Scientist Stories: Sangeeta Bhatia, Multiscale Regenerative Technologies & Human Health

Hi everyone. It's a real honor to be here today. Like, like Gigi, I was going to start with a little bit of my story. So I was born in Boston. I'm the daughter of Indian immigrants. And growing up, the conversation in my household was not so much, what are you going to be when you grow up, but are you going to be an engineer or a doctor or an entrepreneur? And so my father actually pointed me in the direction of an emerging field at the time, biomedical engineering. And I went off to college. And by the time I got to graduate school, the first year of graduate school, I fell in love with the liver. And so what I'm going to tell you today is sort of an engineer's love story about the liver. And actually, the liver has long been appreciated as one of the sort of principal organs of the body. This is an image from Greek mythology. It's actually the story of Prometheus, who stole fire from the gods. And Zeus punished him by sending an eagle to chip away a little bit of his liver every day for eternity. So the liver is actually vital for life. And a more modern-day image of the liver's vitality is this one from Frank Netter, who's the classical anatomist. And here he depicts the liver as a factory. It's a factory of over 500 different functions: protein synthesis, drug detoxification, production of bile, and energy metabolism. And if you actually look, peer inside the architecture of the liver, you can see why I love it so much. It's this gorgeous structure. The hepatocytes, the liver cells in this organ, are arranged in repeating units like spokes on a wheel, over and over and over again, millions of these. And so as a graduate student, I got very interested in the role of the architecture of the liver and how that structure related to its function. And it turned out that there actually was a beautiful set of tools that had emerged from the engineering world and those are the tools known as microfabrication. So microfabrication, for those of you who are not familiar, is a way to pattern transistors and circuits on silicon surfaces. And the way this works, as you can see here in the single transistor, is that you coat a surface with a light-sensitive material and you shine light on that material, and then you develop it like film, and you end up with patterns. And so the field, because of the interest in speeding computation, has gotten better and better at patterning surfaces from what used to be a single transistor you see here. So now on the same footprint in this day and age, we can actually fit a billion transistors. And the core process, that sort of light-based patterning of the surface, is depicted here, is called photolithography. It turns out to be a very useful way to pattern cells, to put them in different configurations and study the relationship between the structure and the function. And so I got interested to do that as part of a community of researchers investigating this. If you peer even further inside the liver, what you can see is that the hepatocytes, the liver cells, have very interesting arrangements. So they have contacts with one another. We call those homotypic interactions. And they have contacts with other cell types in the liver, heterotypic interactions. And they also have interactions with blood that's flowing through the system. So there are mechanical interactions like shear stress.. So there's lots of structural interactions in the liver that we were interested to study. And we knew as a community that if you take these cells out of this architecture, so if you perfuse the liver with an enzyme like collagenase and you disperse it, the liver cells, the hepatocytes, they lose all 500 functions. So the structure is very important in their function. So we set out to sort of study this with microfabrication techniques. And just to give you a sense from the kinds of things that the community had been working on, these are single-cell structures that you can make now by patterning plastic surfaces that cells can grow on in the laboratory instead of silicon surfaces. So here you can make a perfect circle of a cell. Here's a 5-pointed star. Here's a bowtie shape of 2 cells that have been patterned to sort of kiss perfectly right here in the middle. This is a multicellular island that was created to test mechanical tension in this eccentric donut. So the structures that you can create that were borrowed from microfabrication and now brought to cell culture allowed us to start studying studying some of these interactions. In fact, these are completely arbitrary tools. This is a pattern that my postdoc Tal Danino made with an artist in residence at the MIT Media Lab. I mean, you can see that this structure is actually made of cells all the way down to the sort of single-cell level. So this is a completely arbitrary set of tools. So as a graduate student under Mehmet Toner at Mass General Hospital, I got very interested in what I was telling you about before, the homotypic and heterotypic interactions in the liver and how the function of this organ might emerge as it related to that structure. So we did very simple things like make colonies of hepatocytes of different sizes to vary their homotypic interactions. And then we started to add other cell types here to look at their heterotypic interactions. And here, for example, what we did was add another cell type a fibroblast. So this is a cell type that is really thought to be just a simple structural support. And it turned out that we found really empirically that this combination of homotypic and heterotypic interactions could allow you to now stabilize the function of these hepatocytes outside of the architecture of the liver. So now they're sitting on a two-dimensional flat surface in these colonies that actually don't look very much like the architecture I showed you earlier.. But in spite of that, we were able to take these cells and get them to live for weeks in culture, where they used to die— all 500 functions used to die, decline very rapidly. So as a graduate student, I had done this first with rat cells. I was a graduate student in a program at Harvard and MIT where you take a lot of medical school classes, and I ended up finishing my medical degree as well. So when I started my lab, I got very interested in using some of these concepts now to study human disease. So we adapted this model system to human cells that came from patients, from cells from patients where the organs had been consented for donation. Now we wanted to start asking questions