Online Purchasing Account You are logged on as Guest. LoginRegister a New AccountShopping cart (Empty)
United States 

Silencing ROCK Maintains Pluripotency but Modifies Metabolism


Silenced ROCKer
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.

Enzo Life Sciences offers a comprehensive portfolio for phenotyping and studying stem cells including our ROS-ID® ROS/RNS detection kit and MITO-ID® Extracellular pH sensor kit. In addition, Enzo also offers antibodies, ELISA kits, recombinant proteins, and small molecules (e.g. Y-27632), some of which are listed below:

Never miss a new TechNote!

Receive our TechNotes as soon as they are published.


Follow Us!

 

Share this TechNote

 
comments powered by Disqus

Referencse:

  1. Takahashi K, et al. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. (2006) 126: 663.
  2. S.I. Vernardis, et al. Human embryonic and induced pluripotent stem cells maintain phenotype but alter their metabolism after exposure to ROCK inhibitor. Sci. Rep. (2017) 7: 42138.

Related Products

Catalase fluorometric detection kit 

Fluorescent detection, HTS | Print as PDF
 
ADI-907-027 500 tests 350.00 USD
Do you need bulk/larger quantities?
 

Y-27632 . dihydrochloride 

ROCK inhibitor
129830-38-2, ≥98% (HPLC (UV)) | Print as PDF
 
ALX-270-333-M001 1 mg 85.00 USD
 
ALX-270-333-M005 5 mg 174.00 USD
 
ALX-270-333-M025 25 mg 543.00 USD
Do you need bulk/larger quantities?
 

ROS-ID® ROS/RNS detection kit 

Monitor  reactive oxygen species and nitrogen species in live cells
Fluorescence microscopy | Print as PDF
 
ENZ-51001-200 1 Kit 281.00 USD
Do you need bulk/larger quantities?
 

ROS-ID® Total ROS/Superoxide detection kit 

Directly Monitor Global Levels  of Reactive Oxygen Species (ROS), and Specifically Superoxide
Flow Cytometry, Fluorescence microscopy, Fluorescent detection, HTS | Print as PDF
 
ENZ-51010 1 Kit 235.00 USD
Do you need bulk/larger quantities?
 

ROS-ID® Total ROS detection kit 

Flow Cytometry, Fluorescence microscopy, Fluorescent detection, HTS | Print as PDF
 
ENZ-51011 1 Kit 172.00 USD
Do you need bulk/larger quantities?
 

ROS-ID® Superoxide detection kit 

Flow Cytometry, Fluorescence microscopy, Fluorescent detection, HTS | Print as PDF
 
ENZ-51012 1 Kit 172.00 USD
Do you need bulk/larger quantities?
 

ROS-ID® NO Detection kit 

Fluorescence microscopy, Fluorescent detection | Print as PDF
 
ENZ-51013-200 1 Kit 172.00 USD
Do you need bulk/larger quantities?
 

ROS-ID® Hypoxia/Oxidative stress detection kit 

Specific detection system for simultaneous analysis of hypoxia and oxidative stress using microscopy and flow cytometry
Flow Cytometry, Fluorescence microscopy, Fluorescent detection, HTS | Print as PDF
 
ENZ-51042-0125 125 tests 105.00 USD
 
ENZ-51042-K500 500 tests 367.00 USD
Do you need bulk/larger quantities?
 

MITO-ID® Extracellular pH Sensor Kit 

A pH sensitive phosphorescent probe that can monitor cellular lactic acid extrusion, resulting from glycolysis, as a measure of glycolytic flux  (ECA/ECAR)
Fluorescent detection, HTS | Print as PDF
 
ENZ-51048 1 Kit 290.00 USD
Do you need bulk/larger quantities?
 

Nanog monoclonal antibody (23D2-3C6) 

Flow Cytometry, IF, WB | Print as PDF
 
ENZ-ABS270-0100 100 µg 230.00 USD
Do you need bulk/larger quantities?
 

Oct4 monoclonal antibody (9B7) 

Flow Cytometry, IF, WB | Print as PDF
 
ENZ-ABS271-0100 100 µg 230.00 USD
Do you need bulk/larger quantities?
 

Nanog polyclonal antibody 

IF, IHC (PS), WB, ChIP | Print as PDF
 
ENZ-ABS272-0100 100 µg 317.00 USD
Do you need bulk/larger quantities?
 

AMPIGENE® qPCR 1-Step Green Kit Lo-ROX 

Optimized and efficient cDNA synthesis  and dye-based qPCR in a single tube
PCR | Print as PDF
 
ENZ-NUC108-0200 200 Reactions 250.00 USD
 
ENZ-NUC108-1000 1000 Reactions 1,000.00 USD
Do you need bulk/larger quantities?
 

AMPIGENE® qPCR 1-Step Green Kit Hi-ROX 

Optimized and efficient cDNA synthesis and dye-based qPCR in  a single tube
PCR | Print as PDF
 
ENZ-NUC109-0200 200 Reactions 250.00 USD
 
ENZ-NUC109-1000 1000 Reactions 1,000.00 USD
Do you need bulk/larger quantities?
 

AMPIGENE® qPCR 1-Step Probe Kit Lo-ROX 

Optimized and efficient cDNA synthesis and probe-based qPCR in a  single tube
PCR | Print as PDF
 
ENZ-NUC110-0200 200 Reactions 250.00 USD
 
ENZ-NUC110-1000 1000 Reactions 1,000.00 USD
Do you need bulk/larger quantities?
 

AMPIGENE® qPCR 1-Step Probe Kit Hi-ROX 

Optimized and efficient cDNA  synthesis and probe-based qPCR in a single tube
PCR | Print as PDF
 
ENZ-NUC111-0200 200 Reactions 250.00 USD
 
ENZ-NUC111-1000 1000 Reactions 1,000.00 USD
Do you need bulk/larger quantities?
 

AMPIGENE® qPCR 1-Step Probe Kit No-ROX 

Optimized  and efficient cDNA synthesis and probe-based qPCR in a single tube
PCR | Print as PDF
 
ENZ-NUC112-0200 200 Reactions 250.00 USD
 
ENZ-NUC112-1000 1000 Reactions 1,000.00 USD
Do you need bulk/larger quantities?
 

Recommend this page

 
For Research Use Only. Not for use in diagnostic procedures.
Keep in touch

©2017 Enzo Life Sciences, Inc.,