Silencing ROCK Maintains Pluripotency but Modifies Metabolism

Stem cells give rise to the different cells found in tissues. One such type is called pluripotent stem cells (PSCs), which have the capacity to differentiate into almost any cell in the body including heart, muscle or nerve. The most well-known source of PSCs is the embryo and its embryonic stem cells (ESCs). Under the appropriate conditions, ESCs, which have been isolated from the inner cell mass of the blastocyst, can be used to generate almost any cell type. Once the embryonic development stage is completed, the stem cells are no longer pluripotent and can only differentiate into specific types of cells.

Potential Therapeutic Uses for Pluripotent Stem Cells

Pluripotent stem cells are thought to be the driving force of regenerative medicine because of their role in the replacement of damaged or diseased cells. They can be used to treat a wide array of diseases such as autoimmune diseases, burns, cardiovascular disorders and diabetes. However, controversy surrounds the use of ESCs due to the destruction or manipulation of the embryo. In 2012, Professor Shinya Yamanaka and Sir John Gurdon were awarded the 2012 Nobel Prize for the discovery that mature cells can be reverted back to a pluripotent status. These cells were referred to as induced pluripotent stem cells (iPSCs), and can be engineered following the introduction of four specific genes (c-MYC, KLF4, OCT3/4, and SOX2) and subsequent selection for FBX15 expression (Takahashi et al., 2006). This breakthrough discovery for generating iPSCs allows one to bypass the need for embryos. In addition, it eliminates the risk of immune rejection after transplantation since individuals can have their own pluripotent stem cell line administered as a therapeutic.

Inhibition of ROCK by Y-27632

Rho-kinase (ROCK) belongs to the AGC family (protein kinase A, protein kinase G and protein kinase C) of serine/threonine kinases and is an effector of the small GTPase, RhoA. The downstream substrates of ROCK modulate actin cytoskeleton organization, stress fiber formation and smooth muscle cell contraction. Y-27632 is a highly potent, cell permeable, selective, and competitive inhibitor of ROCK1 and ROCK2 (IC₅₀=800nM). It acts as a potent inhibitor of agonist-induced Ca²⁺-sensitization of myosin phosphorylation and smooth muscle contractions. It blocks cell spreading and suppresses RhoA-induced formation of stress fibers in hepatic stellate cells. Y-27632 significantly reduces the increase of inflammatory cytokines after reperfusion, preventing the development of acute renal failure. There are numerous application for this compound including: cardioprotective effects, suppression of tumor cell invasion, inhibition of superoxide production, mimicking the effects of β-agonists on human cells, and an antinociceptive effect. There has been a growing interest in Y-27632 for use in stem cell self-renewal and reprogramming. It is also known to increase the survival rate of human embryonic stem cells undergoing cryopreservation.

Effects of Y-27632 on Metabolism

Although ROCK inhibition by Y-27632 is known to maintain the stem cell phenotype, its effect on metabolism remains unknown. Following treatment with Y-27632, Dr. Vernardis and colleagues from Imperial College London monitored both phenotype and metabolism of pluripotent stem cells, namely ESCs and iPSCs. Conventional markers of pluripotency (SSEA3 and TRA-1-81) and stemness (Nanog, Oct4, and Sox2) were assessed by real time PCR, flow cytometry, and immunocytochemistry. Expression of these markers remained unaltered at both mRNA and protein levels in both ESCs and iPSCs for up to 48 hours following ROCK inhibition by Y-27632, thereby confirming pluripotency. Metabolomic analysis of both ESCs and iPSCs was conducted using gas chromatography-mass spectrometry (GC-MS). Following exposure to Y-27632 for 48 hours, the authors of this study observed a significant reduction in glycolysis, glutaminolysis, the citric acid cycle, and the amino acid pools. Both ESCs and iPSCs adapted to their new environment by reducing their metabolism and this adaptation correlated with the decrease in ROS/RNS levels and the down-regulation of a key metabolic controller called mTORC1, but was independent of caspase-3 or p53 expression. Altogether, these results suggest the importance of metabolomics in characterizing pluripotent stem cells and developing new culture methods in order to improve efficiency, productivity, robustness, and overall stem cell bioprocessing.

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