This podcast is about two scientists, Dr. Patrik Ståhl and Dr. Fredrik Salmén, who are joint first authors of a paper that kickstarted a field. It's about finding work they did with colleagues to enable finding out where in tissue gene expressions is happening. It's called spatially resolved transcriptomics. It is a Nature Methods Method of the Year and I did a story about it here: https://www.nature.com/articles/s41592-020-01033-y . 
This is a podcast series that shares more of what I found out in my reporting. The piece is about patience, stamina, friendship, surfing the Baltic Sea, genomics and imaging. 

[00:00:05.560] - Vivien Marx
Hi and welcome to Conversations with Scientists, I'm Vivien Marx. This podcast is with and about two scientists and about space space in biology. Actually, you'll meet Patrik Ståhl. He's on the faculty of KTH Royal Institute of Technology in Stockholm, Sweden, and Fredrik Salmén, who is currently a postdoctoral fellow at Hubrecht Institute in the Netherlands. They will talk about a field.

[00:00:33.280] - Patrik Ståhl
The whole field. It's really it's it's an awesome field.

[00:00:36.940] - Vivien 
That's Patrik Ståhl. Their work led to a major publication in the journal Science, and they are both joint first authors of this paper,

[00:00:47.710] - Patrik Ståhl
We share the honor

[00:00:47.710] - Fredrik Salmén
and the pain.

[00:00:47.710] - Vivien 
The honor and the pain. That's research for you. Just briefly, before we get to that about this podcast series, in my reporting, I speak with scientists around the world, and this podcast is a way to share more of what I find out. This podcast takes you into the science, and it's about the people doing the science. You can find some of my work, for example, in Nature journals that are part of the nature portfolio. That's where you find studies by working scientists.

[00:01:19.960] - Vivien 
And those are about the latest aspect of their research in a number of these journals offer science journalism. These are pieces by science journalists like me. This podcast episode is one of several I'm producing about space in biology. Months ago, I interviewed researchers who work on Spatially resolved transcriptomics for a story and in my slowpokey DIY podcast production. This is part one in a series about this field of study. So Patrik Stahl and Fredrik Salmen here they are introducing themselves to help me learn how to pronounce their names.

[00:02:02.890] - Patrik Ståhl
Fredrik you go first.

[00:02:03.560] - Fredrik Salmén
Fredrik Salmén. 

[00:02:12.290] - Vivien 
All right. I have to practice. OK, so in

[00:02:16.750] - Patrik Ståhl
English it's Patrick. It's Patrik Stahl.

[00:02:21.650] - Vivien 
Patrick Sahl?  So no t, Stahl

[00:02:29.210] 
all right, you have to brace yourselves.

[00:02:33.980] - Patrik Ståhl
Stahl means steel in English,

[00:02:36.393] - Patrik Ståhl
Patrik Ståhl

[00:02:36.780] - Vivien 
Wow I apologize . Despite their lessons, I am doing the Swedish pronunciation of their names badly. I hope they and Sweden will forgive me. So I interviewed these two Swedish scientists together and when we started to chat, I noticed a poster on the wall behind Fredrik Salmen. It showed a surfer riding a big wave. So I asked about that.

[00:03:03.530] - Patrik Ståhl
Fredrik actually quite advanced surfer, like wave surfer  at the time when we started this project.

[00:03:14.540] - Fredrik Salmén
Yah, it's true. Oh, it's actually me.  It's a little bit self-centered, I guess, to have their own picture on the wall. But it's fun, though. It's

[00:03:27.620] - Vivien 
where was this taken?

[00:03:30.290] - Fredrik Salmén
This is actually Sweden. So it's the Baltic Sea.

[00:03:35.900] - Vivien
The Baltic Sea is cold. You need to wear a special suit if you want to surf there.

[00:03:41.240] - Fredrik Salmén
Yeah. It's like a frog suit with hood and gloves and boots.

[00:03:45.920] - Vivien
So do you still do this or.

[00:03:48.320] - Fredrik Salmén
Yeah, I still do. I'm a little bit, I would say much less nowadays and I'm also a little bit heavier these days, so not as agile anymore. But still when I get the opportunity I try to surf, it's nice. 

[00:04:06.020] - Vivien 
The two researchers worked together along with many others, but their connection was quite intense and you will hear more about that in this podcast.

[00:04:13.260] - Vivien
It was work that took around six years and led to a publication in the journal Science. And that publication kick-started a field. And there was a company spin out to the field of study is called spatially resolved transcriptomics, and it was crowned a Nature Methods method of the year. In this area of spatially resolved transcriptomics, scientists want to know where something takes place. It's part of understanding larger issues, such as why does the head grow where it does?

