Single cell analysis will allow us to understand the amazing regeneration power of salamanders, map all our cells in a biological 'Google Maps' and fight cancer or autoimmune pathologies. That's why it was the Method of the Year for `Nature´ magazine in 2013 and the great scientific breakthrough of 2018 in `Science´.
Cell-by-cell analysis with micro-fluids, which allow each cell to be separated into droplets at the same time as a marker is introduced into its DNA. / ETH Zurich
The room is exciting, but more for what it hides than for what it shows. It really is just a very white space of curved walls plagued with mobile tables. And, in each of them, some black and white blocks that resemble old computers because of their size.
The blocks are actually some of the most modern genetic sequencers of the moment: the driving force behind the National Centre for Genomic Analysis (CNAG), in Barcelona. They are shown to us by Holger Heyn, a German who has been researching in Spain for nearly ten years and who is now in charge of the Single Cell Genomics team at the CNAG itself.
This cell-by-cell analysis is the great scientific hope to unravel the development of organisms -including humans-, to establish the bases for the regeneration of organs, to create a map of all our cells or to uncover keys to some diseases as elusive as cancer. That is why it has been chosen as the scientific advance for 2018 by Science magazine, and that is the reason for our visit.
“Unicellular genomics has reached maturity in an incredible way,” declared Eric Lander, the most influential director of the Broad Institute at MIT and Harvard, during a conference some time ago. “And once you realize that we can do the tests on individual cells, how are you going to accept a milkshake? It's crazy to be doing genomics on milkshakes.”
We visit the main lab dedicated to cell to cell analysis in Spain: the CNAG of Barcelona / CNAG.
Los ‘batidos’ de Lander son los análisis tradicionales donde se recogen un montón de células cuyo ADN (o ARN, el mensajero del ADN) se mezcla antes de pasar por los secuenciadores. La información que resulta es un promedio del conjunto. Habrá árboles particulares que queden ocultos por el bosque y, sobre todo, no permite saber qué información concreta contiene cada célula en particular. El individuo se disuelve en la masa.
Lander’s ‘milkshakes’ are the traditional analyses in which a bunch of cells are collected, their DNA (or RNA, the messenger of DNA) being mixed before going through the sequencers. The resulting information is an average of the set. There will be individual trees which one cannot see for the forest and, above all, the method does not allow us to know which specific information is contained in each particular cell. The individual dissolves into the mass.
These analyses had already been chosen as the Method of the Year in 2013 by Nature magazine. “But by then only ten or twenty cells could be studied; the method was tremendously expensive and very unresolved,” Heyn comments. Now, instead, “we can analyse 10,000 cells in a single experiment and there are projects to do so with up to one million”.
The key technology that has made this leap possible is micro-fluids, a tool that allows each cell to be separated and channelled into tiny droplets while a marker or bar code is introduced into its DNA. This marker makes it possible to identify each one of them once they have been analysed.
In this way, its DNA, its RNA and even its epigenetic information can be studied, because it puts the dots and commas on the genome´s reading. “The resolution is not perfect yet in any case,” Heyn admits, “but the most powerful thing right now is the study of the RNA.” The acknowledgement of the advance of the year was based on some of these studies.
The Schmidtea mediterranea planaria is a very unusual animal. This worm, which is barely a centimetre long, is potentially immortal: its stem cells continuously renew its organs and, if it is split into several pieces, a new worm emerges from each one. The head can regenerate a tail and the tail a head, which even seems to preserve some of its memories. It was discovered by biologist Jaume Baguñà in a Montjuich reservoir in 1968. As far as we know, today it is barely to be found in Barcelona, in certain areas of Menorca... and in around twenty laboratories all over the world.
Photo of a 'Schmidtea mediterranea' with two heads in an experiment carried out during the regeneration of the body/ Dany S Adams
One of them is at the Max Delbrück Molecular Medicine Centre in Germany. There, in 2018, they used cell-by-cell analysis techniques not only to establish a cell atlas of the animal, but also to study the genetic programs that led to its formation and regeneration. Among other surprises, the researchers found that the number of some cells was decreasing very rapidly. This indicated that they could be the reservoir that fed the regeneration process.
Could these discoveries be applied to humans to boost regenerative medicine? “We still have a long way to go,” reply Mireya Plass and Jordi Solana, the first two signatories of the work, in a consensual letter. “The adult planarias have more than 30% of stem cells, which is very far from us. Even so, we hope that some of the mechanisms are the same.”
What is clear to them is that before they were not able to distinguish the different types of cells in embryonic development, and now they are going to collect information “that will be crucial for the laboratory generation of cells, tissues and organs that serve to treat different illnesses.”
There was a time when the writer Julio Cortázar “thought a lot about the axolotl”, to the extent of even writing an amazing tale on these hypnotic amphibians that are often mistaken for salamanders and that, like them, are capable of regenerating their limbs, the skeleton included. Cortázar was obsessed with its gaze, but how do they achieve this immense power of renewal?
Using the new arsenal of techniques, German researchers have ascertained how a particular type of cell is deprogrammed to return to an embryonic-like state and, from there, direct regeneration in the axolotls. Our very limited ability to achieve something similar may be due to the inability to reprogram this type of cells to those states. Our liver or skin may partially regenerate, but they are exceptions. We cannot do the same with a leg, a kidney or a heart.
Here the crucial analysis is that of RNA. DNA does not provide valuable information: it is essentially the same in every cell; what matters is how the code is read in the form of RNA at every time and place. This is what makes it possible to study how, from a single cell, an animal is formed with all its organs and tissues, very different from each other but quite equal in its initial genetics. How we are what we are.
