Skip to main content

Snapshots of Life

Insulin-Producing Organoids Offer Hope for Treating Type 1 Diabetes

Posted on by

Insulin-producing organoid
Caption: Human islet-like organoids express insulin (green). Credit: Salk Institute

For the 1 to 3 million Americans with type 1 diabetes, the immune system destroys insulin-producing beta cells of the pancreas that control the amount of glucose in the bloodstream. As a result, these individuals must monitor their blood glucose often and take replacement doses of insulin to keep it under control. Such constant attention, combined with a strict diet to control sugar intake, is challenging—especially for children.

For some people with type 1 diabetes, there is another option. They can be treated—maybe even cured—with a pancreatic islet cell transplant from an organ donor. These transplanted islet cells, which harbor the needed beta cells, can increase insulin production. But there’s a big catch: there aren’t nearly enough organs to go around, and people who receive a transplant must take lifelong medications to keep their immune system from rejecting the donated organ.

Now, NIH-funded scientists, led by Ronald Evans of the Salk Institute, La Jolla, CA, have devised a possible workaround: human islet-like organoids (HILOs) [1]. These tiny replicas of pancreatic tissue are created in the laboratory, and you can see them above secreting insulin (green) in a lab dish. Remarkably, some of these HILOs have been outfitted with a Harry Potter-esque invisibility cloak to enable them to evade immune attack when transplanted into mice.

Over several years, Doug Melton’s lab at Harvard University, Cambridge, MA, has worked steadily to coax induced pluripotent stem (iPS) cells, which are made from adult skin or blood cells, to form miniature islet-like cells in a lab dish [2]. My own lab at NIH has also been seeing steady progress in this effort, working with collaborators at the New York Stem Cell Foundation.

Although several years ago researchers could get beta cells to make insulin, they wouldn’t secrete the hormone efficiently when transplanted into a living mouse. About four years ago, the Evans lab found a possible solution by uncovering a genetic switch called ERR-gamma that when flipped, powered up the engineered beta cells to respond continuously to glucose and release insulin [3].

In the latest study, Evans and his team developed a method to program HILOs in the lab to resemble actual islets. They did it by growing the insulin-producing cells alongside each other in a gelatinous, three-dimensional chamber. There, the cells combined to form organoid structures resembling the shape and contour of the islet cells seen in an actual 3D human pancreas. After they are switched on with a special recipe of growth factors and hormones, these activated HILOs secrete insulin when exposed to glucose. When transplanted into a living mouse, this process appears to operate just like human beta cells work inside a human pancreas.

Another major advance was the invisibility cloak. The Salk team borrowed the idea from cancer immunotherapy and a type of drug called a checkpoint inhibitor. These drugs harness the body’s own immune T cells to attack cancer. They start with the recognition that T cells display a protein on their surface called PD-1. When T cells interact with other cells in the body, PD-1 binds to a protein on the surface of those cells called PD-L1. This protein tells the T cells not to attack. Checkpoint inhibitors work by blocking the interaction of PD-1 and PD-L1, freeing up immune cells to fight cancer.

Reversing this logic for the pancreas, the Salk team engineered HILOs to express PD-L1 on their surface as a sign to the immune system not to attack. The researchers then transplanted these HILOs into diabetic mice that received no immunosuppressive drugs, as would normally be the case to prevent rejection of these human cells. Not only did the transplanted HILOs produce insulin in response to glucose spikes, they spurred no immune response.

So far, HILOs transplants have been used to treat diabetes for more than 50 days in diabetic mice. More research will be needed to see whether the organoids can function for even longer periods of time.

Still, this is exciting news, and provides an excellent example of how advances in one area of science can provide new possibilities for others. In this case, these insights provide fresh hope for a day when children and adults with type 1 diabetes can live long, healthy lives without the need for frequent insulin injections.

