Polyamines Help Control Embryonic Stem Cell Differentiation

Scientists from the Institute of Medical Biology (IMB), which is a research institute under the Agency for Science, Technology and Research (A*STAR), have made an important discovery about the role of molecules called “polyamines” in embryonic stem cells.

Polyamines are organic molecules that have more than one “amino” group (-NH2). These compounds have several functions inside cells. Since polyamines are highly positively changed, they bind the DNA, which is highly negatively changed. By binding to DNA, they stabilize the structure of DNA and aid with processes that are important for the life of cells, such as DNA replication (see Alm K and Oredssib S. Cells and polyamines do it cyclically. Essays Biochem. 2009 Nov 4;46:63-76). Plants without particular polyamines are more susceptible to drought (Yamaguchi K, et al., A protective role for the polyamine spermine against drought stress in Arabidopsis. Biochem Biophys Res Commun. 2007 Jan 12;352(2):486-90). Polyamine synthesis is extremely heavily regulated, and inhibition of polyamine synthesis causes cell growth to stop or greatly decrease. Polyamines also regulate ion channels in neurons, which mean that they can affect learning and memory.

How do polyamines affect embryonic stem cells? Remember that embryonic stem cells can divide indefinitely and, under the proper culture conditions, can stay in an undifferentiated state. A*STAR scientists found that in mouse embryonic stem cells, an enzyme called Amd1 is essential for maintenance of undifferentiated state and self-renewal. Amd1 catalyzes a reaction that is essential for polyamine synthesis.

For the interested, polyamines are made in a multistep process that begins with a molecule called “ornithine.” Ornithine is made from the amino acid arginine or by related processes. The removal of a carbon dioxide group from ornithine produces putrescine and the enzyme that catalyzes this reaction is ornithine decarboxylase (ODC). Putrescine is used to make two other polyamines called spermine and spermidine. To make spermidine and spermidine, propylamine groups must be added, and these are added by a molecule called S-adenosylmethionine (SAM). SAM is used in cells to add single carbon atoms to molecules, but polyamine synthesis uses SAM in a very unusual manner. The enzyme SAM decarboxylase removes a carbon dioxide from SAM to make dc-SAM, which stands for decarboxylated SAM. Two different enzymes act sequentially to add propylamine groups to putrescine. The first enzyme, spermidine synthase, adds the first propylamine group to make a molecule called spermidine, and the second, spermine synthase, adds the second propylamine to make spermine. Amd1 encodes the enzyme SAM decarboxylase.

According to the researchers at A*STAR, without high levels of Amd1, mouse embryonic stem cells are unable to properly stay in the undifferentiated state and divide. In order to drive embryonic stem cells to differentiate into nerve cells, Amd1 activity must decrease.

This is the first time, polyamines have been linked to embryonic stem cells function. Polyamines have been known for some time to play central roles in cancer and cell growth and division. This novel discovery, links polyamine regulation to ESC biology when the research team conducted a genome-wide screen to look for genes that were differentially controlled during embryonic stem cell differentiation.

The Principle Investigator at IMB, Leah Vardy, who was also the managing author on this paper, said, “The polyamines that Amd1 regulate have the potential to regulate many different aspects of self-renewal and differentiation. The next step is to understand in more detail the molecular targets of these polyamines both in embryonic stem cells and cells differentiating to different cellular lineages. It is possible that manipulation of polyamine levels in embryonic stem cells through inhibitors or activators of the pathway could help direct the differentiation of embryonic stem cells to more clinically useful cell types.”

The Executive Director of IMB, Birgitte Lane, noted, “This is a fine piece of fundamental research that will have breakthrough consequences in many areas and can bring about far-reaching applications. Developing cellular therapies is just one long-term clinical benefit of understanding ESC biology, which can also help develop stem cell systems for disease modeling, developing new drugs as well as a tool for researchers to answer other biological questions.”

Stem Cells used to Transfer Inhibitory RNA Molecules to Neurons to Treat Huntington’s Chorea in Lab Animals

Huntington’s disease is an inherited brain disorder that causes progressive uncontrolled movements, dementia and culminates in death. The symptoms of Huntington’s disease are involuntary jerking or writhing movements (chorea), involuntary, sustained contraction of muscles (dystonia), muscle rigidity, slow, uncoordinated fine movements, slow or abnormal eye movements, impaired gait, posture and balance, difficulty with the physical production of speech, and difficulty swallowing.

