Authors: Li-Pin Kao, Mark Prescott, Thien Tra and Ernst Wolvetang
Little or no mitochondrial biogenesis is thought to occur until implantation of the blastocyst. Nevertheless, human embryonic stem cells (hESCs) that are derived from the inner-cell mass of the blastocyst can be propagated indefinitely in vitro. Prolonged culture of hESCs, however, can result in the accumulation of mutations in the mitochondrial genome , with potentially serious implications for the use of hESC and their differentiated derivatives in regenerative medicine. The number and quality of mitochondria in a cell is determined by the balance between mitochondrial biogenesis and mitochondrial turnover. Mitophagy is a specific form of autophagy whereby the mitochondrion destined for degradation is sequestered within a vesicle of which the membrane is marked by the presence of Light Chain protein III (LC3). Here, we show that immediately prior to and during early spontaneous differentiation of hESC mitophagy is initiated, as indicated by increased levels of co-localisation of Mitotracker Red and LC3-GFP labelled vesicles. Furthermore, we show increased levels of mitophagy in hESCs treated with rapamycin (an mTOR inhibitor), 500ng/ml EtBr (a mitochondrial DNA damaging agent), CCCP (mitochondrial membrane depolarizer), or forced hESC differentiation. Our data demonstrate for the first time the occurrence of mitophagy in hESCs and strongly suggest that mitophagy is involved in the selective turnover and quality control of mitochondria in hESC during or prior to differentiation. A better understanding of the molecular mechanisms that control mitopahgy in hESCs will allow this process to be manipulated. Mitochondrial quality in long-term cultured hESC or iPS cells will benefit their use in future regenerative medicine approaches and is the future focus of this research.
Keywords: imaging: mitophagy, human embryonic, stem cells