What the cell!

Every cell is beholden to a phenomenon called cell fate, a sort of biological preset determined by genetic coding. Burgeoning cells take their developmental cues from a set of core genetic instructions that shape their structure and function and how they interact with other cells in the body.

To you or me, it’s biological law. But to a group of researchers at Stanford Medicine, it’s more of a suggestion. Unconstrained by the rules of evolution, these scientists are instead governed by a question: What if?

What if you could eat a vaccine? Or create a bacterium that could also detect and attack cancer? What if furniture could grow from a seed?

Though these types of questions may sound far-fetched, they consume the minds of researchers who specialize in a field known as cell engineering, which harnesses genetic manipulation to change the essence of a cell. That might mean reprogramming a cell to perform a function it isn’t designed by nature to do, tinkering with its interior machinery or creating an entirely new type of cell.

“As a scientist and engineer, this idea of learning to build biology — to build with the components of living systems — really motivates me,” said Michael Jewett, PhD, professor of bioengineering. “It’s really fun to think about what we can learn from that process — like understanding how the biological world works — but then also apply what we learn to benefit society.”

Those applications could take a multitude of shapes. Maybe it’s building cells that churn out therapeutic drugs — one of Jewett’s projects — or vaccines that stave off a bacterial infection, or enzymes that degrade harmful fungi in the rainforest.

The field of cell engineering is relatively new, but its seeds were planted in the 1950s when Nobelist Arthur Kornberg, MD, who came to Stanford Medicine in 1959, isolated the key enzyme used by cells to synthesize DNA. In the 1970s, researchers, including Stanford Medicine faculty, pioneered cutting and pasting DNA (Nobelist Paul Berg, PhD) and transplanting genes from one organism to another (Stanley Cohen, MD, at Stanford and Herbert Boyer, PhD, at UC San Francisco). This launched genetic engineering and the biotechnology industry. Cell engineering gained momentum about a decade ago, as genetic sequencing and manipulation advanced.
A cell engineer’s approach to learning is generally to design, tinker and see what happens, said Drew Endy, PhD, the Martin Family University Fellow in Undergraduate Education.

“There has been, and continues to be, unbelievably beautiful scientific work to understand cells,” Endy said. “The question for me is, what’s next?”
Stanford Medicine researchers from a variety of disciplines are exploring that question. Some are retooling the insides of immune cells, others are digging out and redesigning the guts of a cell, and one is even building a cell from scratch.

Read on to discover how scientists are rethinking cell biology to benefit humanity.

Teaching cells to count

In the past couple of decades, cancer biologists have developed and refined a powerful new way to vanquish cancer cells floating in the bloodstream. During this treatment, known as chimeric antigen receptor cell therapy, or CAR-T cell therapy, a patient’s immune cells are genetically modified to target specific cancer cells.

Naturally occurring T cells kill cancer cells based on recognition of a molecule on the cell’s surface — an antigen — that acts like a name tag for tumor cells. With CAR-T cells, scientists remove a person’s own T cells and hone their ability to identify specific tumor antigens, heightening their tumor-attacking capabilities. These cells are then returned to the patient.

The therapy has so far proven to be a potent option — but only for blood cancers. The cancer-killing abilities of cell-based therapies like CAR-T generally don’t extend to solid tumors.

“There are little to no unique markers of solid tumors,” said Rogelio Hernandez-Lopez, PhD, assistant professor of bioengineering and of genetics. “Proteins that are known to be markers of cancers, such as HER2 or EGFR, are also shared with other tissues.”

Unleashing T cells engineered to track and kill cells with those markers would wreak havoc on healthy tissues. But there’s a saving grace that is the cornerstone of Hernandez-Lopez’s research: Cancer cells are rich in these markers, and healthy cells are not. “What we’re trying to do is teach these T cells to count and make a ‘decision’ to kill a cell based on the quantity of a particular marker,” Hernandez-Lopez said. That kind of nuanced attack requires an entirely different set of instructions than those that naturally guide a T cell.

Hernandez-Lopez is engineering T cells that detect a high versus low abundance of specific markers, or antigens, by creating circuits of two types of synthetic receptors that embed into a T cell’s surface.

One, called synthetic Notch, has “low-affinity binding,” and when it latches onto its matched antigen, it stimulates the production of the other receptor — which, when bound to an antigen, sparks the T cell to attack. But here’s the kicker: Low-affinity binding means the synthetic Notch receptor doesn’t always latch onto its paired antigen. Only when that antigen is abundant does the circuit switch on.

That’s what gives these T cells the ability to “count and decide.” If the T cell detects healthy cells that harbor just a few antigens also carried by tumor cells, the T cell stays neutral. But if it detects a bunch of these antigens, the receptor latches onto the antigen, triggering a series of molecular steps that ultimately jolt the T cell into action.

Hernandez-Lopez’s team is testing the synthetic receptor in mice, with early results showing promise for the approach.

