Old skins cells reprogrammed to regain youthful function

Close-up of human skin

What is regenerative medicine?

As we age, our cells' ability to function declines and the genome accumulates marks of ageing. Regenerative biology aims to repair or replace cells including old ones. One of the most important tools in regenerative biology is our ability to create 'induced' stem cells. The process is a result of several steps, each erasing some of the marks that make cells specialised. In theory, these stem cells have the potential to become any cell type, but scientists aren't yet able to reliably recreate the conditions to re-differentiate stem cells into all cell types.

Turning back time

The new method, based on the Nobel Prize winning technique scientists use to make stem cells, overcomes the problem of entirely erasing cell identity by halting reprogramming part of the way through the process. This allowed researchers to find the precise balance between reprogramming cells, making them biologically younger, while still being able to regain their specialised cell function.

In 2007, Shinya Yamanaka was the first scientist to turn normal cells, which have a specific function, into stem cells which have the special ability to develop into any cell type. The full process of stem cell reprogramming takes around 50 days using four key molecules called the Yamanaka factors. The new method, called 'maturation phase transient reprogramming', exposes cells to Yamanaka factors for just 13 days. At this point, age-related changes are removed and the cells have temporarily lost their identity. The partly reprogrammed cells were given time to grow under normal conditions, to observe whether their specific skin cell function returned. Genome analysis showed that cells had regained markers characteristic of skin cells (fibroblasts), and this was confirmed by observing collagen production in the reprogrammed cells.

Age isn't just a number

To show that the cells had been rejuvenated, the researchers looked for changes in the hallmarks of ageing. As explained by Dr Diljeet Gill, a postdoc in Wolf Reik's lab at the Institute who conducted the work as a PhD student: "Our understanding of ageing on a molecular level has progressed over the last decade, giving rise to techniques that allow researchers to measure age-related biological changes in human cells. We were able to apply this to our experiment to determine the extent of reprogramming our new method achieved."

Researchers looked at multiple measures of cellular age. The first is the epigenetic clock, where chemical tags present throughout the genome indicate age. The second is the transcriptome, all the gene readouts produced by the cell. By these two measures, the reprogrammed cells matched the profile of cells that were 30 years younger compared to reference data sets.

The potential applications of this technique are dependent on the cells not only appearing younger, but functioning like young cells too. Fibroblasts produce collagen, a molecule found in bones, skin tendons and ligaments, helping provide structure to tissues and heal wounds. The rejuvenated fibroblasts produced more collagen proteins compared to control cells that did not undergo the reprogramming process. Fibroblasts also move into areas that need repairing. Researchers tested the partially rejuvenated cells by creating an artificial cut in a layer of cells in a dish. They found that their treated fibroblasts moved into the gap faster than older cells. This is a promising sign that one day this research could eventually be used to create cells that are better at healing wounds.

In the future, this research may also open up other therapeutic possibilities; the researchers observed that their method also had an effect on other genes linked to age-related diseases and symptoms. The APBA2 gene, associated with Alzheimer's disease, and the MAF gene with a role in the development of cataracts, both showed changes towards youthful levels of transcription.

The mechanism behind the successful transient reprogramming is not yet fully understood, and is the next piece of the puzzle to explore. The researchers speculate that key areas of the genome involved in shaping cell identity might escape the reprogramming process.

 

The first cells might have used temperature to divide

Cell division illustration

 A simple mechanism could underlie the growth and self-replication of protocells -- putative ancestors of modern living cells -- suggests a study publishing September 3 in Biophysical Journal. Protocells are vesicles bounded by a membrane bilayer and are potentially similar to the first unicellular common ancestor (FUCA). On the basis of relatively simple mathematical principles, the proposed model suggests that the main force driving protocell growth and reproduction is the temperature difference that occurs between the inside and outside of the cylindrical protocell as a result of inner chemical activity.

"The initial motivation of our study was to identify the main forces driving cell division," says the study author Romain Attal of Universcience. "This is important because cancer is characterized by uncontrolled cell division. This is also important to understand the origin of life."

The splitting of a cell to form two daughter cells requires the synchronization of numerous biochemical and mechanical processes involving cytoskeletal structures inside the cell. But in the history of life, such complex structures are a high-tech luxury and must have appeared much later than the ability to split. Protocells must have used a simple splitting mechanism to ensure their reproduction, before the appearance of genes, RNA, enzymes, and all the complex organelles present today, even in the most rudimentary forms of autonomous life.

In the new study, Attal proposed a model based on the idea that the early forms of life were simple vesicles containing a particular network of chemical reactions -- a precursor of modern cellular metabolism. The main hypothesis is that molecules composing the membrane bilayer are synthesized inside the protocell through globally exothermic, or energy-releasing, chemical reactions.

The slow increase of the inner temperature forces the hottest molecules to move from the inner leaflet to the outer leaflet of the bilayer. This asymmetric movement makes the outer leaflet grow faster than the inner leaflet. This differential growth increases the mean curvature and amplifies any local shrinking of the protocell until it splits in two. The cut occurs near the hottest zone, around the middle.

"The scenario described can be viewed as the ancestor of mitosis," Attal says. "Having no biological archives as old as 4 billion years, we don't know exactly what FUCA contained, but it was probably a vesicle bounded by a lipid bilayer encapsulating some exothermic chemical reactions."

