Lung Organoids: A Novel Way to Model COVID Infection

Calvin Kuo, MD, PhD, with Shannon Choi, MD, PhD, a student in the Kuo lab. Courtesy Steve Fisch

A year into the pandemic, we’ve all heard the stories. A patient is a little short of breath but appears to have a mild case of COVID-19. The next day, she deteriorates so rapidly that she’s rushed to intensive care, put on a ventilator, and hooked up to a dialysis machine to prevent kidney failure. Her overzealous immune system has gone rogue, attacking healthy cells instead of just fighting off the virus.

What triggers this devastating immune response, called a cytokine storm? Researchers are still struggling to identify the underlying processes that initiate a COVID infection and subsequent cytokine storm.

Biologists use advanced technologies and cell cultures in petri dishes to study severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the coronavirus strain responsible for COVID-19, identifying its key characteristics such as the famous crownlike spikes on their surfaces. But these short-lived cultures don’t act like real organs. And scientists are limited by their samples.

“When you analyze samples from patients, they’re often at the end stage of the disease, and many of the samples are from autopsy. You can’t understand the initiation process because the tissue is essentially destroyed,” says Calvin Kuo, MD, PhD, professor of hematology.

Understanding how the disease develops and testing potential treatments require better ways to model this coronavirus.

Miniature Organs in a Dish

Kuo’s laboratory develops organoids—three-dimensional miniature organs grown in a petri dish that mimic the shape, structure, and tissue organization of real organs.

Grown from human tissue samples using precisely defined ingredients, these organoids are little spheres of gel up to 1 millimeter in diameter. Healthy tissue samples are mechanically minced and enzyme digested to get to single cells, and then the organoids are grown from single stem cells. They last about six months, significantly longer than the few-weeks lifetime of traditional cell cultures.

Kuo initially developed organoids to study stem cell biology and model cancer. His team was the first to use organoids to convert normal tissues to cancer, as previously reported in Nature Medicine.

But he was passionate about using organoids to model infectious diseases. In 2015, he led a National Institute of Allergy and Infectious Diseases U19 research program, recently renewed for an additional five years, in collaboration with Stanford researchers Manuel Amieva, MD, professor of pediatrics and of microbiology and immunology; Harry Greenberg, MD, the Joseph D. Grant Professor in the Stanford University School of Medicine and professor of microbiology and immunology; Elizabeth Mellins, MD, professor of pediatrics; and Sarah Heilshorn, PhD, professor of materials science and engineering. Focusing mainly on the gastrointestinal tract, this multidisciplinary team provided proof of principle that organoids could model infectious diseases.

“With an organoid system, you can start at the infection and look at the very earliest events that occur after infection. And those can give insights as to what needs to be blocked therapeutically,” Kuo explains.

Distal Lung Organoids

After the initial success with gastrointestinal organoids, Ameen Salahudeen, MD, PhD, a hematology and oncology postdoctoral fellow working in Kuo’s lab, led efforts to expand this work by developing distal lung organoids. He partnered with lung stem cell expert Tushar Desai, MD, associate professor of pulmonary, allergy, and critical care medicine at Stanford.

The distal lung is composed of terminal bronchioles and alveolar air sacs, where inhaled air passes through the tiny ducts from the bronchioles into the elastic air sacs. It performs essential respiratory functions that can be compromised by inflammatory or infectious disorders, such as COVID-19 pneumonia.

“Growing distal lung cultures in a pure way that doesn’t require any supporting feeder cells and is in a chemically defined media had not been possible,” Kuo says. “We were able to do this very beautifully—to grow alveoli at the terminal bronchioles as long-term human cultures.”

The team developed two types of distal lung organoids. Both were made from human distal lung samples provided by Stanford cardiothoracic surgeon Joseph Schrager, MD.

They grew the first type, alveolar organoids, from single alveolar type 2 (AT2) stem cells. AT2 cells have several important functions that together help control the immune response to decrease lung injury and repair. The scientists then induced the AT2 cells to produce alveolar type 1 (AT1) cells, which are the thin-walled cells lining the alveolar air sacs; they are essential for the lung’s gas-exchange function.

