Stem Cell for Cystic Fibrosis – Harvard & Boston University Research

Stem cell for cystic fibrosis? Cooperation between Harvard University stem cell researchers at the Massachusetts General Hospital and Boston University School of Medicine scientists — uncharacteristic between typically competing scientific teams — have brought the world closer to the discovery of a drug for cystic fibrosis (CF), a fatal lung disease ranked among the most widespread life-shortening genetic diseases. Their findings can also be used to develop treatments for other major lung diseases.

In the competitive world of science, where being the first to invent or discover something has been the topmost cherished value for centuries, the cooperation between the two teams is unusual but may point the way to the future in science.

For one, both teams say that forging a relationship had helped speed up their individual progress, allowing them to hurdle obstacles and achieve what has eluded stem cell researchers for many years: grow lung stem cells, lung cells and lung tissue.

Growing lung tissue in the laboratory has long been a goal of stem cell scientists. Such lung tissue is valuable because it could be used to probe the causes of major lung diseases like asthma, emphysema and cystic fibrosis, study their development and progression, as well as find and screen potential drugs to combat these. In the long run, such lab-grown lung tissue could also be used to replace tissue damaged by disease and may even be the first step toward creating entire lungs for transplantation.

But lung cells have proven to be technically more difficult to grow than other types of tissues — even brain cells or heart cells.

Working separately but cooperatively, the two teams of scientists have developed new ways to turn stem cells into different types of lung tissue.

The Harvard investigators at the Massachusetts General Hospital grew a cystic fibrosis-in-a-dish model out of lung tissue taken from patients with the genetic mutation that most commonly underlies cystic fibrosis.

The researchers hope the tissue model could be used to study the biology of the rare genetic lung disease — as well as asthma, lung cancer, and other ailments of the airway tissue. They also hope it can also be used to screen drugs.

Meanwhile, the Boston University School of Medicine scientists were able to derive a population of pure lung stem cells from mouse embryonic stem cells, as well as develop a new technique to create the cells and then map the developmental milestones of human lung tissue formation. The researchers hope to refine their technique so that it can be used to derive lung tissue from a bank of 100 stem cell lines of patients with lung disease. Both papers were published April 5 in the journal Cell Stem Cell.

“This frequently happens in science: When you realize you’re working on the same thing, investigators decide to race each other. It’s a competitive field and you worry about getting scooped all the time,” Dr. Darrell Kotton, co-director of the Center for Regenerative Medicine at Boston University and Boston Medical Center, tells the Boston Globe.

Instead, a little over a year ago, when Dr. Kotton first discovered that Dr. Jayaraj Rajagopal of the Harvard Stem Cell Institute was leading the team working at the Massachusetts General Hospital that was working on a similar problem, he decided to propose a cooperative venture. They began holding joint lab meetings and monthly phone calls in which they would discuss their positive and negative results openly and candidly.

The collaboration “really lent confidence to our results,” says Dr. Rajagopal It has “really been an example of having not competition — but collaboration — to push ahead.”

Cystic fibrosis: a rare but fatal disease
A serious genetic disorder affecting the lungs and other organs, cystic fibrosis eventually leads to an early death. The most common fatal genetic disease among Caucasians, it afflicts about 30,000 people in the United States and claims 500 American lives each year.

Aggressive treatments addressing the disease’s symptoms have helped stretch the average lifespan for CF patients over the years. But most people who suffer from CF still don’t live beyond their 30s and 40s.

There have been advances in improving the lives of people with CF. But little progress has been made to address the underlying cause of CF in the vast majority of patients — that is, a defect in a single gene that interferes with the fluid balance in the surface layers of airways in the lung.

When fluid balance is upset, the lungs produce thicker mucus, making breathing more difficult and causing repeated infections and hospitalizations. Patients with the more common form of cystic fibrosis have a delta-508 mutation. This gene is responsible for about 90 percent of all CF cases in the U.S., as well as 70 percent of all cases worldwide.

Cystic fibrosis-in-a-dish
Aiming to create a model of the rare disease that scientists can probe and manipulate to understand the disease better, the Harvard-Massachusetts General Hospital researchers started out by using the skin cells of patients with CF to create induced pluripotent stem cells (iPSCs).

They then couched the cells to develop into functioning lung epithelium — the tissue that lines the lung’s airways. The epithelium is the site where the genes cause irreversible lung disease and unalterable respiratory failure.

The new epithelial tissue contains the delta-508 mutation — as well as another mutation, the G551D mutation, that’s involved in about two percent of CF cases, and for which a drug was approved early this year by U.S., Canadian and European regulators targets. The Harvard researchers can now grow this tissue in unlimited quantities in the lab.

“This work makes it possible to produce millions of cells for drug screening, and for the first time human patients’ cells can be used as the target,” says Dr. Doug Melton, co-director of the Harvard Stem Cell Institute and also co-chair of Harvard’s inter-School Department of Stem Cell and Regenerative Biology. “I would expect to see rapid progress in this area now that human cells, the very cells that are defective in the disease, can be used for screening.”

Scientists may also be able to use the epithelial tissue created by Dr. Rajagopal’s team to study other similar lung conditions — asthma, lung cancer and chronic bronchitis — and new insights from the improved study may help speed the development of treatments.

“We’re not talking about a cure for CF. We’re talking about a drug that hits the major problem in the disease. This is the enabling technology that will allow that to happen in a matter of years,” enthuses Dr. Rajagopal, also a pulmonologist or lung specialist.

