Science and the single cell
Our bodies are made up of trillions of cells - but these aren’t mere biological building blocks, as inert as bricks. They’re constantly communicating and changing. So how do scientists measure this? Plus, you can now take part in an international survey about genetics knowledge, a GIANT study throws up new genes linked to height, and a romantic gene of the month.
In this episode
Spying on single cells
with Stephan Lorenz, Wellcome Trust Sanger Institute
The human body is an enormous conglomeration of trillions of cells, of probably thousands of different types, all working together. Advances in technology now mean that we can spy on their activity and behaviour, right down to the level of a single cell. Kat Arney took a trip to the Wellcome Trust Sanger Institute to meet Stephan Lorenz, head of the single cell genomics core facility, to discover why scientists need to get up close and personal with single cells, and how on earth they manage it.
Stephan - Well, if you think about it, the cell is the fundamental unit of life. By looking at single cells, you can really study the basic fundamental unit of life.
Kat - What can you study in a single cell if a single cell is – it’s got a set of DNA and it’s got some stuff in it? What can you actually look at?
Stephan - It’s an interesting question. I would make the argument that the oldest and most important biological tool has been around for 150 years and is used to study single cells which is the microscope. So, we have looked at single cells for basically over a century.
These days now, when we look at a particular single cell, we can look at its genome, its transcriptome, its epigenome, and to some extent, also the proteome. What's new these days is that we are now able to sequence both the genome and the transcriptome of individual cells.
Kat - Let’s dig into that a little bit more. So the genome is the DNA, that’s looking at the cell’s instructions. What is the transcriptome?
Stephan - So the transcriptome is basically, you read the set of instructions that’s present in the genome and take smaller pieces of these 3 billion base pairs that we have, and use the transcriptome, which has individual transcripts or RNA molecules who then instruct the cellular machinery to make proteins which are the actual things that make a cell work.
Kat - So they're kind of the message between the genes and then the proteins, the stuff of the cell.
Stephan - Exactly.
Kat - How on Earth do you start thinking about how to study all these messages, these RNAs that are in the transcriptome? How do you get them out of a cell? How do you look at them?
Stephan - These days, we look at the transcriptome and these messages by sequencing them. Over the last couple of years, there has been an explosion of methods that allow us to take these tiny quantities of RNA that are present in a single cell which is typically less than 50 pico grams per cell.
Kat - Tiny, tiny!
Stephan - Tiny, tiny! It’s basically not visible and there's probably not that many balances in the world that could actually measure that. So we take this tiny amount of RNA which is still a couple of hundred thousand molecules really and we use methods that have been around for over 20 years but are now applied in a very particular fashion.
So, it’s still at its core, a very old school method. You reverse transcribe your RNA so you copy it over into DNA. And then we use PCR which has been around for a long time to just amplify this material to a point where we can actually sequence it.
Kat - So, it’s effectively the same kind of technique you'd use if you were sequencing the genome of a person or a bacteria or something like that, but you're getting all the RNA, you're turning it all into DNA, and then just reading that – seeing what's in there.
Stephan - Yes. We need to amplify it a lot by probably between 100,000 and a million fold to actually make it compatible with the current sequencing technology.
Kat - So you make loads of copies and then you read all of them.
Stephan - Yes. We make lots of copies which has its own issues because it’s getting to lose some bios and lots of noise which makes the analysis of the top of data very challenging, but at its core, that’s what we do.
Kat - And then you said the analysis. So you’ve got all these reads, all these sequences at the RNA, the messages that a single cell is making, which I guess tells you which of its genes are working. What sort of things can that information tell you?
Stephan - So by looking at transcripts of cells, you could argue that you can infer the function and even the identity of a cell if you think along cell types. All cells in one particular individual share more or less the same genome so they have full instruction set of doing whatever that genome encodes they could do. But as an embryo develops, they differentiate into different cell types and the transcripts reveals what cells are we looking at and which instruction set the cell is actually performing that is encoded in the genome.
So, you can by taking two cell types, mix them and then do single cell sequence, you could tell them perfectly apart. That’s very high level. What you can also see is if you have a better defined cell type, let’s say a T-cell, you could for example then see whether it’s an active or inactive state, whether it’s been exposed to some antigens and some host defence programme working in the cell or whether during the cell cycle of any cell type. You could see which genes are activated as a cell gets ready to divide.