“The second type are the basal organoids, which grow from single basal stem cells. They give rise to the mucus-secreting club cells and the ciliated cells with beating hair. And we can see the beating hair under the microscope—it’s quite dramatic,” describes Kuo. “That’s a very nice reproduction of the differentiation and function of the lung.” The team also grows a mixture of alveolar and basal organoids.

They selected these organoid types to determine which cell types in the bronchioles and alveoli were infectible—in hopes of identifying the different mechanisms for how viruses cause respiratory compromise.

Initially, they tested the distal lung organoids using the H1N1 influenza virus, collaborating with Stanford molecular virology expert Jeff Glenn, MD, PhD.

The team fluorescently labeled the virus and infected the lung organoids, demonstrating that the virus replicated in both basal and alveolar organoids. Next, they did more sophisticated PCR-based testing to show that the virus replicated its genome.

COVID-19 Model

“But then the COVID-19 pandemic hit, so we initiated a fabulous collaboration with infectious disease expert Catherine Blish, MD, PhD, in the Department of Medicine, to infect our lung organoids with SARS-CoV-2. This was driven by a talented MD-PhD student in my lab, Shannon Choi,” says Kuo. “She worked with Arjun Rustagi, an infectious disease fellow in Catherine Blish’s lab, who infected the organoids in a biosafety-level-3 lab.”

Another partnership was critical, though. An important coronavirus receptor, called angiotensin-converting enzyme 2, or ACE2, resides inside the lung organoids. But ACE2 needed to be on the outside of the organoid to get the infection going.

Luckily, Amieva previously devised a way to flip intestinal organoids inside out. Working together, Choi and Amieva turned the lung organoids inside out.

As reported in Nature in November 2020, the team demonstrated that the coronavirus infected their distal lung organoids, including the alveolar air sacs, where COVID-19 pneumonia originates. They also identified a new airway subpopulation as a COVID-19 virus target cell.

“Everyone knew basal cells were stem cells in the lung, but they thought they were all equivalent. Using our organoids, we discovered an unknown basal cell subpopulation containing the stem cell activity. And then we showed this subpopulation actually existed in human lungs in very interesting anatomic locations,” Kuo says.

COVID-19 Applications

According to Kuo, their distal lung organoids have three major applications for COVID-19.

They are using them to screen potential coronavirus therapeutic antibodies and to understand how these treatments work. Although initially focused on COVID-19, this screening will likely expand to other kinds of lung infections in the future.

Because the distal lung with the alveoli is the site of the COVID-19 pneumonia, they also plan to use the organoids to identify the underlying biological mechanisms behind coronavirus infection. Finally, they plan to extend their organoid system to incorporate immune cells and understand more complex processes. In particular, they plan to model the dreaded cytokine storm.

Overall, Kuo emphasizes that this organoid research represents a huge team effort involving many investigators with wide-ranging expertise from various departments at Stanford, as well as an “interesting evolution of events.” “Now we have a human experimental system to model SARS-CoV-2 infection of the distal lung with alveoli, which is the site of the lung disease that kills patients,” he summarizes. “We know patients die because of severe pneumonia and lung failure. We can now recapitulate this in the dish. So, we can study how it works, and also test drug treatments.” 

This is a reposting of my feature article in the recent Stanford Medicine Annual Report. Check it out to see videos of these lung organoids.

Physicists curate list of COVID-19 projects to join

As we continue to deal with the global COVID-19 pandemic, biomedical researchers are racing to understand the virus that causes the disease, to evaluate its spread, and to develop tests, treatments and vaccines.

Physicists are volunteering to assist in these efforts, using their skills in data analytics, machine learning, simulation, software, computing, hardware development and project management. And an organization called Science Responds is helping to match them with projects that need their support.

As Savannah Thais, a postdoctoral researcher in high-energy physics and a co-founder of Science Responds, reported at the April meeting of the American Physical Society, physicists are assisting with a variety of types of projects, divided into the following categories:

Epidemiology

Epidemiology is the branch of medical science that studies public health problems and events in order to understand what causes them, how they are distributed among populations and possible ways to control them. Epidemiologists investigate diverse problems including pollution, foodborne illnesses, natural disasters and infectious diseases such as COVID-19.