[00:04:44.750] - Vivien 
Why does a part of the brain develop where it does? Why does a tumor grow where it does? It's genes that tune such events, genes are turned on or off, they are expressed at high levels or low levels or silenced, their expression can shift. With gene expression, it's like tissues are playing a kind of music, just one you need to find ways to hear. Patrik Stahl and Fredrik Salmen and their colleagues found one way to do just that.

[00:05:15.370] - Vivien 
The work took place in Sweden. It involved surfing the cold waves of the Baltic, as you just heard. It's about friendship. It's about patience, about science, careers. If you're interested in any of that, as well as biology, genomics and imaging, please stick around. So this work in particular took six years and Fredrik Salmen and Patrik Stahl worked intensely together. They are the first authors of this paper in Science published in 2016, and it led to a company called Spatial Transcriptomics.

[00:05:45.790] - Vivien 
What these scientists and their colleagues developed was a way to see where, for example, in a tissue genes are expressed. It's not the first way to do this, but it was a way to analyze a lot of mRNAs, a lot of gene transcripts at the same time. To understand why this matters, we can step back for a moment and consider a practical example that they told me about. A pathologist gets a tissue sample. It might be from a person who was just on the operating table.

[00:06:13.300] - Vivien
The tissue is prepared with chemical stains and then studied. The pathologist interprets what is going on in this tissue. Sometimes pathologists look at many tissue slides from many patients and want to compare them. In other cases, it is information that has to travel quickly to determine how a patient might need to be treated. Or the analysis is for a basic research lab that is studying a particular disease or development. As Patrik Stahl explains, scientists can look at a tissue slide and use stains and dyes to see what is happening there.

[00:06:46.630] - Vivien
Well, sort of. This immunohistochemistry doesn't always answer all the questions of pathologist or other scientists might have

[00:06:55.990] - Patrik Ståhl
So I think this was like late 2009 and it was Jonas Frisen, who is a who is, s stem cell professor working at Karolinska Institute who is subjected to this kind of immunohistorchemistry a lot during his daily work. And I think that he was the one who first grew tired of a lack of spatial information that they could get out of a stain. And so late 2009, he contacted  Joakim Lundeberg and they together in early 2010, initiated this project , trying and then they had this idea basically, I know, putting barcoded reverse transcription primers in an ordered fashion on a surface. And early on, they they brought in Fredrik as a master's student. At the time I was not involved. I was still writing my PhD thesis

[00:08:11.110] - Vivien
At the time. Fredrik Salmen was a master's student at KTH and Patrik Stahl was a Ph.D. student at KTH. He remained at KTH after his dissertation in 2010, then started on this project.

[00:08:24.880] - Vivien 
During the gist of this project, Fredrik Salmen became a PhD student in Joakim Lundeberg lab at KTH and Patrik Stahl was a postdoc in Jonás Frisen's lab  Karolinska Institute. This was a collaboration between University Labs

[00:08:42.460] - Patrik Ståhl
Science for Life Laboratory, where we are sitting, that's a joint effort between the Royal Institute of Technology KTH and then Karolinska Institutet and Stockholm University, which means that we were all sitting together more or less. Jonas Frisen he had a separate lab also sort of up the hill, but quite close to where the rest of us were sitting.

[00:09:10.420] - Vivien
The approach the scientists developed involves working with fixed stained tissue and getting landmarks of gene expression.

[00:09:19.120] - Vivien
This is how it works. The tissue is imaged then treated so it becomes permeabilized. That process releases the mRNAs that move down and attach to an array that is below the tissue. This array holds barcodes. The mRNAs gets stuck in place. At the spots where they are fixed. The mRNAs are reversed transcribed, the tissue is dissolved and what you're left with is spatially barcoded, complementary DNA affixed to an array. Then you can use sequencing. When the complementary DNA is sequenced you get spatially resolved transcriptomics: the barcodes are identifiers for the mRNAs.

[00:09:58.330] - Vivien 
So the platform tells you which genes are where because you have the original imaged tissue slide as a kind of reference. That's the Science paper. The team has set out with ambitious goals. They had wanted to capture them RNA  from every cell in the tissue and they wanted a lot of other things. Here's Fredrik Salmen.

[00:10:19.630] - Fredrik Salmén
We really wanted to aim for single cell at the start. And this is I mean, now you can see this is not what we published in the end in 2016, we went for some kind of larger spots, around hundred micrometers. But early on, we really wanted to go down to that level and was very tricky because we didn't have the technology ourselves or the knowledge how to make them. So we had collaborations with companies and other groups that could do this.