References:

[1] Immune-evasive human islet-like organoids ameliorate diabetes. [published online ahead of print, 2020 Aug 19]. Yoshihara E, O’Connor C, Gasser E, Wei Z, Oh TG, Tseng TW, Wang D, Cayabyab F, Dai Y, Yu RT, Liddle C, Atkins AR, Downes M, Evans RM. Nature. 2020 Aug 19. [Epub ahead of publication]

[2] Generation of Functional Human Pancreatic β Cells In Vitro. Pagliuca FW, Millman JR, Gürtler M, Segel M, Van Dervort A, Ryu JH, Peterson QP, Greiner D, Melton DA. Cell. 2014 Oct 9;159(2):428-39.

[3] ERRγ is required for the metabolic maturation of therapeutically functional glucose-responsive β cells. Yoshihara E, Wei Z, Lin CS, Fang S, Ahmadian M, Kida Y, Tseng T, Dai Y, Yu RT, Liddle C, Atkins AR, Downes M, Evans RM. Cell Metab. 2016 Apr 12; 23(4):622-634.

Links:

Type 1 Diabetes (National Institute of Diabetes and Digestive and Kidney Diseases/NIH)

Pancreatic Islet Transplantation (National Institute of Diabetes and Digestive and Kidney Diseases)

The Nobel Prize in Physiology or Medicine 2012” for Induced Pluripotent Stem Cells, The Nobel Prize news release, October 8, 2012.

Evans Lab (Salk Institute, La Jolla, CA)

NIH Support: National Institute of Diabetes and Digestive and Kidney Diseases; National Cancer Institute


Building a Better Bacterial Trap for Sepsis

Posted on by

NETs
Credit: Kandace Gollomp, MD, The Children’s Hospital of Philadelphia, PA

Spiders spin webs to catch insects for dinner. It turns out certain human immune cells, called neutrophils, do something similar to trap bacteria in people who develop sepsis, an uncontrolled, systemic infection that poses a major challenge in hospitals.

When activated to catch sepsis-causing bacteria or other pathogens, neutrophils rupture and spew sticky, spider-like webs made of DNA and antibacterial proteins. Here in red you see one of these so-called neutrophil extracellular traps (NETs) that’s ensnared Staphylococcus aureus (green), a type of bacteria known for causing a range of illnesses from skin infections to pneumonia.

Yet this image, which comes from Kandace Gollomp and Mortimer Poncz at The Children’s Hospital of Philadelphia, is much more than a fascinating picture. It demonstrates a potentially promising new way to treat sepsis.

The researchers’ strategy involves adding a protein called platelet factor 4 (PF4), which is released by clot-forming blood platelets, to the NETs. PF4 readily binds to NETs and enhances their capture of bacteria. A modified antibody (white), which is a little hard to see, coats the PF4-bound NET above. This antibody makes the NETs even better at catching and holding onto bacteria. Other immune cells then come in to engulf and clean up the mess.

Until recently, most discussions about NETs assumed they were causing trouble, and therefore revolved around how to prevent or get rid of them while treating sepsis. But such strategies faced a major obstacle. By the time most people are diagnosed with sepsis, large swaths of these NETs have already been spun. In fact, destroying them might do more harm than good by releasing entrapped bacteria and other toxins into the bloodstream.

In a recent study published in the journal Blood, Gollomp’s team proposed flipping the script [1]. Rather than prevent or destroy NETs, why not modify them to work even better to fight sepsis? Their idea: Make NETs even stickier to catch more bacteria. This would lower the number of bacteria and help people recover from sepsis.

Gollomp recalled something lab member Anna Kowalska had noted earlier in unrelated mouse studies. She’d observed that high levels of PF4 were protective in mice with sepsis. Gollomp and her colleagues wondered if the PF4 might also be used to reinforce NETs. Sure enough, Gollomp’s studies showed that PF4 will bind to NETs, causing them to condense and resist break down.

Subsequent studies in mice and with human NETs cast in a synthetic blood vessel suggest that this approach might work. Treatment with PF4 greatly increased the number of bacteria captured by NETs. It also kept NETs intact and holding tightly onto their toxic contents. As a result, mice with sepsis fared better.