Neutralizing the neutrophil

When bacteria or viruses sneak into your bloodstream, a brigade of immune cells known as neutrophils attack, and they’re equipped with a slew of molecular weaponry. They kill the infected cells by engulfing and destroying them; by releasing toxic chemicals toward them; or through a dramatic demonstration of demolition — like a microscopic supernova, a neutrophil can explode, spewing its DNA and enzymes that devour surrounding cells.

This process, known as NETosis (with NET standing for neutrophil extracellular traps), is unique to innate immune cells.

The cells’ variety of attack modalities, while effective, can be too much. Neutrophils respond to signals of inflammation (which is one of the ways the immune system counters infection), but not all inflammation is caused by a microbe or virus.

Even in cases in which infection is not the culprit, such as in autoimmune diseases, neutrophils see the inflammatory signal as a summons, and they dutifully report to the ailing site.

But this time, their defense mechanisms worsen symptoms, exacerbating an already problematic situation. Hawa Racine Thiam, PhD, an assistant professor of bioengineering and of microbiology and immunology and a neutrophil expert (and enthusiast) is keenly aware of the problem.

Part of how neutrophils contribute to heightened inflammation comes down to some technical details of NETosis. “When the neutrophil ‘nets,’ it simultaneously releases DNA and cytotoxic proteins that can kill the pathogen,” Thiam said. Those toxic molecules can inflict additional damage even after the invader has been killed.

Thiam is exploring whether it’s possible to quell an overzealous neutrophil attack while maintaining its ability to kill off real threats. But to answer that question, she needs to know more about how NETosis plays out in the first place, one of the big goals of her research.

There are a few theories, one of which Thiam is testing. “For the cell to net there needs to be a breakdown of the nuclear membrane, and for that to happen we think the cell needs to generate force,” she said.

She and others in the field suspect that push comes from genetic structures in the cell known as chromatin. Chromatin, the bundles of DNA and protein that form chromosomes, can change conformation, depending on the environment or cellular conditions.

“Think of a cord coiled up in a blown-up balloon,” Thiam said. “If the cord unfurls and takes up more space, it puts pressure on the balloon, which can make it pop.”

In a neutrophil, that is part of the process that ruptures the cell and expels pathogen-degrading enzymes.

“This is a working hypothesis that our early data supports,” she said of the research. “But there’s a lot more to understand.” To that end, she’s using genetic and biophysical experimentation to study how the cell bursts and to determine which components of the expelled content damage the host.

Building from the bottom up

Imagine you are an alien presented with a chocolate layer cake. You’re now asked to bake one from scratch.

How does one work backward from cake to ingredients (and the amounts necessary) to make the confection? Even broken into its component parts — cake, frosting, sprinkles — it’s still not clear what the ingredients are nor how to blend them into that decadent baked good. And so ensues a lengthy process of trial-and-error experimentation.

Such is Endy’s conundrum — only it’s not a cake. It’s a cell. And it’s much more complicated. Endy is on a mission to build a cell from scratch. After more than a decade of work by him and a crew of Stanford students, they have built a prototype, which he calls a “precursor cell.”

A successful synthetic cell will need to satisfy a few generally agreed upon (though sometimes debated) criteria: compartmentalization, formed by some sort of lipid bilayer or the like; self replication, during which a cell duplicates its innards and divides on its own; and metabolism or energy production, to power the former.

Attempting to build a synthetic cell in Endy’s lab starts with a commercially available set of molecules that, together, carry out one of the cell’s main jobs — the synthesis of proteins. Endy’s team has toyed with and manipulated this mixture to boost its capabilities and move it from test tube to a biological capsule, while maintaining its ability to create proteins.

That’s not as simple as it might sound. So, to guide the process, Endy’s group of researchers is implementing a model of cellular behavior based in something called “colloidal hydrodynamics,” to predict how a synthetic cell might form and react under certain laboratory conditions. Put together, experimentation and modeling have yielded some interesting molecular concoctions. But perhaps more impressively, some precursor cells are exhibiting core functions of a cell, churning out proteins when they are fed DNA.

The final step is visualization. Through a type of microscopy called cryogenic electron microscopy, or cryo-EM, which images frozen molecules by bombarding electrons at the specimen and measuring refraction, Endy and his team can glimpse the precursor cells they’re making. Some look like cells — a lipid bilayer that surrounds machinery on the inside — but some go wonky, absorbing one another like a Russian nesting doll.

Either way, Endy is excited to see his lab’s progress. “It’s so heartening to see these things. It took six years for it to come together.”

Endy hopes that his synthetic-cell building will fuel his larger goal as a bioengineer: the broad and accessible dissemination of bioengineering capabilities that can one day support solutions to the world’s biggest threats, such as hunger, insufficient access to medicine in every country and climate change. Synthetic cells, he hopes, will be able to generate new solutions. Exactly what they will look like isn’t yet clear, but Endy believes his progress has laid a foundation. “That sets us up for the next generation of synthetic cell building,” he said.