[00:10:48.520] - Fredrik Salmén
And it yeah, it turned out to be extremely hard to make arrays that has this small spots to capture single cells and at the same time have great quality, so great quality on this reverse transcription probes and a lot of them on the surface that we needed. And that is one of the issues. Another one was the diffusion, so we were we were worried that we might have diffusion. So to get RNA out from the cells, you had to permeabilize them somehow.

[00:11:25.020] - Fredrik Salmén
And we didn't know if they would if the cells burst and if everything just floats away and hybridizes is wherever on the chip, or if it actually went locally and just to the to the closest spot.

[00:11:40.740] - Vivien
They also had to worry about transcripts floating away and not drifting down onto the array from the location they had in the tissue. Here's Fredrik Salmen and Patrik Stahl.

[00:11:51.090] - Fredrik Salmén
Yeah. So if they would move horizontal. Right. You have a problem because then the spatial information, is gone. Right. Because if one cell here and the capture areas here and RNA go like this, then you have expression of this cell, this cell over there. But I mean, it will probably spread more everywhere. So you have a mixed expression pattern across several cells or larger areas.

[00:12:19.140] - Patrik Ståhl
When we when we started doing this, I mean, there were not because of the things Fredrik explained, there were not many people that thought this was going to work because essentially you put tissue onto a microscopic glass slide. And you treat it to make essentially the molecules go out of it or at least not stick so hard. And obviously everyone thought that this diffusion was going to go crazy. But then we early Fredrik actually came up with a very good trick.

[00:12:51.990] - Patrik Ståhl
And that was to do the initial reaction where the mRNA from cells meet the probes on the surface was to do, this initial reaction, reverse transcription reaction using fluorescent nucleotides. So when you do that, you actually get a very nice, which is figure one in the Science paper, you actually get a very nice fluorescent footprint, of where everything went. So where you actually but the captured. That was for us, that was a gigantic stepping stone to get the rest to work and into getting everyone's kind of appreciation that this actually was going to work. And that was a very big sort of point for us.

[00:13:45.070] - Fredrik Salmén
I remember what you say, Patrik, it's nice. Because I remember the nonbelievers in the lab and, you know, they were all just at that point convinced more or less that it might work. And that was was nice.

[00:14:01.300] - Vivien
The approach they developed brings together imaging and genomics, computing an an old way of capturing gene expression, namely microarrays. The new approach melds all of these together.

[00:14:13.750] - Patrik Ståhl
So, I mean, we were essentially bringing together, kind of the best in microarray  technology, with the best in imaging, with the best in sequencing and with the best in actually bioninformatics analysis as well. We were doing barcoding, we were doing unique molecular identifiers so we can have everything at once. And as you say, I mean, I think that gave us a huge headstart in a way, in the field. The melding,

[00:14:42.640] - Fredrik Salmén
The microarray provides expression. Right. But they don't provide the spatial about but what we did in the project was combining the two. And like Patrik Stahl with the sequencing, which is what took over a little bit after the microarrays for the actual expression.

[00:15:02.140] - Vivien
Because the approach was so new and different. And unlike other methods, the team didn't have an easy time to publish it.

[00:15:10.480] - Patrik Ståhl
I mean, we had a pretty rough kind of run to publish. I mean, it could have been smoother. We had some pretty critical reviewers at one point as well. So, I mean, Science was a pretty regular process. But we were actually, we had submitted to another magazine before, and I think it was we experienced a little bit of like this what every scientist fears, which is this peer review process where you're ayou're unsure about everyone's motives because it looks like I mean, this was clearly very novel. But apart from that, I think everything was very positive reaction to the publication.

[00:16:01.690] - Vivien
This project took a long time and the two scientists were both starting out. And I wondered how this is all shaped their career.

[00:16:10.990] - Fredrik Salmén
Yeah, I definitely feel it was a career-maker for sure. I would say so. Not only the outcome of it, but the whole process. So how much should we actually tested, you know, how much things we learned in the different fields, working with tissues and sequencing simultaneously, like you say, traditionally two separated areas. And then, of course, also in this field is always counted the scientific output, right. So if you publish something high, you're very likely to to get to know your position in the ladder.

[00:16:45.520] - Fredrik Salmén
So I think if you only look at output, what's on the paper, but you didn't actually learn anything, then I think your career probably is going to slow down or stop a little bit after that because you don't have any anything to build it on. But I think at least for me, I learned so much during these years.

