The Analysis of Mitochondria and Mitochondrial DNA in Human Embryonic Stem Cells

Abstract

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).

Downregulation of Multiple Stress Defense Mechanisms During Differentiation of Human Embryonic Stem Cells

“…There are low numbers of mitochondria in liver and hematopoietic stem cells, which also show low oxygen consumption [12,13]. Human ESC (hESC) also contain very few mitochondria [14]. During the differentiation process, mitochondrial proliferation and transcription increases significantly, which suggests that mitochondrial activity might play an important role in the balance between self-renewal and differentiation in ESC [15].…”

A Mechanism by Which Propofol Induces Cytotoxicity

“…Mitochondrial membrane potential (∆Ψm) was assessed using 5,59,6,69-tetrachloro-1,19,3,39-tetraethylbenzimidazolocarbocyan ine iodide (JC-1; Molecular Probes, OR, USA) [13,14]. Cells were loaded for 30 minutes with 1 µM JC-1 at 37°C.…”

Mitochondrial Transcription Factor A's role in heart failure.

“…Additionally, increased TFAM expression reduced neuronal pathologies and increased patient survival rate, deeming it clinically beneficial (Correia et al 2011). A significant component of mitochondrial transcription, the mitochondrial RNA polymerase, requires TFAM and TFBM2 for efficient initiation (Morozov et al 2014;St John et al 2006).…”