It’s what’s inside that counts

Not all cell-based engineering has to take place in a cell — that’s the grounding philosophy of much of the work that comes out of Jewett’s lab. What he cares about is on the inside. “We basically take cells, rip off their cell walls, collect the insides and build with that machinery, which has all the information necessary to support information flow in biology,” Jewett said.

He’s particularly focused on the ribosome — the little protein-making machines that operate inside cells and turn RNA into proteins, which then carry out a variety of biological functions. “We’re trying to boot up ribosomes in a test tube,” he said.

Unhoused ribosomes offer a lot of potential advantages. For instance, they could be shipped to faraway places (without the need to maintain the rest of the cell) where they could churn out proteins, which often are fundamental to therapeutics.

For now, synthetic ribosomes’ potential remains to be realized — the current goal is to build a foundation that can be tweaked so that one day engineered ribosomes might assume new powers, such as the ability to create proteins in unnatural abundance or under extreme conditions.

For his experiments, Jewett often makes use of the ribosomes of the bacterium Escherichia coli, which are made of 54 different proteins and three strands of RNA. Together, those molecules translate RNA genetic templates into proteins, including those that make other ribosomes.

“Creating new, functional ribosomes in a test tube has really been a challenge, in part because it’s kind of like the chicken or the egg paradox. The ribosome produces proteins that, in turn, are required to build ribosomes.”

So far, Jewett and his team have figured out how to co-assemble all of the ribosomal proteins with the ribosomal RNA in a test tube and use that mixture to create new proteins. To get all the pieces working takes more than just swirling them all together, however. Biological processes require energy to be a self-sustaining system.

“It’s like building a house. You need materials, you need energy and information,” Jewett said.

“In this case, the information is DNA instead of house blueprints, the energy for the biological systems is the chemical compound adenosine triphosphate (or ATP) instead of human labor, and the materials are amino acids or nucleotides, rather than wood or brick.”

But they’ve yet to have a ribosome beget another ribosome. “Our test tube ribosomes are good enough to make all of the chemical bonds necessary to synthesize another ribosome, but we’re missing a step to get it to self-assemble,” Jewett said. “Our next task is to figure out why.”

Assistant to assassin

There’s a code switch happening in the lab of Kyle Daniels, PhD, an assistant professor of genetics who has an express goal of coaxing out the unnatural side of immune cells, experimentally encouraging them to exhibit new capabilities. “We’re engineering cells to get them to do things they don’t normally do,” he said. “If you can understand how to do that, you open up a whole world of possibilities in the future to tackle problems that we may not even know about yet.”

Much of Daniels’ work focuses on immune cells, with one project homing in on T cells, which are considered the cancer killers. Generally speaking, there are two types of T cells in the body: helper T cells, CD4, and killer T cells, CD8 — so named to denote the receptors embedded in the cells’ outer surface. CD4 helper cells play an organizational role, stimulating other immune cells to do the dirty work and act against pathogens or tumor cells. CD8 cells, however, are natural born killers, built to destroy at the behest of CD4s. That, at least, is the traditional understanding of the role these cells have in human biology.

While T cells often function this way, Daniels and his team are using synthetic biology tools to reveal a recently discovered secret about both types of T cells: In every CD4 T cell, a killer lurks, and in every CD8, a mediator. But the hidden ability of CD4s to facilitate death has caught Daniels’ attention the most. His team is creating a variety of synthetic receptors that can lodge in the outer layer of T cells and, when bound to the target molecule, guide the cells’ behavior and activity.

Those receptors adhere to certain antigens, and their binding sets off a flurry of events that result in the T cell expelling toxic molecules that kill the cells around it.

“I think we assumed that the CD8 cells were doing all the killing in our experiments, but it turns out if you have CD4 cells alone, they’re really good at killing leukemias and lymphomas with the synthetic receptor,” Daniels said.

And the modified CD4 cells can maintain their killing spree longer than CD8 cells that are modified with the same receptor. “It’s been a big surprise to us.”

His team has even found that, depending on the type of synthetic receptor, it can selectively activate a CD4 cell’s killing program, triggering the same destruction of killer cells without tampering with the function of CD8 cells.

Exactly how these CD4s go from assistant to assassin is still a question. Are they equipped with the tools to kill all along? Or do the synthetic receptors reprogram a new pathway that generates its killing ability? Daniels is exploring that question in his lab.

He also hopes to test the engineered CD4 cells in mice as a next step. “We’re seeing that CD4s might be a major driver of the killing. I think there’s some appreciation of that, but I don’t think it’s most people’s assumption,” Daniels said. “It’s clear that this is happening. Now we’re trying to find cell signaling programs that maximize this effect so that we can really take advantage of it.