Of course, mice are not humans. More study is needed to see if the same strategy can help people with sepsis. For example, it will be important to determine if modified NETs are difficult for the human body to clear. Also, Gollomp thinks this approach might be explored for treating other types of bacterial infections.

Still, the group’s initial findings come as encouraging news for hospital staff and administrators. If all goes well, a future treatment based on this intriguing strategy may one day help to reduce the 270,000 sepsis-related deaths in the U.S. and its estimated more than $24 billion annual price tag for our nation’s hospitals [2, 3].

References:

[1] Fc-modified HIT-like monoclonal antibody as a novel treatment for sepsis. Gollomp K, Sarkar A, Harikumar S, Seeholzer SH, Arepally GM, Hudock K, Rauova L, Kowalska MA, Poncz M. Blood. 2020 Mar 5;135(10):743-754.

[2] Sepsis, Data & Reports, Centers for Disease Control and Prevention, Feb. 14, 2020.

[3] National inpatient hospital costs: The most expensive conditions by payer, 2013: Statistical Brief #204. Torio CM, Moore BJ. Healthcare Cost and Utilization Project (HCUP) Statistical Briefs. Agency for Healthcare Research and Quality (US); 2016 May.

Links:

Sepsis (National Institute of General Medical Sciences/NIH)

Kandace Gollomp (The Children’s Hospital of Philadelphia, PA)

Mortimer Poncz (The Children’s Hospital of Philadelphia, PA)

NIH Support: National Heart, Lung, and Blood Institute


Seeing Coronavirus Replicate in Kidney Cells

Posted on by

Bernbaum Imaging Post
Credit: NIAID Integrated Research Facility, Fort Detrick, MD

You’ve probably seen pictures of SARS-CoV-2—the novel coronavirus that causes COVID-19—that look alarming. But the high-resolution micrograph above paints a rather different picture, using rich pseudo-colors to show how newly assembled viral particles cause infected cells to bulge, or bleb, and then self-destruct.

This image depicts a common primate kidney cell line (green) infected with SARS-CoV-2. Notice the bulging, spherical cellular blebs, seen best in the upper right and bottom left corners. These badly damaged cells, which are filled to the point of bursting with viral particles, are beginning to self-destruct. Some cells have apparently already burst open, allowing hundreds of viral particles (purple) to spill out and potentially infect other cells.

This stunning picture was taken by John Bernbaum, an electron microscopist with NIH’s National Institute of Allergy and Infectious Diseases (NIAID). Bernbaum works at NIAID’s Integrated Research Facility (IRF), Fort Detrick, MD, a specialized, high-level biocontainment facility equipped with unique medical imaging capabilities. In this special environment, Bernbaum and his colleagues can safely visualize SARS-CoV-2, as well as other viruses and microbes that pose serious risks to human health.

To get this shot of SARS-CoV-2, Bernbaum relied on a conventional scanning electron microscope (SEM). First, a sample of kidney cells that had been exposed to SARS-CoV-2 was dehydrated, chemically preserved, and coated with a thin layer of metal. Once everything was ready, the SEM was used to focus a high-energy beam of electrons onto the sample. As electrons bounced off the metal surface, they revealed spatial variations and properties in the sample that were used to generate this 3D image.

Originally, this image was in gray scale. To better highlight the destructive powers of SARS-CoV-2, Jiro Wada, a skilled graphic illustrator at the IRF, used a computer program to colorize key features in exquisite detail. By studying these 3D images, researchers can learn about things such as the rate of infection and the prodigious number of particles each infected cell produces. They can also learn about how the infection affects the conditions inside cells.

Interestingly, what Bernbaum finds most striking about SARS-CoV-2 is what you don’t see in his images. Uninfected kidney cells look like a flat, delicately interwoven quilt (not pictured). When Bernbaum used SEM to study this sample of kidney cells, about 80 to 90 percent of the cells appeared flat and unremarkable. Yet, as the scan progressed, he came across a small subset of cells that appeared to be deformed by SARS-CoV-2 infection. Those abnormalities include the spherical bulges that I pointed out earlier, along with some worm-like protrusions that you can see in the top left.