Kat - I was going to say, so a T-cell is a type of immune cell. Why can't you just look at a bunch of immune cells or a bunch of liver cells? Why do you need to look at just one?
Stephan - We’re not just looking at one. We’re looking at thousands, but each cell individually might be in a different state at a point in time. What we used to do in the old days was look at 100,000 cells and average them. You only get an average response from the tissue of interest you're looking at.
So, what we see more and more is that these tissues are actually complex mixtures of cells with different function or states. So, that response really gets diluted if you just look at the average.
Kat - I guess it’s like doing an opinion poll and you could say, “Well, on average, everyone likes cake” but actually, no. Some people like cakes, some people like biscuits, some people want fruit.
Stephan - Yeah, if you want to go with the food analogy. You could ask people, “Do you like cake?” and they would say yes and “Do you like steak?” and they would also say yes. So your average response is everybody likes cake and steak although if you look at each individual response, you can actually say, “Oh, people that like cake are less likely to like steak.”
Kat - There's been huge advances in this technology over the past couple of years. Where do you think it’s going to go next? Are there more developments that could happen?
Stephan - Absolutely. I would make the argument that we are still in a kind of infant stage when it comes to single cell sequencing. So over the last 3 to 4 years, there has been a lot of attention when it comes to single cell transcriptome sequencing. But the interesting thing to note is that in Nature Methods, which is a journal, the Method of the Year was called Single cell genomics.
So actually, most people at the moment focus on the transcriptome and the functional state of the cell was just the genome of the single cell which we start to realise can actually be quite different across cells of the same individuals. That’s deeply linked to cancer for example.
Then we have epigenetics which are imprinting mechanisms active in cells. So, there will be a lot more attention in the coming years on understanding what information we could gather from looking at genomes of single cells, and at the epigenome.
In our institute, we’ve developed quite a few methods in collaboration with others that allow us to look at multiple things at the same time of the same cells. So we have now methods to look at the genome and the transcriptome so we can then see how certain in the genome actually change the biological programme of the cell. We have similar methods that allows to look at epigenetic markers and the transcriptome. And there's a lot of focus and effort on enabling more multi-omic techniques.
Ideally at the end of the day, what we would like to do is look at the genome, the epigenome, the transcriptome, and the proteome of cells so that we have the full cascade of the classical paradigm of biology.
Kat - The sort of the Ome-some.
Stephan - Yes.
Kat - Stephan Lorenz from the Wellcome Trust Sanger Institute.
09:36 - All genes great and small
All genes great and small
An international collaboration of researchers has discovered 83 new genetic variations linked to human height, according to a paper published in the journal Nature. So far 700 genetic variations have been found that affect height, and these new discoveries add yet more information to the picture. The new findings come from the appropriately named GIANT study, or Genetic Investigation of Anthropometric Traits, involving more than 700,000 people.
The GIANT team previously used a technique called GWAS, or genome-wide association study, to link genetic variations at single DNA letters, called SNPs to height. In this study, they’ve turned to a more in-depth genetic analysis technique called exome sequencing - which looks at the whole DNA sequence of genes rather than snapshots of single letters - to find rare variations linked to height, which are found in less than 5 per cent of the population.
The study is an important proof of principle that this more detailed method can find new and rare genetic variations, as well as revealing the genetic pathways that help to control height.
10:50 - Gene editing fights leukaemia
Gene editing fights leukaemia
Doctors at Great Ormond Street children’s hospital in London are thrilled to announce that two baby girls with cancer, who were both treated with gene-edited immune cells, are doing well more than a year later, according to a report published in the journal Science Translational Medicine.
Back in November 2015, the team announced that a baby girl with leukaemia had been treated with an experimental therapy made from immune cells modified using new gene editing technique. Another baby girl was also treated in December that year, and both received the treatment as a last-ditch attempt when all other options had failed.
