“HIV is a solvable issue in our lifetime,” said Quin Wills, an Oxford research fellow developing experimental computation techniques for single-cell genomics. Citing high drug prices and lag time to market as barriers, he posited that the way to solve the issue is by making single-cell investigative technology available to more scientists working to understand biological processes in order to develop molecular drug targets.
The team at Oxford used the Polaris™ system to isolate singe cells, expose them separately to different sets of factors and analyze each cell’s unique genetic responses. “Polaris is a powerful democratizing technology that could be used to quickly and cheaply develop drugs to target particular cell mechanisms,” said Wills, who works at the University of Oxford’s Single Cell Biology Consortium (OSCBC). “I see single-cell analysis as a form of molecular microscopy to study how cells behave. Macrophages can be pro-inflammatory or anti-inflammatory, but there’s a whole spectrum in between that we don’t fully understand. The cells adopt a certain phenotype or behavior in a certain microenvironment.”
“When Polaris was released I immediately saw the future of the single-cell science I want to do.”
—Quin Wills, University of Oxford
Wills studies within-patient variability using reverse genetics in lieu of traditional cross-patient genetic variability to analyze function. “Like a lot of scientists, I realized the samples we were studying was so cell-type-specific that studying them at a bulk level was hopeless,” Wills said. “While most geneticists study the differences between people, I study the differences within people.”
“Macrophages are great examples of cells that talk to each other all the time. Their behavior is determined by one macrophage telling another macrophage that there’s too much or too little inflammation out there. They regulate each other’s behavior.” Wills pointed out, “It’s almost like humans talking to each other: How would you be able to react to your environment if I put you in a dark room that completely blocked your opportunity for allowing anyone to talk to you?”
Understanding this communication process may yield important information about the broader role of macrophages in the pathogenesis of HIV. As Wills described it, “These macrophages are like a leaky tap. HIV doesn’t kill macrophages; it just sits there. These are long-lived cells that can go to little corners of your body and slowly drip, drip, drip HIV.” He continued, “The question in our minds with macrophages and HIV is: Does the tap need to be fixed, or does the tap need to be replaced? That is the important point in terms of thinking about how we treat, fundamentally—how we get rid of HIV in people’s bodies.”
For Wills, the impetus to invest in single-cell analysis to study HIV resistance at the Oxford lab was personal. “First and foremost, my interest is in HIV,” he said. “As a gay South African, I’ve seen how devastating AIDS has been in patients and in friends.”To that end, he and a team of OSCBC researchers successfully used the Polaris system to combine single-cell perturbation, imaging and single-cell sequencing on one platform to support their detailed examination of heterogeneous inflammatory cell states.
Four lab members at OSCBC spoke with Fluidigm about their findings around mutant cells carrying the SAMHD1 gene. Kenny Moore, Quin Wills, Esther Mellado and Rory Bowden set out to identify the relationship between this genotype and HIV latency. The confounding factor? Macrophages exist in microenvironments within the body, signaling back and forth.
In addition to isolating single cells individually in chambers for analysis, Polaris monitors cell-to-cell communication. Smart technology zooms molecular biology to the single-cell level to explore heterogeneity and function in utterly new ways.
“At Oxford, we call Polaris our ‘10 Postdocs in a Box’ because what we can do on Polaris now would have taken an entire lab—an entire room and a small army of people—to do, and in a much longer time.”
—Quin Wills, University of Oxford
A virology lecturer and research fellow at the Dunn School at Oxford, Kenny Moore works with macrophages derived from stem cells to research interactions and genetic manipulation between the HIV host and pathogen to learn why some macrophages get infected and others don’t. “Macrophages are studied much less than T cells because they’re non-dividing and difficult to obtain and infect.” Cell lines are notably easier to work with, he explained. “There’s an intrinsic inability to infect all the macrophages.”
Macrophages have predetermined inflammatory phenotypes sensitive to microenvironmental cues. Polaris single-cell approaches enabled the group to collect cell-signaling results to provide clues about the role of macrophages in HIV pathogenesis. HIV infects the macrophage immune cells and either incubates there or acts as a reservoir for later disease. It also contributes to the systemic chronic inflammation seen in infected individuals.
Examining HIV infection in SAMHD1 knockout cells is important for determining the role of macrophages and how genotype and cytokine microenvironments and signaling molecules impact their function. To do this, the scientists individually dosed separate macrophages with culture medium prepared with cytokines and inflammatory signaling molecules. They then measured the macrophages’ responses to their cellular microenvironment and inflammatory conditions.