This has been done by other research groups in 2018 with fishes and frogs, seeing how the genes in each cell are turned on and off during their development. And so they have been able to track the development of the brain and spinal cord of a mouse until the 11th day after its birth, identifying along the way more than a hundred different cell types.
“These are very nice studies,” Heyn admits. “They allow us to see things that we couldn't see before and follow the growth without previous hypotheses: how a cell divides and starts doing different things in each division.” However, “that is only a small part of the possibilities it offers.” Heyn refers to the possibility of drawing up an entire human atlas and its possible applications in medicine.
Axolotl, the amphibian that intrigued Cortázar and is capable of self-regeneration / Research Institute of Molecular Pathology
Eric Lander explains it as follows: “If we had a periodic table of cells, we would be able to find out the composition of any sample.” That theoretical periodic table that once revolutionized chemistry has adopted another image, that of a cellular atlas, and has given rise to a project that gathers the idea: the “Human Cell Atlas”.
Its aim is to identify each cell in the body, where it is located and how it acts together with the rest to form organs and tissues. Something like a cell Google Maps that serves as a reference so that “we can then place the houses and buildings on top,” as visualized by Heyn, who has no doubt that it will be something that “will change the rules of the game, as the Genome Project did in its day.” The next meeting of the initiative will take place in October, in Barcelona.
Although it is a voluntary consortium with no specific funding, it has had such popular sponsors as Mark Zuckerberg, the founder of Facebook, who “drove its inception and collaborates by co-financing specific studies.”
Heyn's group is involved in two ways: it is responsible for the quality control of the project and is in charge of studying all types of B lymphocytes - the cells that make antibodies - that exist in the human body. “We want to see how they develop and activate in each place, how they work and how they change as they travel through the body.”
Previously, it was thought that there were 500 different cell types; now it is said that there are at least ten times as many. “But the numbers dance a lot, and they're probably even higher,” Heyn acknowledges. “The key is to think that the cells are incredibly plastic, that there's much more variety than we could have studied before.
In just two years of life, the project has already produced advances: it has served to identify a new type of neurons, which they have called ‘rosehip’ and which, for the time being, seem exclusive to humans; it has made it possible to establish a map of the cells the placental barrier and discover those that modulate the response of the defences and prevent their rejection; they have even found a new type of lung cell that seems to be involved in cystic fibrosis, a potentially fatal hereditary disease.
Once the function of a cell has been defined, when that function fails in the organism we will know who is responsible and what cell needs to be studied. The LifeTime project proposes focusing this type of technique on the study of the disease. The initiative aims to achieve - and has reached the final phase of evaluation - a funding of 1,000 million Euros from the European Commission as a Flagship project (as were the Graphene Project or the Human Brain Project).
One of the diseases where the new tools will be most talked about is cancer. If these techniques make sense “where there is heterogeneity,” as Heyn points out, cancer is the perfect candidate.
The German researcher Holger Heyn is responsible for the Single Cell Genomics team at the CNAG. / CNAG
A tumour is, in essence, an evolutionary machine. It accumulates changes and mutations in an unbridled way. But the changes can be different in one cell or another. Some can lead to metastases; others are able to resist treatments and regenerate the tumour and those beyond simply grow without apparent control. If healthy cells essentially share the same DNA, tumour cells can be very distant cousins.
The heterogeneity and evolutionary capacity of cancer are one of the great challenges of medicine and, surely, the great obstacle of the new precision medicine. The fact that some cells acquire new key mutations - or that a few are capable of resisting the chosen treatment - means that the efficacy of the therapies is generally only temporary. Cell-by-cell analysis techniques can be used to gain a better understanding of the biology of cancer and thus overcome some of these obstacles.
“In the past, we had to deduce that evolution, now we can directly see the tree it builds,” Heyn explains. The applications - although incipient and not without difficulties - are remarkable. For example: major precision medicine projects are based on giving treatments if they find a certain mutation in the tumour, but they do not take into account the amount of cells that contain it and, above all, they cannot determine what type of cells they are and what their function is. Thus, it is not only more difficult to predict their efficacy, but also to prevent relapses of the disease.
Another example: the type of cells that give rise to many tumours is not known. In the case of retinoblastoma, a hereditary cancer, this analysis has identified the small population of cells in the retina that initiates it. By isolating them and studying their metabolism, researchers have proposed a treatment to prevent it. “Since the effects occur only in these cells, we couldn't see them when we analysed the entire tissue of the eye,” they explain.
The powerful software of these analyses will also contribute to the development of the liquid biopsy, the possibility of detecting or following the evolution of a tumour through its trace in the blood. And the technology is already being used to study in the response of our defences and what specific type of cells act in the promising immunotherapy against cancer. Knowing them is a key step towards improving it.
“Other diseases that can benefit from these techniques are autoimmune diseases,” Heyn states, “because we still don't know what kind of cells produce the attacks.” These include inflammatory diseases such as Crohn's disease, lupus or multiple sclerosis. “And it will also serve to know, in the case of Alzheimer’s, what type of cells die at each stage.”
The visit ends with an enthusiastic statement in which, however, it is difficult to find any affectation: “These techniques allow us to see what we didn't even know existed,” Heyn says. “They're going to help us study the complexity of life, to know how we're built. And once we have an atlas, we will be able to compare it with that which fails, in us and between us.”
Were or weren't the blocks in the initial room exciting?