Although purely theoretical, the model could be tested experimentally. For example, one could use fluorescent molecules to measure temperature variations inside eukaryotic cells, in which mitochondria are the main source of heat. These fluctuations could be correlated with the onset of mitosis and with the shape of the mitochondrial network.

If borne out by future investigations, the model would have several important implications, Attal says. "An important message is that the forces driving the development of life are fundamentally simple," he explains. "A second lesson is that temperature gradients matter in biochemical processes and cells can function like thermal machines."

Origin of life: The chicken-and-egg problem

RNA abstract concept illustration 

A Ludwig-Maximilians-Universitaet (LMU) in Munich team has shown that slight alterations in transfer-RNA molecules (tRNAs) allow them to self-assemble into a functional unit that can replicate information exponentially. tRNAs are key elements in the evolution of early life-forms.

Life as we know it is based on a complex network of interactions, which take place at microscopic scales in biological cells, and involve thousands of distinct molecular species. In our bodies, one fundamental process is repeated countless times every day. In an operation known as replication, proteins duplicate the genetic information encoded in the DNA molecules stored in the cell nucleus -- before distributing them equally to the two daughter cells during cell division. The information is then selectively copied ('transcribed') into what are called messenger RNA molecules (mRNAs), which direct the synthesis of the many different proteins required by the cell type concerned. A second type of RNA -- transfer RNA (tRNA) -- plays a central role in the 'translation' of mRNAs into proteins. Transfer RNAs act as intermediaries between mRNAs and proteins: they ensure that the amino-acid subunits of which each particular protein consists are put together in the sequence specified by the corresponding mRNA.

How could such a complex interplay between DNA replication and the translation of mRNAs into proteins have arisen when living systems first evolved on the early Earth? We have here a classical example of the chicken-and-the-egg problem: Proteins are required for transcription of the genetic information, but their synthesis itself depends on transcription.

LMU physicists led by Professor Dieter Braun have now demonstrated how this conundrum could have been resolved. They have shown that minor modifications in the structures of modern tRNA molecules permit them to autonomously interact to form a kind of replication module, which is capable of exponentially replicating information. This finding implies that tRNAs -- the key intermediaries between transcription and translation in modern cells -- could also have been the crucial link between replication and translation in the earliest living systems. It could therefore provide a neat solution to the question of which came first -- genetic information or proteins?

Strikingly, in terms of their sequences and overall structure, tRNAs are highly conserved in all three domains of life, i.e. the unicellular Archaea and Bacteria (which lack a cell nucleus) and the Eukaryota (organisms whose cells contain a true nucleus). This fact in itself suggests that tRNAs are among the most ancient molecules in the biosphere.

Like the later steps in the evolution of life, the evolution of replication and translation -- and the complex relationship between them -- was not the result of a sudden single step. It is better understood as the culmination of an evolutionary journey. "Fundamental phenomena such as self-replication, autocatalysis, self-organization and compartmentalization are likely to have played important roles in these developments," says Dieter Braun. "And on a more general note, such physical and chemical processes are wholly dependent on the availability of environments that provide non-equilibrium conditions."

In their experiments, Braun and his colleagues used a set of reciprocally complementary DNA strands modeled on the characteristic form of modern tRNAs. Each was made up of two 'hairpins' (so called because each strand could partially pair with itself and form an elongated loop structure), separated by an informational sequence in the middle. Eight such strands can interact via complementary base-pairing to form a complex. Depending on the pairing patterns dictated by the central informational regions, this complex was able to encode a 4-digit binary code.

Each experiment began with a template -- an informational structure made up of two types of the central informational sequences that define a binary sequence. This sequence dictated the form of the complementary molecule with which it can interact in the pool of available strands. The researchers went on to demonstrate that the templated binary structure can be repeatedly copied, i.e. amplified, by applying a repeating sequence of temperature fluctuations between warm and cold. "It is therefore conceivable that such a replication mechanism could have taken place on a hydrothermal microsystem on the early Earth," says Braun. In particular, aqueous solutions trapped in porous rocks on the seafloor would have provided a favorable environment for such reaction cycles, since natural temperature oscillations, generated by convection currents, are known to occur in such settings.

During the copying process, complementary strands (drawn from the pool of molecules) pair up with the informational segment of the template strands. In the course of time, the adjacent hairpins of these strands also pair up to form a stable backbone, and temperature oscillations continue to drive the amplification process. If the temperature is increased for a brief period, the template strands are separated from the newly formed replicate, and both can then serve as template strands in the next round of replication.

The team was able to show that the system is capable of exponential replication. This is an important finding, as it shows that the replication mechanism is particularly resistant to collapse owing to the accumulation of errors. The fact that the structure of the replicator complex itself resembles that of modern tRNAs suggests that early forms of tRNA could have participated in molecular replication processes, before tRNA molecules assumed their modern role in the translation of messenger RNA sequences into proteins. "This link between replication and translation in an early evolutionary scenario could provide a solution to the chicken-and-the-egg problem," says Alexandra Kühnlein. It could also account for the characteristic form of proto-tRNAs, and elucidate the role of tRNAs before they were co-opted for use in translation.