More than a quarter of a million Americans are affected by Huntington’s disease. Huntington’s disease is passed through families even if only one parent has the abnormal huntingtin gene, since it is inherited as an autosomal dominant. The huntingtin gene is found on the fourth chromosome, and Huntington’s disease-causing mutations result from the expansion of a trinucleotide (CAG) repeat (Jones L, Hughes A. Int Rev Neurobiol.2011;98:373-418 & Reiner A, Dragatsis I, Dietrich P. Int Rev Neurobiol. 2011;98:325-72). This trinucleotide repeat is normally repeated up to 28 times on the chromosome, but polymerase slip during DNA replication can expand the number of these repeats so that an abnormal form of the Huntingtin protein to be made. The abnormal Huntingtin protein accumulates in the brain and this cause the disease’s devastating progression. Individuals usually develop symptoms in middle age if there are more than 35 copies of the CAG repeats. A more rare form of the disease occurs in youth when the number of CAG repeats occurs many more times.

Huntington’s disease can be managed with medications. For example Terabenazine (Xenazine) suppresses the involuntary jerking and writhing movements associated with Huntington’s diseases. Antipsychotic drugs such as Haloperidol (Haldol) and Clozapine (Clozaril) can suppress movements but they can also increase muscle rigidity and involuntary contractions. Other medications like clonazepam (Klonopin) and diazepam (Valium) can suppress the chorea, dystonia and muscle rigidity.

Even though brain grafts in laboratory animals have shown some promise, these experiments used a chemically induced form of Huntington’s disease. Because the surrounding tissue was genetically normal, implanted brain tissue simply integrated into the damaged brain tissue and healed it. However, clinical Huntington’s disease is due to mutations in the huntingtingene, and the surrounding brain tissue is not genetically normal. Therefore grafted stem cells are killed off by the toxic environment in the brain (Clelland CD, Barker RA, Watts C. Neurosurg Focus.2008;24(3-4):E9 & Dunnett SB, Rosser AE. Exp Neurol. 2007 Feb;203(2):279-92). To overcome this problem, researchers have developed a technique for that used stem cells to deliver therapeutic agents that specifically target the genetic abnormality found in Huntington’s disease.

Scientists at the UC Davis Institute for Regenerative Cures have developed a novel, and promising approach that might prevent the disease from advancing. Jan A. Nolta, principal investigator of the study and director of the UC Davis stem cell program and the UC Davis Institute for Regenerative Cures, thinks that the best chance to halt the disease’s progression will be to reduce or eliminate the mutant Huntingtin (Htt) protein found in the neurons of those with the disease. RNA interference (RNAi) technology has been shown to be highly effective at reducing Htt protein levels and reversing disease symptoms in mouse models.

Nolta said: “For the first time, we have been able to successfully deliver inhibitory RNA sequences from stem cells directly into neurons, significantly decreasing the synthesis of the abnormal Huntingtin protein. Our team has made a breakthrough that gives families affected by this disease hope that genetic therapy may one day become a reality.” She continued: “Our challenge with RNA interference technology is to figure out how to deliver it into the human brain in a sustained, safe and effective manner,” said Nolta. “We’re exploring how to use human stem cells to create RNAi production factories within the brain.”

The research team from UC Davis showed for the first time that inhibitory RNA sequences are directly transferable from donor cells into target cells to greatly reduce unwanted protein synthesis from the mutant huntingtin gene. To transfer these inhibitory RNA sequences into their targets, Nolta’s team genetically engineered mesenchymal stem cells (MSCs) from bone marrow that had been collected from unaffected human donors. Over the past two decades, Nolta and her colleagues have shown MSCs are safe and effective vehicles for the transfer of enzymes and proteins to other cells. According to Nolta, MSCs can also transfer RNA molecules directly from cell to cell, in amounts sufficient to reduce levels of a mutant protein by over 50% in the target cells. This discovery has never been reported before and offers great promise for a variety of disorders.

Nolta has recently received a Transformative Research Grant from the National Institutes of Health (NIH) to study how MSCs can transfer microRNA and other factors into the cells of damaged tissues, and how that process can be harnessed to treat injuries and disease. Nolta said: “Not only is finding new treatments for Huntington’s disease a worthwhile pursuit on its own, but the lessons we are learning are applicable to developing new therapies for other genetic disorders that involve excessive protein development and the need to reduce it. We have high hopes that these techniques may also be utilized in the fight against some forms of amyotrophic lateral sclerosis (Lou Gehrig’s disease) as well as Parkinson’s and other conditions.”

Published – Scott D. Olson, Jan Nolta et al.; Examination of mesenchymal stem cell-mediated RNAi transfer to Huntington’s disease affected neuronal cells for reduction of huntingtin;”  Molecular and Cellular Neuroscience,2011; DOI: 10.1016/j.mcn.2011.12.001.