[00:17:05.170] - Patrik Ståhl
I agree, you definitely learn like what it's like to be on that level to try and try and do a really high level publication all the effort that goes into it.

[00:17:17.760] - Patrik Ståhl
And then also like learning, because being a technology developer can sometimes be kind of difficult. You're not getting that many grants for pure technology development. You can do a super nice method, but it's not going to be used anyway. so this was like really. Yeah. This was kind of a testament for us to know that actually can pay off. And sure like for me also definitely a career maker, I got my postdoc grant based on this.

[00:17:52.330] - Patrik Ståhl
I got the starting grant based on this, a position at KTH, now I'm associate of. So, yeah, it's like, of course, it's. You can't complain, although although I mean as Fredrik says, it's a lot of work and it took a long time, actually,

[00:18:16.730] - Vivien
When you develop a method, you want it to be used. You don't want to be the only researcher using it. There is one circle of users, Fredrik Salmen and Patrik Stahl would love to help. They dream about helping pathology use this kind of gene expression analysis, maybe even use it to the point at which pathologists no longer need imaging. That is a tall order. Of course,

[00:18:40.100] - Patrik Ståhl
The big thing as a technology developer is to see your technology used somewhere else. I mean, that's the ultimate proof that, you know, that you did something that that that's actually good. And I guess that that came after a while and people were able to start using these arrays in other other parts of the world.

[00:19:00.080] - Fredrik Salmén
So, yeah, it sounds a little bit like a cliche, but I would like to see it be used in pathology. And so I think that was a little bit of that of the idea when it was, you know, when when the concept was created. It would replace or at least complement the pathological analysis to the instead of the staining in the future. So obviously, it's not the case at the moment, but this is where I would love to see this actually take place and would be nice if that could add another another level to how the treatment is selected and help out in the future. That's what I would like to see.

[00:19:43.490] - Patrik Ståhl
We have this little dream or we still do about like something we call digital pathology, essentially. And then I would like this technology allowing an unbiased analysis of the tissue almost with the appropriate resolution, almost without having an image of the tissue you could actually be able to like just computational, deduce. Based on the gene expression patterns, you know, what's areas without even staining tissue what areas are actually part of what subclone of a tumor , for instance?

[00:20:23.630] - Patrik Ståhl
But we also like we have a lot of discussions early on. We also realized that for this concept to come alive, you know, that it would have to be kind of adopted by the pathologists. And making them, I guess, lenient to rely on this type of data because. And that's going to be a difficult trick. And they are used to kind of being in control of the annotation and not leaving a machine  do it. Although we think and we also think that we have shown in a few of the papers now and that sometimes spatial data can actually be more accurate than the manual annotation. 

[00:21:13.520] - Vivien
This is something for the future, not the present. A lot is needed to get there, less tissue staining and more use of just gene expression data and a certain resolution and a cultural shift. Here's Patrik Stahl.

[00:21:32.660] - Patrik Ståhl
One obvious one is to have resolution at a suitable level. I'm not necessarily a fan of having a maximum resolution like one, two micrometers. I think that that may even be like counter-productive. I think that maybe like single cell  like 10 micrometers, may be the sweet spot, because you want also to have enough data  linked to every pixel. So I think that's like the lowest hanging on the wish list, I guess. Then if you look at the commercial version that 10X released, the Visium.  Obviously they have improved the efficiency, like the sensitivity quite a lot. And with some upgrades enzymes, I think. So so that's kind of been partly taken care of, I guess. And yeah, I guess people would like an additional stains and  additional applications.

[00:22:38.190] - Vivien
The technology led to a spinout called Spatial Trascriptomics. That company was bought by 10X Genomics. That was in 2018. And there is a continued connection to 10X Genomics.

[00:22:52.710] - Patrik Ståhl
Yeah. So 10X and KTH, we have like a collaboration. It's like an academic collaboration essentially. So we have some involvement based on that.

[00:23:09.360] - Vivien
The technology is commercialized and out there and it seems to the team, many labs are using it. When mRNA is synthesized in a cell, it is processed one and ends up becoming polyadenlyated. At one end, the RNA molecule will have a series of adenine molecules, maybe 50 or even 200 of them. It's called a poly (A) tail. This tail helps to stabilize mRNA. Patrik Stahl talks about how labs are using the technology, his lab and others, and about the importance of the poly (A) tail.

[00:23:42.880] - Patrik Ståhl
Yes, I think like on and on the level of like current application areas, you know, I mean, obviously internally we like we essentially have projects within everything. I mean, everything from, you know, organelles to cancer to to plants, to developmental tissues. So really everything and and possibly even other  vertebrates and obviously mouse and so on but even others. And so I guess, you know, statistically, I guess, 10X will have the figures on where they are selling  their products.

