Teaching Old Cells New Tricks

The laboratory of Helen Blau at Stanford University has devised a technique to lengthen the sequences that cap the ends of chromosomes in skin cells. This treatment enlivens the cells and makes them behave as though they were younger.

In order to properly protect linear chromosomes from loosing DNA at their ends, chromosomes have a special set of sequences called “telomeres” at their ends. Telomeres consists of short sequences that are repeated many times. A special enzyme called the telomerase replicates the telomeres and maintains them. As we age, telomerase activity wanes and the telomeres shorten. This threatens the genetic integrity of the chromosomes, since a loss of genes from the ends of chromosomes can be deleterious for cells. In young humans, the telomeres may be 8,000 to 10,000 bases long. When the telomeres shorten to a particular length, growth stops and the cells become quiescent.

Human telomeres

Embryonic stem cells have long telomeres at the ends of their chromosomes and they also have robust telomerase activity. Adult stem cells have varied telomerase activity and telomere length, but it seems that the length of the telomeres and the activity of the telomerase correlates with the vitality of the stem cell population and its capacity to heal (see H. Saeed and M. Iqtedar (2013). J. Biosci. 38, 641–649). As we age our stem cell quality decreases as their telomeres shorten.

Blau and her colleagues used a modified type of RNA to lengthen the telomeres of large numbers of cells. According to Blau: “Now we have found a way to lengthen human telomeres by as much as 1,000 nucleotides, turning back the internal clock in these cells by the equivalent of many years of human life. This greatly increases the number of cells available for studies such as drug testing or disease modeling.”

In these experiments, Blau and her coworkers used chemically modified messenger RNA molecules that code for TERT, which is the protein component of the telomerase. The expression of these messenger RNAs in human cells greatly increased the levels of telomerase activity.

This technique devised by Blau and her team have distinct advantages of previously described protocols. First, this technique boosts telomerase activity temporarily. The modified messenger RNA sticks around for several hours and is translated into TERT protein, but this protein only lasts for about 48 hours, after which its activity dissipates. After the telomerase have lengthened the telomeres, they will shorten again after each cell division as before.

“This new approach paves the way toward preventing or treating diseases of aging,” said Blau. “There are also highly debilitating genetic diseases associated with telomere shortening that could benefit from such a potential treatment.”

Blau and her team are testing their technique in other cell types besides skin cells.

Making Safer Induced Pluripotent Stem Cells

Induced Pluripotent Stem Cells or iPSCs are made from mature, adult cells by means of genetic engineering techniques that introduce pluripotency genes into the adult cells. These introduced genes drive the adult cells to de-differentiate into an embryonic stem cell-like cells that have the ability to differentiate into almost all of the cells of the adult human body.

Despite the attractiveness of these cells for regenerative medicine, there is a dark side to iPSCs, since the production of iPSCs introduces new mutations into them. While not all iPSC lines are created equal and the methods by which they are derived also influences the degree of genetic damage to them, there are serious questions about the safety of iPSCs for clinical use.

A new paper from a Spanish group has discovered that a protein called SIRT1 is required to protect the chromosomal integrity of iPSCs during reprogramming.

Linear chromosomes are capped at their ends by special structures called telomeres. These telomeres shorten over time, but during reprogramming, the telomeres lengthen. This lengthening requires the SIRT1 protein, but SIRT1 is also required to maintain the telomeres at this elongated length. In this way, SIRT1 helps safeguard the chromosomes during reprogramming. The SIRT1 protein is also up-regulated in embryonic stem cells.

Using a mouse model system, researchers from the Spanish National Cancer Research Center’s Telomeres and Telomerase Group made cells that completely lacked any functional SIRT1 protein. Maria Luigia De Bonis, Sagrario Ortega, and Maria A. Blasco from CNIO discovered that SIRT1-deficient mouse cells could be reprogrammed, but the telomeres of these cells lengthened much less efficiently, and eventually experienced chromosome abnormalities and DNA damage. SIRT1-deficient iPSCs also formed larger, poorly differentiated tumors when transplanted into nude mice.  Thus SIRT1 seems to keep the chromosomes of iPSCs healthy.

Interestingly, the c-MYC protein, which is encoded by the c-myc gene – one of the four genes required to reprogram adult cells – is stabilized by SIRT1. Normally, the c-MYC protein has a very short half-life, but SIRT1 protects c-MYC from degradation, and c-MYC increases the production of the enzyme that replicates and elongates the telomeres; telomerase.

This work could very possibly lead to protocols that will stabilize the chromosomes of iPSCs during reprogramming. This will make iPSCs safer for possible use in the clinic.

TRF1 Gene Necessary for Reprogramming

In order to convert cells from almost any tissue in our bodies into induced pluripotent stem cells (iPSCs) requires a detailed knowledge of the reprogramming process. Initiating the reprogramming process differs from one cell type to another, but the cellular and genetic mechanics of reprogramming might be largely the same.

A research team at the Spanish National Cancer Research Center headquartered in Madrid, Spain, and headed by Ralph P. Schneider from the Telomeres and Telomerase Group, which is led by Maria A. Blasco, have discovered that a gene called TRF1 is essential for nuclear reprogramming.

TRF1 or telomere repeat binding factor 1 is a member of a complex of proteins called the “shelterin complex” that binds to the ends of chromosomes (known as telomeres) and protects them. Mouse embryos that lack TRF1 die very early during embryonic life and if an adult tissue is missing TRF1, it shrinks and stops working (organ atrophy).

Shelterin Complex

A variety of observations have established that pluripotent cells have long, intact telomeres. Furthermore, pluripotent cells have a very active telomerase enzyme, which is the enzyme that synthesizes the telomere ends of each chromosome. Telomeres not only protect the structural integrity of the chromosomes, but they also serve as a template or starting point for the replication and extension of the telomerase by telomerase.

In the cell, the telomere does not exist in isolation, but it is embedded in a complex of DNA and the shelterin complex proteins, of which TRF1 is a member. Pluripotent cells have very long telomeres, but it is uncertain if the shelterin complex components are necessary to maintain the pluripotent state (see Marión RM, Blasco MA. Curr Opin Genet Dev. 2010 Apr;20(2):190-6).

To investigate this question, Schneider and others constructed a version of TRF1 that was fused to a glowing proteins in order to track its function during reprogramming. Then they injected this construct into mouse embryonic stem cells and made genetically engineered mice that carried this glowing version of TRF1.

When they tracked TRF1 function in adult cells, embryonic cells, and stem cells, it was clear that TRF1 is a superb marker for stem cells. It distinguishes adult stem cells from non-stem and is also indispensable for stem cell function. In fact, TRF1 is such a good marker for stem cells that it can be used to isolated stem cells from surrounding cells.

Pluripotent stem cells show the highest levels of TRF1 expression. In fact, in iPSCs, the expression of TRF1 goes from very low to rather high. This led Schneider and his colleagues to suggest that TRF1 is an indicator of pluripotency. To corroborate their hypothesis, Schneider and others showed that the more pluripotent the iPSC stem cell line, the higher the levels of expression of TRF1. Also, TRF1 is required to maintain pluripotency and is also required for the induction of pluripotency. TRF1 inhibits cell death and the expression of TRF1 is directly activated by the pro-pluripotency gene Oct4.

Thus TRF1 is another gene required for iPSC production.  It also seems to be required for iPSC production regardless of the tissue from which is comes from.