Known as CAR-T cells, the therapy involves modifying immune cells, known as T-cells, so that they spot cancer cells and destroy them. Many trials are underway around the world using modified versions of a patient’s own T cells, in order to avoid provoking an immune response against the therapy, although this level of personalisation makes the treatment very expensive and challenging to do. By using gene editing to knock out a gene in T cells that provokes the immune system, bulk-produced modified CAR-T cells can be given to any patient.
It’s not perfect, and just under 1 per cent of the modified T cells still stimulated an immune response, which can be very serious. But once it had settled down in the two treated babies, the modified T cells got to work killing their cancer cells. Further trials are now underway in other parts of the world, making this a hugely exciting area to watch for potential future cancer cures.
12:25 - Revealing rare disorders
Revealing rare disorders
Fourteen new developmental disorders have been discovered by a team led by researchers at the Wellcome Trust Sanger Institute, publishing their findings in the journal Nature this month. The Deciphering Developmental Disorders study - the largest of its kind in the world - has been analysing the DNA of thousands of children affected by previously undiagnosed rare genetic conditions, such as intellectual disability, epilepsy, autism or heart defects, along with their parents, in order to uncover the gene faults responsible.
On average, 1 in 300 children born in the UK have a rare developmental disorder caused by a new fault in a gene, adding up to 2,000 children a year in the UK. To find the genes responsible, the team screened all 20,000 or so human genes from more than 4,000 affected UK and Irish families, focusing their attention on new gene faults, or mutations, that crop up randomly as DNA is passed from parents to their child.
By matching up this genetic information with clinical records, the researchers were able to find children with new mutations in genes that had previously been linked to developmental disorders, but also managed to to spot 14 new developmental disorders that had been caused by spontaneous mutations in a child’s DNA and weren’t found in their parents.
Importantly, the study also revealed that older parents have a higher risk of having a child with a developmental disorder due to this kind of new, spontaneous mutation, with the chances rising from 1 in 450 for 20-year-old parents to 1 in 210 for 45-year-old parents. As part of the study, the researchers managed to provide a firm diagnosis for rare conditions affecting over a thousand children and their families - something that is very important for investigating potential treatments, informing the best clinical care, and getting access to additional health and educational support.
14:41 - Test your genetic knowledge
Test your genetic knowledge
with Robert Chapman, Goldsmith's University of London
Cast your mind back to the Naked Genetics podcast from June 2016, and you’d have heard Kat Arney interviewing Robert Chapman from Goldsmiths University of London about a pilot study he was carrying out to look at public knowledge and understanding about genetics - aiming to find out what people know, what they don't know, and what they think they know, as well as finding out if there are any gaps or areas of concern in specific age or ethnic groups.
Robert - So for example, do people from certain ethnic groups have different concerns to other groups - are there age differences, are there international differences, is the way that genetics is taught at school a predictor of concerns and things like that. So, we're really trying to do an empirical quantitative study, I believe for the first time, in the broad area of genetics. There has been research in this area, focusing on medical genetics, but not generally the issues that they apply across society so that's the new thing hopefully.
Kat - So everything from pea plants to pandas.
Robert - Exactly, yeah. I couldn't say it better.
Kat - So tell me a bit more about the study. What are you actually doing? What are you asking? Who are you asking?
Robert - So I can't give too much away because we've just piloted it and I'm hoping that some or all of your readers will be interested enough to engage with the study when it is published. But we're looking at what people know about genetics so there are general knowledge questions. We're looking at how they feel about genetics. So do they have concerns for example about genetically modified foods? We're also asking for information about their demographics. So this is very much a first stage. We're hoping to talk to as many people as possible. We're aiming for about 5,000 participants and stratified by profession and country. We're asking people whether they're parents or students so we can really build-up a picture of the demographics of our participants and see if there are any trends that we can spot which might help us target training material information more effectively.
Kat - I’m pleased to tell you that Robert’s full survey, the International Genetic Literacy and Attitude Survey, is now online at http://tagc.world/iglas/ - not only is it a fun quiz to test your genetics knowledge, but it’s providing important data to help the scientific community communicate genetics better in the future, so please do take a few minutes to fill it in. That’s http://tagc.world/iglas/
17:34 - Social lives of cells
Social lives of cells
with Alpha Yap, University of Queensland
Our bodies are made up of trillions of single cells all working together - common idea is that they are inert, almost like bricks. Alpha Yap, professor of cell biology at the university of Queensland in Brisbane, Australia, is keen to show that this idea of static cells simply isn’t true. Kat Arney caught up with him at the Royal Institution, where he was giving a lecture, sponsored by the Company of Biologists, entitled “Touching and holding: The social lives of our cells” to find out why.