Bernbaum has been producing amazing images like this one for 32 years—the last 11 of them at the IRF. If you’d like to see even more of his impressive work and that of the IRF team, check out the NIAID’s image gallery.

Links:

Coronavirus (NIH)

Integrated Research Facility (National Institute of Allergy and Infectious Diseases/NIH)

NIH Support: National Institute of Allergy and Infectious Diseases


3D Printing the Novel Coronavirus

Posted on by

Credit: 3D Print Exchange, NIAID, NIH

The coronavirus disease 2019 (COVID-19) pandemic has truly been an all-hands-on-deck moment for the nation. Among the responders are many with NIH affiliations, who are lending their expertise to deploy new and emerging technologies to address myriad research challenges. That’s certainly the case for the dedicated team from the National Institute of Allergy and Infectious Diseases (NIAID) at the NIH 3D Print Exchange (3DPX), Rockville, MD.

A remarkable example of the team’s work is this 3D-printed physical model of SARS-CoV-2, the novel coronavirus that causes COVID-19. This model shows the viral surface (blue) and the spike proteins studded proportionally to the right size and shape. These proteins are essential for SARS-CoV-2 to attach to human cells and infect them. Here, the spike proteins are represented in their open, active form (orange) that’s capable of attaching to a human cell, as well as in their closed, inactive form (red).

The model is about 5 inches in diameter. It takes more than 5 hours to print using an “ink” of thin layers of a gypsum plaster-based powder fused with a colored binder solution. When completed, the plaster model is coated in epoxy for strength and a glossy, ceramic-like finish. For these models, NIAID uses commercial-grade, full-color 3D printers. However, the same 3D files can be used in any type of 3D printer, including “desktop” models available on the consumer market.

Darrell Hurt and Meghan McCarthy lead the 3DPX team. Kristen Browne, Phil Cruz, and Victor Starr Kramer, the team members who helped to produce this remarkable model, created it as part of a collaboration with the imaging team at NIAID’s Rocky Mountain Laboratories (RML), Hamilton, MT.

The RML’s Electron Microscopy Unit captured the microscopic 3D images of the virus, which was cultured from one of the first COVID-19 patients in the country. The unit handed off these and other data to its in-house visual specialist to convert into a preliminary 3D model. The model was then forwarded to the 3DPX team in Maryland to colorize and optimize in preparation for 3D printing.

This model is especially unique because it’s based exclusively on SARS-CoV-2 data. For example, the model is assembled from data showing that the virus is frequently oval, not perfectly round. The spike proteins also aren’t evenly spaced, but pop up more randomly from the surface. Another nice feature of 3D printing is the models can be constantly updated to incorporate the latest structural discoveries.

That’s why 3D models are such an excellent teaching tools to share among scientists and the public. Folks can hold the plaster virus and closely examine its structure. In fact, the team recently printed out a model and delivered it to me for exactly this educational purpose.

In addition to this complete model, the researchers also are populating the online 3D print exchange with atomic-level structures of the various SARS-CoV-2 proteins that have been deposited by researchers around the world into protein and electron microscopy databanks. The number of these structures and plans currently stands at well over 100—and counting.

As impressive as this modeling work is, 3DPX has found yet another essential way to aid in the COVID-19 fight. In March, the Food and Drug Administration (FDA) announced a public-private partnership with the NIH 3D Print Exchange, Department of Veterans Affairs (VA) Innovation Ecosystem, and the non-profit America Makes, Youngstown, OH [1]. The partnership will develop a curated collection of designs for 3D-printable personal protective equipment (PPE), as well as other necessary medical devices that are in short supply due to the COVID-19 pandemic.

You can explore the partnership’s growing collection of COVID-19-related medical supplies online. And, if you happen to have a 3D printer handy, you could even try making them for yourself.

