Role Of Mitochondria In Mesenchymal Stem Cells

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1 Role Of Mitochondria In Mesenchymal Stem Cells Roman Eliseev, MD, PhD, Tamara Raymond, Regis O'Keefe, MD, PhD. University of Rochester, Rochester, NY, USA. Disclosures: R. Eliseev: None. T. Raymond: None. R. O'Keefe: None. Introduction: Mesenchymal stem cells (MSCs) are multipotent progenitors that reside in the marrow of long bones and give rise to osteoblasts, adipocytes and chondrocytes. In undifferentiated MSCs, glycolysis is the predominant mechanism of energy production while mitochondrial oxidative phosphorylation (OxPhos) is inactive. During the process of osteogenic differentiation mitochondria are activated via yet unknown mechanism and OxPhos becomes the major source of ATP. Our data and the literature indicate that the disruption of this bioenergetics switch leads to decreased osteogenicity of MSCs during aging and other pathologies and may contribute to osteoporosis, delayed fracture healing and osteoarthritis. Our goal is to determine the mechanism of activation of mitochondria during differentiation of MSCs and find ways to manipulate this mechanism for the purposes of therapy and prevention. Methods: MSC culture. We have used human MSCs (hmscs) and C3H10T1/2 cells (C3H). MSCs were also isolated from mouse bone marrow (BM MSC). Multilineage differentiation of MSCs. Cells were incubated in osteogenic or adipogenic media. Differentiation was confirmed with specific staining and by measuring expression of either osteogenic or adipogenic markers. Subsets of cells were differentiated in the media containing antimycin A (AntA). Cell bioenergetics. Mitochondrial function and glycolysis were assessed by simultaneously measuring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) respectively using the Seahorse apparatus. ATP was measured using CellTiter-Glo luminescence kit from Roche. Mitochondrial membrane potential (ΔΨm) was measured with TMRE fluorescent probe and FACS. Mitochondrial DNA (mtdna) was measured via qpcr and normalized to genomic DNA. Mitochondrial morphology was assessed via MitoTracker Green (MTG) and fluorescence imaging or via electron microscopy (EM). Gene expression analysis. RNA-seq technology was used to assay gene expression changes during hmsc differentiation. Results: Activation of mitochondria during osteogenic but not adipogenic differentiation of hmscs. Osteogenic or adipogenic differentiation of hmscs was confirmed by specific staining (Fig. 1A) and by expression of specific markers. During osteogenic differentiation of hmscs, mitochondrial function is activated as evident from the increase in OCR and ΔΨm while glycolysis (ECAR) does not change. During adipogenic differentiation of hmscs, mitochondria remain inactive while glycolysis is activated (Fig. 1B, C). Similar results were obtained with C3H cells and BM MSCs. Mitochondrial biogenesis assessed by mtdna content shows similar dynamics (Fig. 1D). Mitochondria in differentiated cells become elongated (Fig. 1E) which usually indicates higher functionality. Presence

2 of a mitochondrial inhibitor, AntA, disrupts osteogenic differentiation as evident from the decreased alkaline phosphatase (ALP) staining but does not affect adipogenic differentiation as evident from the unchanged Oil Red staining (Fig. 2). These data confirm that mitochondria are activated during osteogenic differentiation of MSCs; and such activation is required for MSC osteogenic differentiation while mitochondrial activation is not required for adipogenic differentiation. Metabolic gene signature changes during differentiation of MSCs. To determine possible signaling pathways involved in regulation of MSC metabolism during differentiation, we have performed RNAseq analysis of gene expression in undifferentiated and differentiated hmscs and found that genes associated with glycolysis are down- or up-regulated during osteogenic or adipogenic differentiation respectively (Fig. 3). These genes are under the transcriptional control of various signaling systems, such as Akt and Hif. Contribution of these pathways will be further evaluated and discussed during our presentation. MSC mitochondrial function is disrupted in aging. We isolated BM MSCs from young and aged mice and measured mitochondrial function and osteogenic potential of these cells. Our data indicate both mitochondrial dysfunction and decreased osteogenicity in aged BM MSCs (Fig. 4). Discussion: The above data present evidence of activation of different metabolic pathways during osteogenic or adipogenic differentiation of MSCs: mitochondrial OxPhos or glycolysis respectively. Coordinated changes in expression of genes regulating glycolysis during MSC differentiation likely indicate transcriptional regulation by various signaling systems, such as Akt and Hif. Our data also suggest that active mitochondria are required for MSC osteogenicity. Any changes in regulation of MSC energy metabolism will potentially affect cell fate and cell ability to undergo differentiation. Our data and the literature indicate that MSC mitochondrial function is compromised during aging which may be one of the key reasons for their decreased osteogenicity leading to osteoporosis. Significance: Our data suggest that there is a strong connection between the mitochondrial function and MSC cell fate and that cell bioenergetics may be a new target to increase osteogenic potential of MSCs and improve bone health. Acknowledgments: References:

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5 ORS 2014 Annual Meeting Poster No: 0591

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