“When we talk about research and advances, donors and patients ask: ‘When? How soon?’ And we usually hesitate to answer. But we now have every single piece we need for the final push. So I have every hope that we’ll have a therapy in a matter of years,” he says.

The team achieved tremendous progress in only two years, and this is credited to their study of the underlying developmental biology of mice as by postdoctoral fellow Hongmei Mou, first study author and colleague of study senior author Dr. Rajagopal.

“I was able to apply these lessons to the iPS cell systems,” she says. “I was pleasantly surprised the research went so fast, and it makes me excited to think important things are within reach. It opens up the door to identifying new small molecules [drugs] to treat lung disease.”

Creating pure lung — and thyroid — stem cell populations
For some year now, scientists have been growing mature tissues from embryonic stem cells. Some tissues — muscle and nerves, for instance — have proven easy to grow. But others — pancreas, thyroid, liver and lung — have been more difficult.

All these tissues spring from the endoderm — the innermost layer of an embryo which forms when it’s about three weeks old. As early as five weeks, the tissues differentiate into organs — the endoderm differentiates into organs as diverse as the lungs and the stomach in a period of two weeks.

To derive a population of pure lung stem cells in the lab, the Boston University School of Medicine researchers first had to indentify the specific factors that trigger the development of endoderm cells into lung cells — as well as the specific series of steps that takes them from endoderm to lung cells. And then, the candidate lung progenitors had to be purified. While that seems like relatively simple procedures, these goals have eluded scientists until now. “The trick,” Dr. Kotton says, “was in the engineering.”

What Dr. Kotton’s team did was engineer the endoderm cells — attaching a fluorescent tag that glowed fluorescent green at the moment the stem cells expressed a gene called Nkx2-1, which was the moment that they were taking a step toward becoming lung cells. This allowed the team to track the cells as they developed, and at the end of their study, the researchers were able to map each of the six critical decisions on the path to lung tissue.

In the end, 160 lung progenitor cells could be generated for each starting stem cell and these progenitors could be purified easily since the fluorescent tag glowed only when the cells had become lung cells.

One positive side effect of the discovery was that the Boston University investigators were also able to map out the road from stem cell to thyroid. Also coming from the endoderm layer, the thyroid derives from a progenitor that expresses the same key gene as lung progenitors.

“We succeeded in capturing a cell fate decision in cultured stem cells that is normally very transient during the earliest stages of lung and thyroid development,” says Dr. Kotton, who also is an associate professor of medicine at BUSM.

“Most importantly, our results emphasize that the precise inhibition of certain pathways at defined stages is as important as the addition of pathway stimulators at different developmental stages during lung and thyroid specification,” he says.

To demonstrate that the cells they purified were indeed lung progenitors, Kotton’s team took samples of mouse lungs and rinsed them with detergent until they became cell-free lung-shaped scaffolds. They then proceeded to seed one lung with 15-day-old lab-grown lung cells that they had purified from stem cells.

As a control, they seeded another lung with undifferentiated embryonic stem cells. Ten days after seeding, the lung cells populated the lung, and organized themselves to create a pattern that Dr. Kotton recognized as lung tissue. The stem cells in the control group grew and clumped into an unrecognizable mass.

This new technology can be used to grow new primordial lung progenitors to study human lung diseases in the lab.

The findings “lay the groundwork for studying the mechanisms and programming of cells during lung development, which, in turn, will help develop new treatments,” says Dr. James Kiley, director of the Division of Lung Diseases at the National Heart, Lung, and Blood Institute (NHLBI), which funded the study. “The ability to generate a supply of progenitor cells with the potential to differentiate into lung cells will be a huge boon to several research fields,” he says.

Speeding the development of a cystic fibrosis drug
Earlier this year, Cambridge, Massachusetts-based biotech company Vertex Pharmaceuticals received federal approval for Kalydeco (chemical name: ivacaftor) — the first drug to target directly the underlying genetic cause of cystic fibrosis.

Vertex says it found Kalydeco after screening thousands of drugs on a far less than ideal cell line. Then, in the end, many of the drugs that functioned well on the cell line proved ineffective when used on genuine human airway tissue.

Before the lung drug could be tested on people in a clinical trial, testing them on genuine human airway tissue was the gold standard. But Vertex had found it been extremely difficult to obtain the tissue from patients — and when it could be obtained, the tissue rarely survived long in the lab. Together, these factors created a major bottleneck in the drug screening.

“We had to rely on donor tissue obtained from patients with cystic fibrosis, and it’s a bit more challenging, because the number of donor lungs you can get and the number of cells you can derive from there” are more limited, Dr. Frederick Van Goor, head of biology for Vertex’s cystic fibrosis research program, recounts.

This is the problem that both stem cell research teams — the one from the Boston University and that from Harvard — seems to have solved, although it’s still too early to say when drug companies would be able to use the new technology in screening,

“It’s a significant event for the lung field,” Dr. Thiennu Vu, associate professor of medicine at the University of California San Francisco, tells the Boston Globe. Dr. Vu wasn’t involved in either of the two researches.

But much work has to be done before the new lung tissue can be used as lung-disease-in-a-dish models to study major pulmonary conditions — or to repair or replace damaged tissue. The next step is for both teams to refine their techniques to ensure that they yields pure populations of the specific types of functional lung cells.

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