Mitochondrial transfer from muscle-derived mesenchymal stem cells to restore keratinocytes metabolism in an vitro laminitis model
Serteyn D1, Storms N*2, Ceusters J1, Sandersen C1, Franck T1
1University of Liege/Equine clinic, Liege, Belgium, 2Via Nova, Bree, Belgium.
Objectives:
The activation of polymorphonuclear neutrophils in lamellar tissue and skin was demonstrated in experimental models of laminitis and a mitochondrial dysfunction may lead to the failure of the dermal-epidermal interface.
Three main objectives were addressed:
1/ to design an in vitro laminitis model using keratinocytes submitted to anoxia-reoxygenation (A/R) combined with activated neutrophils;
2/ to evaluate a possible curative role of muscle-derived mesenchymal stem cells (mdMSCs) assessed by a recovery of cellular metabolism,
3/ to show a transfer of mitochondria from mdMSCs to keratinocytes.
Methods:
The laminitis stress model consists of a continuous cell line of keratinocytes (HaCaT) exposed to anoxia for 48 h in the presence of a supernatant of activated neutrophils. Reoxygenation was performed by adding new media complemented with or without mdMSCs during 24h. Cell metabolism was evaluated using cell proliferation assay (MTS). Mitochondrial transfer from mdMSC to HaCaT was evaluated by live imaging assay (Nanolive, CXA), using PK MitoRed as fluorescent tracer. Statistical analysis was performed by a Mann-Whitney non-parametric test (n=5; P<0.01).
Results:
HaCaT cells exposed to anoxia-reoxygenation, in the presence of activated neutrophil supernatant, exhibited a strong significant decrease of their metabolic activity.
MdMSCs co-cultured with HaCaT showed a significant protective effect against stress conditions.
HaCaT cells showed a red staining likely as a result of exchange with mdMSCs, previously stained by pk-Mito Red.
Conclusions:
The anti-inflammatory potential of mdMSCs and their ability to restore the metabolism of keratinocytes by mitochondrial transfer could constitute a promising future for cell therapy in laminitis.