[00:24:27.580] - Patrik Ståhl
But my general view is that it seems to be kind of very widely adopted at the moment. Maybe because you have this kind of general capture capability, right. It's about capturing anything that's poly (A) tail. So, so and I guess people are still kind of trying it out for anything and obviously and then you have this kind of crossover from, you know, people that have been running like single cell stuff and now they want to do a spatial. And then you have know a lot of people kind of integrating, I think, starting to integrate spatial and single cell data, which is really cool because single cell data works as a very neat validation or even resolution enhancer of the spatial data. And so they're working very well together .

[00:25:30.080] - Vivien
The technique they developed is a wet lab process and it also has a computational side to analyze these data, take software and computational power.

[00:25:40.120] - Patrik Ståhl
I think we could easily have as many computational people as we have in the lab. I mean, there are enough things to do. I mean, it's really I think it's structured differently in different research groups; like in our group, we we try to let all the PhDs can learn in all parts of the process so they get proficient in bioinformatics part as well. And but we can say that, like, once they generate the data, set a couple of data sets, then they can sit for a while, you know, with the data processing, it's not that straightforward. Especially for new tissues, you have to come up with like, what's the best way of doing this? Before Fredrik left we also had this project and we're going to predict like immune cell patterns in different issues. So, yeah, there are tons of things to do on both ends.

[00:26:42.680] - Vivien
Developing a technology takes patience and stamina.

[00:26:47.300] - Patrik Ståhl
I think what's interesting maybe for listeners to hear is kind of a little bit like I'm in the process of actually developing a technology or a protocol like this and kind of how how hard we struggled, because it was a long struggle. And we have like there are a couple of anecdotes around this. I don't know, Fredrik, if you want to say anything around there because you were constantly in the lab.

[00:27:15.830] - Fredrik Salmén
Yeah, it's true. I mean, as a PhD student you're you're expected to be in the lab long hours. Right. And late. But also, if you have a passion for what you're doing, then it's then you don't mind to do it. But like Patrik said, method development is, it is tough. It's 95 percent of your stuff are going to fail, 95 percent of what you what you try are going to fail. And we're not talking about fail for six months.

[00:27:45.800] - Fredrik Salmén
We're talking about fail for like four or five years maybe. And so so you have to keep on at it. I think if you have the motivation, it's easier if you don't have the motivation, of course, it's it's much tougher.

[00:28:03.260] - Vivien
This kind of science is about people working together.

[00:28:06.830] - Fredrik Salmén
You need a good group. And I think we had. Patrik and me and we had so much brainstorming meetings all the time. And obviously other people also. And I think it was a very supportive environment in general for the project, and that's very important. But if you're by yourself, if you be only one person driving the whole project, then it's probably not going to go far, I would. We had many talented, people PhD students, postdocs, PIs  working very hard on this for a very long time. We kind of said, not to mention anyone, so that we don't forget anyone. But I think everyone else who they are. An anecdote, which I think is fun, Fredrik had like a few of these like office binders, filled with bio analyzer biomolecular traces. And everyone who is running BIoanalyzers know you get one or two pages with your traces .

[00:29:16.460] - Patrik Ståhl
He was essentially filling these up,  there must have been like hundreds of I mean, it's crazy, really, when you think about it, because, you know, it's one thing to make it work, but it's another thing to make it efficient enough that you want to publish and you want to, like, go out there and show the world what you've done. So in that way, maybe we were too perfectionist, but I think it paid off in the in this case. Because people could adopt this, you know, from from the like the first day.

[00:29:47.120] - Vivien
Looking back, they see how the field has evolved.

[00:29:50.330] - Patrik Ståhl
But I mean, the whole field is really , it's an awesome field.

[00:29:54.470] - Vivien 
And looking back at the process, they are both proud of the work and their continued connection. They share first authorship of this paper.

[00:30:04.520] - Patrik Ståhl
We share the honor.

[00:30:08.270] - Fredrik Salmén
And the pain.

[00:30:11.160] - Vivien 
That was conversations with scientists. Today's episode was with Dr. Patrik Stahl at KTH Royal Institute of Technology in Sweden and Dr. Fredrik Salmen, a postdoctoral fellow at Hubrecht Institute in the Netherlands. And I just wanted to say, because there's confusion about these things sometimes, these scientists and their institutions did not pay to be in this podcast. This is independent journalism produced by me in my living room. I'm Vivien Marx. Thanks for listening.