Alpha - The cell is a fundamental biological building block of our bodies. But the problem we’re thinking about as brick, is that it gives the impression it’s a very static thing and that’s not true. The cells themselves are very dynamic. They're born, they live, they die, and the tissues that are made of cells are themselves dynamic in which the cells will rearrange.
They need to organise themselves. They need to respond to stresses such as the risk of infection or toxins in the environment. They need to therefore be able to perceive as it were whether things are good and healthy, or whether they're under stress, and need to compensate in some way.
Although it’s a wonderful idea to think of cells of our body like the bricks of a house, you would have to think about it as bricks that are busy, active, and actually know where they're need to be.
Kat - So how does this work then? How does say, the cells in my skin talk to each other, know that they're skin, know what they're doing and which way up they're meant to be?
Alpha - Absolutely, so what we’re talking about is communication, biological information. The truth of the matter is, there are many ways in which cells communicate with one another.
The best understood are chemical ways in which one cell will send a signal to another cell. It might be a signal that goes long distance such as a hormone in the body like insulin or cortisone, or it might be a chemical signal that goes a short distance – perhaps 1 or 2 or 4 cell diameters. That’s really quite well understood and it’s been studied extensively over decades.
What is interesting now is that we started to realise that there's another level of communication which is a level of physical communication in which cells use mechanical force to communicate with one another. They push, they pull upon one another and that constitutes as it were a complimentary level of communication that has advantages and disadvantages to the better understood chemical modes of communication.
Kat - So you're telling me that my cells are kind of poking each other?
Alpha - They're sometimes poking each other but especially the cells say of your skin, are actually constantly pulling on one another. That generates tension at the junctions between them. It’s a little bit like a handshake.
For better or worse, we interpret or we infer a lot of information from handshakes – the physicality of those handshakes. And to an extent, our cells do as well. Although my guess is they're probably more intelligent than we are.
Kat - So, they're feeling a nice firm handshake or a limp-wristed handshake or they're missing the handshake altogether?
Alpha - Absolutely, yes. And so, one idea that is starting to develop especially in tissues that are constantly, physically interconnected is that they're constantly pulling upon one another. They can feel the constant tension from their surroundings. They can sense whether that tension changes – whether it increases or whether it decreases – and they respond to that to try and restore the balance of tension.
The point is that sometimes potential stresses in the body - like a cell that’s been infected, a cell that’s been fatally injured by cigarette smoke - that cell generates different patterns of force, and its neighbours respond to that, and respond in a way that ultimately helps to protect the tissue of which they're a part.
Kat - It’s a lovely image to think of all these cells kind of holding hands together, ganging up together, and then when someone breaks that chain, you know that something is wrong. How quickly do these physical signals, these kind of shape signals pass through our tissues?
Alpha - That’s an extremely interesting point. What's I think important is that those physical cues, those physical signals can be propagated very quickly. Theoretically, depending upon the materials involved, they can be propagated as quickly as the speed of sound.
Kat - Doinggggg!
Alpha - Exactly! You could think about it like a neighbourhood watch system. It’s very sensitive. If I were a cell, I could detect something going wrong really quickly. What you sacrifice for speed is specificity. I may know something has gone wrong, but I may not know exactly what the nature of that is, which is why you have other communication cues – chemical cues that we know that provide a different form of information, albeit somewhat more slowly.
Kat - So if this physical connection is broken, you might not know if it’s something really bad like the cell has gone wrong or if it’s just a little shuffle.
Alpha - You could think about it like this – if my neighbour is ill, I sense because of the loss or change in physical connection, I sense that they're ill. I don’t know whether they're ill because they’ve been poisoned, whether they're ill because they’ve actually started to become cancerously transformed. Eventually, I need to know that. But as a first warning, all I need to know is that something is not right.