Science Responds is connecting physicists with epidemiological projects that are working to model how the virus that causes COVID-19 might spread. Physicists hope to help address a major problem that the experts making these models face: incorporating data from a multitude of dissimilar sources.

Thais says physicists have the background and experience needed to provide epidemiologists this kind of support.

“We don’t think physicists should be building their own epidemiological models from scratch, because they don’t have the domain expertise of an epidemiologist or biologist about infectious diseases,” she says. But “physicists can be most effective by providing their computing and statistics skills to interdisciplinary research.”

One epidemiology project Science Responds encourages volunteers to join is HealthMap, which displays data about COVID-19 cases across the globe over time via an openly accessible website and mobile app. HealthMap integrates and filters data from diverse, publicly available sources—including online news aggregators and reports from governments and agencies such as the World Health Organization—and then creates intuitive visualizations of the state of the outbreak by location.

Other modeling projects use analyses of the genomic features of previously studied viruses to help estimate unreported COVID-19 cases; integrate health and hospital resource data to inform localized risk predictions; and incorporate information from previous animal and human outbreaks to improve model accuracy.

Diagnosis

An important part of dealing with an epidemic is determining who has the disease, but shortages of testing supplies have made diagnosis a challenge. Science Responds promotes projects that are trying to address this gap in different ways.

Some projects use artificial intelligence to process visual or audio data. The project CAD4COVID, for example, builds off an existing technology that has been highly successful in diagnosing tuberculosis through the analysis of chest X-rays. The project COVID Voice Detector, on the other hand, is collecting audio recordings to develop an AI that can recognize signs of COVID-19 infection in a patient’s voice.

Other projects are building tools to predict who is likely to experience the most severe effects of COVID-19. These machine-learning-based efforts identify indicators such as markers that appear in blood tests or specific features from lung biopsies to predict the likelihood of long-term hospitalization or death.

Treatments and cures

The race to develop COVID-19 vaccines and treatments begins with understanding the physical structure of the virus. On this front, Science Responds collaborators are providing key support for an effort called Folding@Home, which uses computer simulations to map out the proteins the SARS-CoV-2 virus uses to reproduce and suppress a patient’s immune system. Physicists are helping to develop the protein-folding simulations, but they are also playing a pivotal role in looking for help from anyone with a computer that Folding@Home can use remotely to run folding simulations.

In addition, physicists are helping process the massive amount of data related to the SARS-CoV-2 genome. They’re hoping to identify molecules that are important to the growth and spread of the virus and to understand its mutations.

Science Responds collaborators are also aiding efforts to use machine learning to identify drugs that could be repurposed to treat COVID-19. For example, they are using natural-language-processing algorithms to comb through a massive database of scholarly articles, called CORD-19, for relevant ideas. Other projects are using deep-learning-based models with existing data to predict how commercially available drugs will interact with the virus.

Supporting hospitals and healthcare systems

Science Responds participants are volunteering on projects to support frontline workers who are providing medical care to COVID-19 patients. These efforts include developing models to help predict hospital overload and to allow for the sharing of resources such as mechanical ventilators and personal protective equipment.

One example is the CHIME project, which gathers information on hospital resources and predicts when the needs of patients will exceed an institution’s capacity. CHIME has already been deployed in several hospitals, including the University of Pennsylvania Health System.

Another project in this area is COVID Care Map, which is using open-source data to map existing supplies of hospital beds, ventilators and other resources needed to care for COVID-19 patients such as available staff.

Other projects highlighted by Science Responds are aimed at improving telehealth. Enhanced at-home care could reduce the spread of COVID-19 by eliminating unnecessary hospital visits and improving access to care for rural areas.

Researchers are helping to develop AI-based chatbots that can be used to assess possible infections, educate patients and call on human providers when necessary. Other projects are working to combine in-home sensors and cameras with AI-assisted technologies to remotely monitor the health of vulnerable populations.

Socio-economic response

Finally, Science Responds volunteers are also working to address what they call “second-order effects,” not directly related to healthcare.

