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.”

Induced Pluripotent Stem Cells Form Red Blood Cells

Concerns over the mutations that occur when adult cells are reprogrammed into induced pluripotent stem cells has caused scientists to step back and take a second look at this technology. Can such a technology be used to treat human patients safely?

Some cells in our bodies lack nuclei. For example, platelets and red blood cells do not have nuclei, and therefore, they lack a human genome. If red blood cells can be made from pluripotent stem cells, they could potentially treat patients who suffer from anemia. The red blood cells will not harbor any mutations because they do not have DNA. Thus, induced pluripotent stem cells could potentially be used to treat patients.

A paper in Stem Cells and Development by Jessica Dias and colleagues in the laboratory of Igor Slukvin at the University of Wisconsin, Madison has reported the generation of red blood cells from human induced pluripotent stem cells (J. Dias, et al., Stem Cells and Dev 20, no 9 (2011): 1639-47).

To make red blood cells from induced pluripotent stem cells (iPSCs), they made human iPSCs from skin cells called “fibroblasts” that were taken from new-born babies.  They made they iPSCs with methods that did not use viruses.  Instead they placed in the fibroblasts, small circles of DNA that contained all the genes necessary to create iPSCs.  These small circles of DNA are called “episomes.” and they can create iPSCs without maintaining themselves in the cells.  That is to say, once the episomes convert the adult cells into iPSCs, they are lost and do contaminate the genome of the iPSCs.

After making iPSCs, they grew them for seven days with two other cells; human embryonic stem cells and a mouse bone marrow cell line called OP9.  This combination converted the iPSCs into bone marrow stem cells.  The bone marrow stem cells were isolated and cultured for five days with chemicals that are known to push bone marrow stem cells to become red blood cells.  These chemicals (erythropoietin, stem cell factor, thrombopoietin, interleukin-3, dexamethasone, insulin, interleukin-6, and iron), drove the stem cells to become red blood cell-like cells.  Because these cells were also grown under conditions that prevented them from attaching they grew and differentiated.  After five days, the cells were maintained on another mouse bone marrow cells line called MS5 cells.

Dias and her colleagues also used an alternative technique that worked just as well that did not include isolating the bone marrow stem cells, but subjected the cells to a Percoll centrifugation that also isolated the differentiating cells from the other cells.  This technique seemed faster and less troublesome.

Neither of these techniques could be employed if these cells were to be used for human treatments.  The use of animal cells lines could contaminate the iPSCs with animal viruses or animal proteins.  Both of these would cause the human immune system to react adversely to the cells (Martin MJ, Muotri A, Gage F, Varki A. Human embryonic stem cells express an immunogenic nonhuman sialic acid.Nat Med. 2005 Feb;11(2):228-32).  Therefore, some other protocol will need to be devised if this type of treatment is employed for anemic humans.

Nevertheless, this culture did generate red blood cells that expressed mainly embryonic and fetal types of hemoglobin.  While there was some adult hemoglobin made, it was the minority molecule.  All of the cells produced by this cell culture system were of the same type as those that produce red blood cells (erythroid), and not of those that make white blood cells (myeloid).  This shows that it is feasible to make red blood cells from iPSCs, and it might even be feasible to produce them in a culture system that makes large quantities of them.  Other uses for culture systems like this could include making red blood cells to grow malarial parasites for drug research.  Clearly this is a remarkable discovery that may lead to a source of red blood cells for patients and laboratories alike.