Mitochondrial metabolism directs stemness and differentiation in P19 embryonal carcinoma stem cells

The relationship between mitochondrial metabolism and cell viability and differentiation in stem cells (SCs) remains poorly understood. In the present study, we compared mitochondrial physiology and metabolism between P19SCs before/after differentiation and present a unique fingerprint of the association between mitochondrial activity, cell differentiation and stemness. In comparison with their differentiated counterparts, pluripotency of P19SCs was correlated with a strong glycolytic profile and decreased mitochondrial biogenesis and complexity: round, low-polarized and inactive mitochondria with a closed permeability transition pore. This decreased mitochondrial capacity increased their resistance against dichloroacetate. Thus, stimulation of mitochondrial function by growing P19SCs in glutamine/pyruvate-containing medium reduced their glycolytic phenotype, induced loss of pluripotent potential, compromised differentiation and became P19SCs sensitive to dichloroacetate. Because of the central role of this type of SCs in teratocarcinoma development, our findings highlight the importance of mitochondrial metabolism in stemness, proliferation, differentiation and chemoresistance. In addition, the present work suggests the regulation of mitochondrial metabolism as a tool for inducing cell differentiation in stem line therapies.Embryonal carcinoma cells, including the P19 cell line, are pluripotent cancer stem cells (CSCs) derived from pluripotent germ cell tumors called teratocarcinomas. These have been described as the malignant counterparts of embryonic stem cells (ESCs) and are considered a good model to study stem cell (SC) differentiation. The P19 cell line can be maintained as undifferentiated cells (P19SCs) or differentiated (P19dCs) to any cell type of the three germ layers. Similar to ESCs, P19 cells differentiate with retinoic acid (RA) in a dose-dependent manner and depending on growth conditions.¹ Although differentiation generally yields a mixed population of differentiated cells, P19 cells grown in monolayer and treated with 1 μM RA primarily differentiate in endoderm or mesoderm, while retaining their immortality.^{2, 3}Although some therapeutic approaches for regenerative medicine and to targeting CSCs are based on differentiation⁴ and mitochondrial-targeted therapies,^{5, 6} very little is known about the role of mitochondrial metabolism in SC maintenance and differentiation.⁷ Several mitochondrial characteristics that distinguish transformed cells from healthy cells have been described,⁸ including increased mitochondrial transmembrane electric potential (Δψm), which may result from decreased mitochondrial ATP production under normoxia.⁹ Similarly, normal SCs primarily rely on glycolysis for energy supply, although the exact mechanism how this occurs in the presence of oxygen and the relationship between SC metabolism and cell fate control is not yet completely understood.¹⁰Given the mitochondrial involvement in stemness and differentiation,¹¹ one can ask whether manipulation of mitochondrial physiology results in an improvement of therapy efficacy. Therefore, characterizing the metabolic and mitochondrial profiles of both SCs and differentiated cells holds promise in order to explain the resistance of cancer cells expressing an embryonic signature to mitochondrial-targeted therapies. In the present work, we have two tandem hypotheses: (a) metabolic and mitochondrial remodeling accompanies P19SC differentiation and (b) P19SC differentiation results in a higher susceptibility to mitochondrial-directed therapies.