Some projects deal with infodemiology, research into what we can learn from user-contributed, health-related content on the internet. Researchers are analyzing millions of real-time tweets related to COVID-19 to answer questions like: How are people reacting to the outbreak? How is Twitter being used to pass on vital information? How is Twitter being abused to spread false information, panic and hate?

Physicists with data-analysis and data-engineering expertise can volunteer for projects aimed at bringing attention to at-risk populations. Thais leads a project that is developing a COVID-19 Vulnerability Index, an AI-based predictive model used to identify communities at high risk of socio-economic and health impacts associated with the spread of COVID-19.

The index looks at a wide range of measures, such as whether community members have access to home Wi-Fi, whether they are affected by non-COVID health issues such as diabetes, and whether healthcare resources are available to them.

Are you a physicist looking to volunteer? Thais recommends checking out the Science Responds website, which lists projects organized by their required skills, highlights available data sources, computing resources and funding opportunities, and provides instructions for getting connected.

Illustration by Sandbox Studio, Chicago with Ana Kova

This is a reposting of my news feature, courtesy of Symmetry magazine.

 

Why do viruses like the coronavirus sometimes steal our sense of smell?

When you catch a severe cold, your nose stuffs up, you can’t smell anything and food tastes funny. Fortunately, most people regain their sense of smell once the cold runs its course. But for others, the complete (anosmia) or partial (hyposmia) loss of the sense of smell is permanent.

I spoke with Zara Patel, MD, a Stanford associate professor of otolaryngology, head and neck surgery, and director of endoscopic skull base surgery, to learn more about her research on olfactory disorders. In particular, we discussed her recent study on the possible association between post-viral olfactory loss and other cranial neuropathies, which are disorders that impair your nerves and ultimately your ability to feel or move. She also described how her work pertains to the COVID-19 pandemic.  

How does a virus impair someone’s sense of smell?

A variety of viruses can attack the cranial nerves related to smell or the mucosal tissue that surrounds those nerves. Cranial nerves control things in our head and neck — such as the nerves that allow us to speak by using our vocal cords, control our facial motion, hear and smell.

For example, COVID-19 is just one type of disease caused by a coronavirus. There are many other types of coronaviruses that cause colds and upper respiratory illnesses, as well as rhinoviruses and influenza viruses. Any of these viruses are known to cause inflammation, either directly around the nerve in the nasal lining or within the nerve itself. When the nerve is either surrounded by inflammatory molecules or has a lot of inflammation within the nerve cell body, it cannot function correctly — and that is what causes the loss or dysfunction of smell. And it can happen to anyone: young and old, healthy and sick.

How did your study investigate olfactory loss?

In my practice, I see patients who have smell dysfunction. But I’m also a sinus and skull base surgeon, so I have a whole host of other patients with sinus problems and skull-based tumors who don’t have an olfactory loss. So we did a case-control study to compare the incidence of cranial neuropathies — conditions in which nerves in the brain or brain stem are damaged — in two patient groups. Ninety-one patients had post-viral olfactory loss and 100 were controls; and they were matched as closely as possible for age and gender.

We also looked at family history of neurologic diseases — such as Alzheimer’s disease, Parkinson’s disease and stroke.

What did you find?

Patients with post-viral olfactory loss had six-times higher odds of having other cranial neuropathies than the control group — with an incidence rate of other cranial nerve deficits of 11% and 2%, respectively. Family history of neurologic diseases was associated with more than two-fold greater odds of having a cranial nerve deficit. Although we had a small sample size, the striking difference between the groups implies that it is worthwhile to research this with a larger population.

Our findings suggest that patients experiencing these pathologies may have inherent vulnerabilities to neural damage or decreased ability of nerve recovery — something beyond known risk factors like age, body mass index, co-morbidities and the duration of the loss before intervention. For example, there may be a genetic predisposition, but that is just an untested theory at this point.

How does this work pertain to COVID-19?

Smell loss can be one of the earliest signs of a COVID-19 infection. It can sometimes be the only sign. Or it can present after other symptoms. Although it may not affect every patient with COVID-19, loss of smell and taste is definitely associated with the disease. In some countries, including France, they’ve used this as a triage mechanism. People need to know that these symptoms can be related to the COVID-19 disease process so they aren’t going about their lives like normal and spreading the virus.