about human livers that you could ask in a dish where you had a stable human liver for, let's say, a month. We wanted to ask questions that were very challenging to ask in model systems where, for example, rodent livers didn't reflect that reflect the human liver biology. So in order to do that, we needed to develop another set of engineering tools. This is a tool that we created where we could create these micro-patterned co-cultures that we call them, now in a multi-well format that biologists could use. And I'm going to skip the engineering piece of this, but there was quite a bit of engineering and sort of adapting the patterning methods of microfabrication now to these sort of plastic multi-well formats. And you could make— here's a 24-well version, a 96-well version, a 384-well version. And if you peer inside these, you can see that we have about 40 little human micro-livers in each well of this plate. Over the years then, what we went on to do was study human liver diseases, as I said, for which there are not good model systems. And so this is just a snapshot of some of the things that we've done. Here we're looking at drug-induced liver injury. This is so-called DILI. So human-specific drug toxicity that was not apparent in rodent or primate studies and only showed up in patients. This is sort of a very well-known case of a drug in the '90s called thalidomide. We grew hepatitis C in collaboration with Charlie Rice at the Rockefeller and studied antivirals. And as many of you may well know, hepatitis C has actually recently been cured, excitingly. And so we went on then to study hepatitis B.. And so these model systems of the human liver can now be perturbed with drugs or with pathogens in vitro and to get a glimpse into the human biology. So we published this paper in 2008, and right about that time I got invited to go to the Gates Foundation. They saw the paper and they asked us to work on another pathogen, which many of us don't think of as a liver hepatotropic pathogen, but in fact it is. And that's the malaria parasite. So this is an example of what happens when you get bitten by a parasite-ridden mosquito. The parasite actually first homes in on the liver inside a hepatocyte where it grows for about a week to 10 days silently, amplifies, and then it bursts out into the blood and infects the blood cells and gives the symptoms of fever that we all recognize malaria to be associated with. Associated with. At that stage then it has a sexual cycle and a naive mosquito can bite this person and transmit the disease. And so what I learned in 2008 was that there's a renewed call for malaria eradication and there was an increased focus on the liver stage of the parasite. It's actually a really attractive place to think about battling the disease. There are no symptoms at that stage. It's prior to amplification, sort of 10,000 times that it grows in the liver. And if you could kill it in the liver stage, you would also prevent transmission. And the challenge in the field is that there were no good models of the liver stage organism. So we set out to tackle that. In particular, there's a kind of malaria— there's falciparum and vivax. Those are the two main kinds of malaria. Vivax actually has a dormant form in the liver. It's called the hypnozoite because it's hypnotized. And it is the single major barrier to eradication of this We don't have any drugs that kill it. We knew very little about it until recently. In fact, when it was first described in 1982, which is really not that long ago, this is really all we knew about it, these little glimpses of micrographs. This is a liver biopsy of a chimpanzee that's been infected with Vibax. So we knew that this was the dormant reservoir, that this could wake up again from its hypnotized state and reinfect the patient and reinfect the population, but we didn't know much more about it. And so we set out to sort of capture this whole life cycle of the parasite in our in vitro model system. So it took us about 10 years. 2 months ago we accomplished this. I mean, this is just a snapshot of that. So now you're looking inside these multi-well plates of the micro-patterned co-cultures. This work is done in Bangkok with our collaborators at Mahidol University. So my graduate students fly over there and infect these microlivers with vivax from patients. And here you can see this little green form is malaria parasite inside a hepatocyte that's been stabilized in one of our cultures. If you wait a little while, the dormant forms stay dormant and the big forms grow and burst out into the blood. And after 3 weeks, all you have left now are these dormant forms. And so now we've gone from those little glimpses on the early 1982 micrograph that I showed you of the hypnozoite to actually growing hundreds and hundreds and hundreds of these elusive forms. And we've just published now the first molecular description of this parasite, so the transcriptome. We can ask questions about anti-malarials, about vaccine development, and then importantly about what it is about the host cell that allows it to harbor this dormant form. So we've sort of found our way to the science of malaria through the engineering of the architecture of the liver. There's much more to do. You can see just sort of the time scale of science here. It took us 5 years to grow the first form. That was falciparum. And another 5 years to grow vivax. But once you get the organism growing, the science sort of opens up. So that's been really exciting. So the second thing about the liver, which I love, which I didn't mention earlier, it's actually going back to this picture. There's a remarkable thing about this, and we sort of argue in the liver community about whether this was prophetic or not or accidental, is that the, the eagle comes back every day to eat the liver, and every day the liver grows back, and the eagle comes again. And in fact, we know now that the liver has an enormous regenerative capacity. Regenerative capacity. So this was first described in the 1930s in this paper where if you can cut out 70% of a rodent liver and it will grow back in weeks. This is regeneration without a stem cell. It's actually the adult hepatocyte that undergoes mitosis. And this was beautifully described even further by Nancy Booker who, she's an amazing scientist based in Boston. We lost her last year at the age of 104. And in the 1950s, she did this incredible experiment where she showed that the factors that drive regeneration and expansion of the liver are actually blood-borne. And the way she did the experiment was to hook up two