Reference:

[1] FDA Efforts to Connect Manufacturers and Health Care Entities: The FDA, Department of Veterans Affairs, National Institutes of Health, and America Makes Form a COVID-19 response Public-Private Partnership (Food and Drug Administration)

Links:

Coronavirus (COVID-19) (NIH)

NIH 3D Print Exchange (National Institute of Allergy and Infectious Diseases/NIH, Rockville, MD)

Rocky Mountain Laboratories (NIAID/NIH, Hamilton, MT)

Department of Veterans Affairs (VA) Innovation Ecosystem (Washington, D.C.)

America Makes (Youngstown, OH)

NIH Support: National Institute of Allergy and Infectious Diseases


Capturing Viral Shedding in Action

Posted on by

Credit: Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, MT

You’ve probably seen some amazing high-resolution images of SARS-CoV-2, the novel coronavirus that causes COVID-19, on television and the web. What you might not know is that many of these images, including the ones shown here, were produced at Rocky Mountain Laboratories (RML), a part of NIH’s National Institute of Allergy and Infectious Diseases (NIAID) that’s located in the small Montana town of Hamilton.

The head of RML’s Electron Microscopy Unit, Elizabeth Fischer, was the researcher who took this portrait of SARS-CoV-2. For more than 25 years, Fischer has snapped stunning images of dangerous viruses and microbes, including some remarkable shots of the deadly Ebola virus. She also took some of the first pictures of the coronavirus that causes Middle East respiratory syndrome (MERS), which arose from camels and continues to circulate at low levels in people.

The NIAID facility uses a variety of microscopy techniques, including state-of-the-art cryo-electron microscopy (cryo-EM). But the eye-catching image you see here was taken with a classic scanning electron microscope (SEM).

SEM enables visualization of particles, including viruses, that are too small to be seen with traditional light microscopy. It does so by focusing electrons, instead of light, into a beam that scans the surface of a sample that’s first been dehydrated, chemically preserved, and then coated with a thin layer of metal. As electrons bounce off the sample’s surface, microscopists such as Fischer are able to capture its precise topology. The result is a gray-scale micrograph like the one you see above on the left. To make the image easier to interpret, Fischer hands the originals off to RML’s Visual Medical Arts Department, which uses colorization to make key features pop like they do in the image on the right.

So, what exactly are you seeing in this image? The orange-brown folds and protrusions are part of the surface of a single cell that’s been infected with SARS-CoV-2. This particular cell comes from a commonly studied primate kidney epithelial cell line. The small, blue spheres emerging from the cell surface are SARS-CoV-2 particles.

This picture is quite literally a snapshot of viral shedding, a process in which viral particles are released from a dying cell. This image gives us a window into how devastatingly effective SARS-CoV-2 appears to be at co-opting a host’s cellular machinery: just one infected cell is capable of releasing thousands of new virus particles that can, in turn, be transmitted to others.

While capturing a fixed sample on the microscope is fairly straightforward for a pro like Fischer, developing a sample like this one involves plenty of behind-the-scenes trial and error by NIAID investigators. As you might imagine, to see the moment that viruses emerge from an infected cell, you have to get the timing just right.

By capturing many shots of the coronavirus using the arsenal of microscopes available at RML and elsewhere, researchers are learning more every day about how SARS-CoV-2 enters a cell, moves inside it, and then emerges to infect other cells. In addition to advancing scientific knowledge, Fischer notes that images like these also hold the remarkable power to make an invisible enemy visible to the world at large.

Making SARS-CoV-2 tangible helps to demystify the challenges that all of us now face as a result of the COVID-19 pandemic. The hope is it will encourage each and every one of us to do our part to fight it, whether that means digging into the research, working on the front lines, or staying at home to prevent transmission and flatten the curve. And, if you could use some additional inspiration, don’t miss the NIAID’s image gallery on Flickr, which includes some of Fischer’s finest work.

Links:

Coronavirus (COVID-19) (NIH)

Rocky Mountain Laboratories (National Institute of Allergy and Infectious Diseases/NIH)

Elizabeth Fischer (National Institute of Allergy and Infectious Diseases/NIH)

NIH Support: National Institute of Allergy and Infectious Diseases


Next Page