability and function improvement.
Methods: Cryopreserved hepatocytes were thawed and stained with CFSE cytoplasmic green fluorescent dye (2μM, 10min, 37°C), and MSC were stained with a mitochondria-targeted red fluorescent dye (0.1µM, 15min, 37°C). Mitochondrial transfer from MSC to hepatocytes was detected over 24h by co-localization of green and red fluorescent signals using Fluorescent Activated Cell Sorting (FACS), live-and fixed-cell microscopy. The presence of TNT in fixed 2, 4, 6 and 24h co-cultures of CFSE-labelled hepatocytes and mitochondria-labelled MSC was studied by fluorescent and phase contrast microscopy. Immunofluorescence was performed to identify actin and tubulin as potential TNT structural components. TNT-disruption was tested by dosing 2 actin polymerization inhibitors: 0.5µM- 5µM lantrunculin A (LatA) or 2µM- 10µM cytochalasyn D (CytD).
Results: Mitochondrial transfer from MSC to hepatocytes was progressively detected after 2h using live microscopy (n=3) and confirmed by FACS (n=3). There was a significant increase in the percentage of double stained hepatocytes in the co-cultures after 4 and 6h (21±6 and 27.6±10% respectively, p<0.05 vs monocultures). TNT structures containing red fluorescence-labelled mitochondria were observed between MSC in mono-cultures, as well as MSCs/HCs in co-cultures. Actin was identified as the main structural component of TNT, whose inhibition was achieved by adding 0.5µM LatA or 10µM Cyt D in the culture media.
Conclusion: Our preliminary studies show that MSC do transfer mitochondria to primary human hepatocytes through actin-based TNT. 0.5µM LatA or 10µM CytD provide efficient inhibition of the actin-based TNT formation, which will be used to assess the importance of this phenomenon on MSC-induced beneficial effects on hepatocyte viability and function.
RFD’s work is funded by the Ananda Trust through a Khosla PhD Studentship.
 Dhawan, A. et al., unpublished data
 Terry,C. et al.Cryopreservation of isolated human hepatocytes for transplantation: State of the art. Cryobiology 53, 149–159 (2006)
 Fitzpatrick, E. et al.(2013). Co-culture with mesenchymal stem cells results in improved viability and function of human hepatocytes. Cell Transplant. 1–29 (2013).
 Li, X. et al. Mitochondrial transfer of induced pluripotent stem cell-derived mesenchymal stem cells to airway epithelial cells attenuates cigarette smoke-induced damage. American Journal of Respiratory Cell and Molecular Biology, 51(3), 455–65 (2014)
17:30 - 18:30
|Young Investigators Best Mini-Orals Session||Investigation of the mitochondrial transfer from human mesenchymal stem/stromal cells to human primary hepatocytes through tunnelling nanotubes as a potential mechanism for cell survival and function enhancement during hepatocyte transplantation||Plenary Room 1|