Mesenchymal Stem Cells Shift Mitochondrial Dynamics and Enhance Oxidative Phosphorylation in Recipient Cells

Abstract

Mesenchymal stem cells (MSCs) are the most commonly used cells in tissue engineering and regenerative medicine. MSCs can promote host tissue repair through several different mechanisms including donor cell engraftment, release of cell signaling factors, and the transfer of healthy organelles to the host. In the present study, we examine the specific impacts of MSCs on mitochondrial morphology and function in host tissues. Employing in vitro cell culture of inherited mitochondrial disease and an in vivo animal experimental model of low-grade inflammation (high fat feeding), we show human-derived MSCs to alter mitochondrial function. MSC co-culture with skin fibroblasts from mitochondrial disease patients rescued aberrant mitochondrial morphology from a fission state to a more fused appearance indicating an effect of MSC co-culture on host cell mitochondrial network formation. In vivo experiments confirmed mitochondrial abundance and mitochondrial oxygen consumption rates were elevated in host tissues following MSC treatment. Furthermore, microarray profiling identified 226 genes with differential expression in the liver of animals treated with MSC, with cellular signaling, and actin cytoskeleton regulation as key upregulated processes. Collectively, our data indicate that MSC therapy rescues impaired mitochondrial morphology, enhances host metabolic capacity, and induces widespread host gene shifting. These results highlight the potential of MSCs to modulate mitochondria in both inherited and pathological disease states.

Keywords: hepatic; high-fat diet; metabolic inflammation; metabolism; mitochondrial regulation.

Figure 1

Mitochondrial morphology and mtDNA of fixed fibroblasts and live fibroblasts co-cultured with MSCs. (A) Representative images demonstrating assessment of mitochondrial morphology and mtDNA of fixed fibroblasts from controls and patients with clinically diagnosed mitochondrial disease. Images were collected using immunofluorescence against TOMM20 (mitochondria) and DNA (DNA). (B) Representative images demonstrating assessment of mitochondrial morphology and mtDNA of live fibroblasts (from controls and patients with a clinically diagnosed mitochondrial disease) co-cultured with MSCs. Images were collected following fluorescent labeling of fibroblasts with MitoTracker Deep Red (mitochondria) and PicoGreen (DNA). MSCs were separately fluorescently labeled with MitoTracker Red (mitochondria) prior to co-culture. Images from both panels were collected using confocal microscopy.

Figure 2

Quantification of mitochondrial morphology scores between patient and control cells. Skin fibroblasts from healthy controls (A) and patients with a clinically diagnosed mitochondrial disease (B) were manually classified into one of five mitochondrial morphology categories. Category 1 (red) corresponds to a fully fragmented morphology and category 5 (green) corresponds to a fusion morphology. Results from baseline were compared following contact co-culture with mesenchymal stem cells (MSCs). A minimum of 150 cells were quantified per metric and per condition from 2 to 3 independent experiments. All data are mean ± SEM with n = 4 for each group. ^*Denotes statistical significance at p < 0.05.

Figure 3

Quantification of mitochondrial mtDNA nucleoid clusters between patient and control cells. Skin fibroblasts from healthy controls (A) and patients with a clinically diagnosed mitochondrial disease (B) were manually classified into one of twocategories for mtDNA structure. Results from baseline were compared following contact co-culture with mesenchymal stem cells (MSCs). A minimum of 150 cells were quantified per metric and per condition from 2 to 3 independent experiments. All data are mean ± SEM with n = 4 for each group. ^*Denotes statistical significance at p < 0.05.

Figure 4

MSC detection using sequence-specific qualitative PCR. Liver tissue homogenates were used to detect mouse and human genomic DNA 24 h following control (saline) or MSC therapy into C57BL/6 mice. (A) Mouse—forward and common reverse PCR primers for prostaglandin E receptor 2 (PTGER2) were amplified to detect the presence of mouse-specific genetic material. (B) Human—forward and common reverse PCR primers for a non-homologous region of PTGER2 were amplified to detect the presence of human-specific genetic material. Human control samples were isolated from a cultured human cell line. NTC, no template control.

Figure 5

Detection of reactive oxygen species production and superoxide dismutase enzyme activity from liver homogenates. Following control (saline) or MSC therapy, liver homogenates were used to quantify the generation of reactive oxygen species (ROS) and the free radical scavenging enzyme superoxide dismutase (SOD). (A) Relative rates of H₂O₂ production as a function of ROS generation. Mitochondria isolated from liver tissue were stimulated in the presence of ADP under a variety of conditions including: glutamate and malate as substrates (G & M; complex I), rotenone as an inhibitor (complex I), and antimycin as an inhibitor (complex III). (B) SOD enzyme activity measured from liver homogenates. SOD activity is stratified into manganese (Mn) or zinc and copper (Zn and Cu) fractions, with total representing both portions combined. SOD data are normalized to mg of liver protein and ROS data are normalized to mg of mitochondrial protein. All data are mean ± SEM with n = 8 for both groups. ^*Denotes statistical significance at p < 0.05.

Figure 6

Heat map of functional categories of closely related genes with differential gene expression following MSC administration. Enriched gene ontology terms of MSC treated liver tissues compared to control (saline) liver tissues using z-scores computed from microarray gene expression profiling. Orange: upregulated gene expression (z-score >0), colorless: equal gene expression (z-score = 0), blue: downregulated gene expression (z-score <0). Data were generated from microarray gene expression data with each square corresponding to a single functional category. n = 7 tissues per condition. p < 0.001 for each (MSC treated vs. control).

Figure 7

Heat map of lipid liver metabolites/indices, measured by nuclear magnetic resonance. Yellow, High concentrations; Black, Medium concentrations; Blue, Low concentrations. SI, Saturation Index; UI, Unsaturation Index; PUI, Polyunsaturation Index; PUFA/MUFA, Polyunsaturated Fatty Acids/Monounsaturated Fatty Acids. High-fat fed animals were separated into two groups for analysis: MSC treated (HFM) and saline control (HFS) ^*denotes statistical significance at p < 0.05.