The team focused on a dNTPase SAMHD1 in macrophages—poorly understood reservoirs of latent HIV infection. Since signaling macrophages influence one another, these must be eliminated to cure patients. “It’s early days in this field,” mused Moore, “but the role macrophages play in the pathogenesis of HIV is a massive question that’s important for clearing infection. Polaris was significant in understanding the SAMHD1 findings and looking at HIV latency in real time.”
Oxford’s data on macrophage response to immune challenge highlights the importance of studying interactions between genes and their environment in the absence of extracellular signaling. “It’s at the heart of investigating virus-host interactions. What heterogeneity allows one cell to become infected but not the other? What effect does the infection have—does it make the cell more inflammatory? The more we learn about why macrophages become infected, the more we see potential drug targets.”
University research lecturer Rory Bowden is deputy head of High-Throughput Genomics at the Wellcome Trust Centre for Human Genetics. “The thing people latch onto with Polaris is capturing cells and setting rules with the temporal aspect,” he said. “You get tight control of cell comparability and fantastic robustness for querying interactions that’s not possible with any other approach.” Bowden described how different cell behaviors make hidden heterogeneity important at the whole-organism level. “The study we designed proved you can do more complicated single-cell experiments with Polaris than by any other means.”
“When Polaris was released,” Wills said, “I immediately saw the future of the single-cell science I want to do: Answer questions when we knock out particular genes we think are relevant; understand how the cells are talking to each other, how they’re shaping the microenvironment and how that ultimately affects the biology.” He added, “We can see important mechanisms and start commenting on potential therapeutic angles for blocking signals the macrophages send each other.”
“We want to study population dynamics as they unfold, not just the input or the endpoints. This is an important thing that Polaris allows us to do. If there’s no temporal history, it’s like looking at a Google map without Google telling you the routes to go.” Wills noted that an important RNA-seq shortfall is that it’s a descriptive catalog of heterogeneity but doesn’t tell you how the heterogeneity got there or what’s driving it.
“We were excited about Polaris when we learned that we could culture cells in individual chambers for up to 24 hours,” recalled Esther Mellado, a research assistant at OSCBC. “You can pre-select cells and see them move in a controlled, real-time process.” Mellado found the dosing rate to be a highlight. “On your own, you can isolate single cells in a closed microenvironment and treat different cells on the same IFC and experiment,” she said. “You can’t do that on other platforms. After using Polaris a couple of times, I found it easy.”
“At Oxford, we call Polaris our ‘10 Postdocs in a Box’ because what we can do on Polaris now would have taken an entire lab—an entire room and a small army of people—to do, and in a much longer time,” said Wills. He described how “Polaris selects cells, puts individual cells in chambers, cultures them, images them and perturbs them—all automatically. Then at the end of the experiment, Polaris analyzes the cells and you can sequence them.”
“The immediate follow-on is that we’re infecting these macrophages now with HIV and observing the infectious dynamics in real time while we knock out genes to see if it makes a difference. That is happening right now and it’s fantastic,” he remarked.
“We would never have begun that without Polaris because a lot of cell processes are tough to synchronize. With Polaris we could see meaningful signals, so we’re directly infecting cells and watching them play out. That’s been the big step forward for HIV work. Polaris allows us to directly control cell signaling and study individual cell microenvironments. We can see at a single-cell level what signal those cells are sending, what they’re secreting.”
Moore speculated about promising follow-up advancements such as the ability to precisely dissect extracellular macrophage signaling components. “Polaris could give unique insights into how different tissue macrophages respond to HIV. That’s where we’d like to go.”
“When people see what they can do with Polaris, they’ll be excited by it. The main thing is the ability to isolate cells and prevent them from talking to each other. That has incredible power for numerous life science applications.” It’s heady science. The OSCBC group has submitted a paper, “The nature and nurture of cell heterogeneity: Accounting for macrophage gene–environment interactions with single-cell RNA-seq,” for publication. The team’s next step is to apply similar methods to the study and better understand the adaptive immune system as a whole.
For a more in-depth look at the project, download Wills’ slide presentation The nature and nurture of 500 gene edited macrophages.
Case Study
Goal
Identify the role of the SAMHD1 gene in HIV pathogenesis and inflammation using iPSC-derived macrophages.
Challenge
The microenvironment influences cellular response in individual macrophages.
Solution
Use Polaris to isolate discrete iPSC-derived macrophages, dose them to simulate microenvironments and perform whole transcriptome analysis to differentiate treated cells.
It’s the only system for simulating and controlling biological conditions to study single-cell responses. Select, manipulate and measure cells over a 24-hour period; specify immunophenotype, viability or fluorescent marker characteristics; and design and execute multifactorial cell studies at variable concentrations and timeframes.