rats— that's called parabiosis— and injure the liver of one rat and watch the liver of the second animal grow. And so since that time, it's been clear that there are blood-borne factors that could create expansion of the liver. So we were inspired by this experiment and wanted to take our micro-livers, which now have stabilized function, to the next level to think about, could we now think about what architectural cues would allow them to proliferate and expand? And then, of course, could we potentially do this for patients? So here we borrowed another set of engineering techniques. This came from the rapid prototyping field of additive fabrication. This is commonly known now as 3D printing. And again, it's light-based patterning. You just shine a light now on a vat of a material and a layer-by-layer process, and you end up with a 3-dimensional part. And we thought sort of in the early 2000s, wouldn't this be interesting to do for livers? So it took us about 5 years, but we figured out a material system that hepatocytes could live in that could similarly be photocrosslinked. And I'm going to sort of just whisk through this, but to show you, we have made all kinds of structures now. Using this combination of microfabrication and light and biomaterials and here electric fields and molding processes and not just one cell type but multiple cell types to sort of explore the architecture of liver cells now in 3D materials and their dependence on function. And now that we're in 3D, we can ask— we can introduce another item, which is the effect of space. Space and blood flow. And so here what we've done in collaboration with Chris Chen is now actually 3D print a sacrificial system that will become the voids through which we want blood to flow. And so here you see that here. So this is a sugar layer that has been dissolved. These are endothelial cells that line it, and these are hepatocyte liver cells in 3D aggregates around it. And so this becomes— turns out to be a structure that's ideal both for preserving hepatocyte function and hooking up to the blood vessels of the host when you implant it. And then we set up to actually do the experiment that I described that Nancy did initially in her parabiosis experiments. And so here what we've done— oops. Okay. Here what we've done is create a scaled-up version of that structure that I described to you. We called these optimistically seeds, hoping that they would grow. We put them in a modern-day version of Nancy's experiment, which is a genetic version of the liver injury. And here you can see the control experiment where the red aggregates are the liver cells. And when you injure the liver of that animal, hoping that the blood-borne growth factors will allow the expansion of the graft, you see that they grow about 50 times. So again here, the engineering now has given us a framework now to explore the science of regeneration. So I've told you just really two vignettes of the work that I've done and the sort of field I represent, but actually it turns out there's many, many tools coming out of engineering that allow us to sort of probe this sort of sub-100-micron length scale. And in our lab, we're now going even down to the nanoscale and making materials that are so small that they can enter into human patients into an intact liver and interrogate the liver in situ. So that's sort of a whirlwind love story of an engineer in love with the liver. And I just wanted to conclude by saying that in recent years I've been very aware that as I progress in my career that there are fewer and fewer sort of people like me, woman, engineer, physician, scientist, mother, daughter of immigrants. And so I've been working hard on diversification of the pipeline. These are actually my two daughters with my husband. They're in our lab. She knows to wear gloves. This is a middle school outreach program that we, I helped to start at MIT. It's now about 20 years old. This is a postdoc program to help postdocs actually engage in public policy. This is an art project to change the dialogue around vaccination. This is a social media campaign to change perceptions around stereotypes of engineers. And I don't know if you can see it, but it says #ILookLikeAnEngineer. And finally, I know that this is sort of a controversial venue for disseminating science, but actually I used it as a way to talk to the public about how nanoscale materials can impact human health. And so far it's reached about a million people. So I think, you know, it's something that we should all sort of engage in and think about in terms of how to engage the public with the work we do. So I want to just close by saying thank you. Thank you for inviting me here. Thank you to family, mentors, trainees, team members. And just to say that I feel very lucky to have been trained here in the public school as the daughter of immigrants. And actually, in the end, I got to be an engineer and a doctor and an entrepreneur and a scientist. Thank you. Go ahead. Thanks. That was great. So if you develop a drug, which stops the parasites going to the liver and/or stops them growing from the dormant state? How will you know who to treat with it? No, it's an excellent question. So it turns out that another thing that you could do, which is not on my list, is try and use this system to develop biomarkers. And so that's exactly what will have to happen hand in hand. What is the nature of the structure-function relationship? How much structure do you have to add back to recover functions? With 500 functions, do you need a linear proportion of the structure to recapture those 500 functions? Yeah, that's a great question. And I think we are just finding our way through the 500 functions. So I told you we bucket them in 4 major classes. And as a hepatology community, we prioritize them by energy metabolism because that's probably the most vital for life. So protein synthesis, you probably can give those functions with, with biologics. That's less important. Production of bile, not so important unless it kills the hepatocyte. So mostly we focus on the energy metabolism and drug detoxification functions of the liver.. And for those functions, so far we found that you need two other cell types in addition to the hepatocyte to recover them. But the more we probe, the more we find. So for example, the immune system is really important in the liver, and I ignored that completely. So now we're adding those back and asking, do you now get the tolerance function of the liver, which it's known for? The deeper you go, the more you probably need to capture. Thank you.