João Facucho-Oliveira

BACKGROUND: Mitochondrial DNA (mtDNA) encodes key proteins associated with the process of oxidative phosphorylation. Defects to mtDNA cause severe disease phenotypes that can affect offspring survival. The aim of this review is to identify how mtDNA is replicated as it transits from the fertilized oocyte into the preimplantation embryo, the fetus and offspring. Approaches for deriving offspring and embryonic stem cells (ESCs) are analysed to determine their potential application for the prevention and treatment of mtDNA disease. METHODS: The scientific literature was investigated to determine how mtDNA is transmitted, replicated and segregated during pluripotency, differentiation and development. It was also probed to understand how the mtDNA nucleoid is regulated in somatic cells. RESULTS: mtDNA replication is strictly down-regulated from the fertilized oocyte through the preimplantation embryo. At the blastocyst stage, the onset of mtDNA replication is specific to the trophectodermal cells. The inner cell mass cells restrict mtDNA replication until they receive the key signals to commit to specific cell types. However, it is necessary to determine whether somatic cells reprogrammed through somatic cell nuclear transfer, induced pluripotency or fusion to an ESC are able to regulate mtDNA replication so that they can be used for patient-specific cell therapies and to model disease. CONCLUSIONS: Prevention of the transmission of mtDNA disease from one generation to the next is still restricted by our lack of understanding as to how to ensure that a donor karyoplast transferred to an enucleated oocyte is free of accompanying mutant mtDNA. Techniques still need to be developed if stem cells are to be used to treat mtDNA disease in those patients already suffering from the phenotype.

Oxidative phosphorylation (OXPHOS), the intracellular process that generates the majority of the ATP of a cell through the electron-transfer chain, is highly dependent on proteins encoded by the mitochondrial genome (mtDNA). MtDNA replication is regulated by the nuclear-encoded mitochondrial transcription factor A (TFAM) and the mitochondrial-specific DNA polymerase gamma, which consists of a catalytic (POLG) and an accessory (POLG2) subunit. Differentiation of pluripotent embryonic stem cells (ESCs) into specific cell types requires expansion of discrete populations of mitochondria and mtDNA replication to meet the specific metabolic requirements of the cell. We determined by real-time PCR that expression of pluripotent markers is reduced before the upregulation of Polg, Polg2 and Tfam in spontaneously differentiating R1 murine (m)ESCs, along with transient increases in mtDNA copy number. In D3 mESCs, the initial transient increase did not take place. However, precursors of neuronal and cardiomyocyte differentiation were positive for both POLG and TFAM. Similar-stage ESCs also showed active mtDNA replication, identified by 5-bromo-2'-deoxy-uridine labelling, as mtDNA copy number increased. Retinoic-acid-induced differentiation resulted in more consistent patterns of replication and upregulation of Polg, Polg2 and Tfam, whereas siRNA knockdown demonstrated that steady-state expression of POLG is essential for maintaining pluripotency.

As human embryonic stem cells (hESCs) undergo differentiation, they express genes characteristic of the lineage for which they are destined. However, fully differentiated individual cell types can be characterized by the number of mitochondria they possess and the copies of the mitochondrial genome per mitochondrion. These characteristics are indicative of a specific cell's requirement for adenosine triphosphate (ATP) and therefore cellular viability and function. Consequently, failure for an ESC to possess the full complement of mitochondria and mitochondrial DNA (mtDNA) could limit its final commitment to a particular fate. We describe a series of protocols that analyze the process of cellular mitochondrial and mtDNA differentiation during hESC differentiation. In addition, mtDNA transcription and replication are key events in cellular differentiation that require interaction between the nucleus and the mitochondrion. To this extent, we describe a series of protocols that analyze the initiation of these key events as hESCs progress from their undifferentiated state to the fully committed cell. Last, we describe real-time polymerase chain reaction protocols that allow both the identification of mtDNA copy number and determine whether mtDNA copy is uniform (homoplasmy) in its transmission or heterogeneous (heteroplasmy).