The pandemic also might impact how we treat patients with olfactory dysfunction in general. When someone has a viral-induced inflammation of the nerve, we sometimes treat it with steroids to decrease the inflammation. But treating COVID-19 patients with steroids might be a bad idea because of its effect on the inflammatory processes going on in their heart and lungs.

What advice do you have for people who have an impaired sense of smell?  

First, if you lose your sense of smell and it isn’t coming back after all the other symptoms have gone away, seek care as soon as possible. If you wait too long, there is much less that we can do to help you. Interventions, including olfactory training and medications, are more effective when you are treated early.

Second, if you lose your sense of smell or taste during this pandemic and you don’t have any other symptoms, contact your doctor. The doctor can decide whether you need to be tested for COVID-19 or whether you need to self-isolate to avoid being a vector of the virus in your family or community.

Image by carles

This is a reposting of my Scope story, courtesy of Stanford School of Medicine.

Defend or delay? Grad students must decide whether to present their thesis virtually

Graduate students who are trying to finish their degrees amid the COVID-19 pandemic are finding, after years of research and months of preparation, that the big day of defending their thesis has to be delayed or done remotely.

Faced with a new order to shelter at her off-campus home, Anjali Bisaria, a graduate student in chemical and systems biology at Stanford, decided to forge ahead. She works in the lab of Tobias Meyer, PhD,  where they study how human cells move and divide to build, maintain and repair tissues and organs.

On the scheduled date and time, Bisaria logged into a Zoom session and defended her research to a virtual audience of advisors, classmates, friends and family members. She then virtually met with just her faculty examinees. After being declared a doctor, she celebrated with her lab via yet another Zoom session.

“I know it was the right thing to do to keep the community safe,” she said in a Stanford news story. “But it was a little bit sad because this is likely my last quarter on campus. So to not be able to interact with my classmates and not be able to enjoy that honeymoon phase of grad school felt unceremonious.”

Soon, microbiology and immunology graduate student Kali Pruss will face the same decision. Her in-person PhD oral is currently scheduled for May 22 at Munzer Auditorium on Stanford campus.

“I haven’t yet decided whether I’ll proceed with my defense via Zoom or delay my defense to later in the summer, in hopes that I would be able to have an in-person defense,” Pruss told me. “I was planning on staying through the summer, taking a writing quarter anyway. Thankfully, this gives me some flexibility in terms of timing.”

As a member of the lab run by Justin Sonnenburg, PhD, Pruss studies how Clostridium difficile — a bacteria that commonly causes diarrhea and colitis — adapts to the inflammation that it generates, she said.

Pruss is currently writing a paper on her research, but the pandemic is impacting that too. She told me that she’s doing more data analysis and relying less on experiments than she normally would — and she’s a bit worried about how this approach will be received.

“I’m concerned with how this is going to affect the review process, and whether I’ll be able to successfully address reviewer comments asking for additional experiments for my papers,” she said.

She added, “Ultimately, though, I feel incredibly privileged and grateful to be able to continue working remotely towards my dissertation. The question of how my research is being impacted, and whether to postpone my defense, has been a minor concern in the scope of what is currently happening at Stanford and around the world.”

Given the extension of the Bay Area’s shelter-at-home order to last through at least May 3, Pruss’s hopes of defending in-person on May 22 may not be realized. So, her extended family — from Wisconsin, Indiana and Illinois — canceled their travel arrangements. They hope to come in late summer if she delays her defense and sheltering orders have been lifted.  

Regardless of how she defends her thesis, she plans to celebrate her upcoming educational milestone.

“This is the one time we, as PhD students, get to celebrate our time in grad school as an accomplishment,” she said.

After graduation, Pruss plans to join Jeffrey Gordon’s lab at Washington University School of Medicine in St. Louis as a postdoc. Ultimately, she plans to run her own academic lab.

Photo by Anjali Bisaria

This is a reposting of my Scope story, courtesy of Stanford School of Medicine.