Kat - How do you measure these forces? Because as a geneticist, I know that we can measure genes being turned on and off, we look for the messages coming from our genes. If you're interested in the molecules being produced by cells you can measure levels of molecules in the blood or in cells themselves. How do you start measuring these tiny, tiny forces that our cells are generating and poking each other with?
Alpha - That’s actually a non-trivial problem because in fact, it’s very hard to measure forces. What you choose to measure depends a little bit upon what you're interested in. If for example, I was interested in a molecule that I think is responding to force, then we now have tools in which we engineer the molecules so that when they become stretched, they change the amount of light that might be emitted from a sensor.
If on the other hand, I'm interested in something much larger like a cell - the extent to which it’s being pulled or pushed - then we have tools to infer the forces. So for example, if I'm interested in the amount of tension between two cells, I can cut the junction between two cells with the laser and I can measure the recoil of that cut zone. A little bit like, if you can imagine a rubber band – the amount of tension that is present in the band can be inferred by cutting it and measuring how quickly that band snaps back.
Those are not very good measures of force directly. And so, we’re at a stage where I think we’re still developing new tools. But the other that’s important is that this is really a multidisciplinary problem. Biologists are terribly bad – at least biologists like me are terribly bad - with physics. Some of us are innumerate and really, what we need to do is to collaborate with physicists, mathematicians, engineers. Because each of the different disciplines brings a different expertise to address the problem.
Kat - And for biologists themselves, is it going to be more and more important that we think of cells as physical objects of the forces, the squishing, the connections, the communications, rather than just being sort of blobs that are turning genes on and off?
Alpha - I think that’s absolutely the case. I think that in a way, what we’re talking about here is a very old science - the impact of forces on biological systems and bodies really appeared in the late 19th century. One of the classic texts is D’Arcy Wentworth Thompson’s great book On Growth and Form, how geometry and physical factors influence body form and shape.
But all of that went out of favour with the genetic revolution and the sheer power of molecular genetics. That’s not to say that that isn’t extraordinarily powerful. But as you say, at one level, we are physical objects – we hurt when we fall, we move through space, and that physicality is something that also coordinates biological behaviour.
Kat - Alpha Yap, from the University of Queensland in Australia, speaking to me at the Royal Institution. Thanks again to the Company of Biologists for sponsoring his talk.
27:07 - Gene of the Month - Panton-Valentine leukocidin
Gene of the Month - Panton-Valentine leukocidin
To celebrate Valentine’s day, for this episode's Gene of the Month we’ve picked the most romantic molecule we could find - Panton-Valentine leukocidin, or PVL. Unlike flowers, chocolate or sexy underwear, this is definitely not a gift that you’ll want to receive from your beloved, as it’s a potent bacterial toxin.
PVL was first discovered by Belgian researcher Honore Van de Velde in 1894, who was searching for chemicals produced by bacteria that could damage white blood cells, or leukocytes - important cells in the immune system. But it’s named after It’s named after Philip Panton and Francis Valentine, two scientists working in the 1930s who discovered that it was produced by strains of Staphylococcus aureus bacteria that caused the most severe infections in rabbits.
Further research revealed that PVL is made up of two proteins LukS-PV and LukF-PV, encoded by two separate genes. Together, they form a small pore in the wall of immune cells, causing their insides to leak out - something known as cell lysis. The contents of these sadly exploded cells than acts as tasty nutrients to feed the bacteria as the infection spreads. Intriguingly, the genes seem to have originally come from tiny viruses that infect bacteria, called bacteriophages.
Just as Panton and Valentine found that PVL-producing Staph. aureus produce nasty infections in bunnies, they also bring a lot of harm to humans, causing a type of necrotising pneumonia that can kill up to three quarters of patients. PVL-producing bacterial strains have also been pinpointed in many fatal bacterial outbreaks.
Given that PVL is found in the majority of antibiotic resistant Staph. aureus - the infamous MRSA superbugs - there’s a lot of interest in developing new treatments that block the toxin, which could combat the growing problem of antibiotic resistance. So although it may have Valentine in its name, PVL definitely isn’t something you’ll want to give the one you love this February.