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The Gift of Life

After Decades of Intense Research, Dr. Shahin Rafii and His Group Are Making Dramatic Advances in the Quest to Grow Human Blood Cells Outside the Body

By John Hubbell

The proof they were seeking was close; the researchers knew that much. After years of trying—years in which the lights on the west side of the Ansary Stem Cell Institute and Division of Regenerative Medicine deep inside Weill Cornell Medicine made a small and unyielding dot on the predawn Manhattan skyline—their experiment had unfolded as Dr. Shahin Rafii  and his team had long theorized. Inside a small Petri dish yards away from Dr. Rafii’s office, human blood stem cells capable of forming into any type of blood cell had multiplied time and again in a carefully curated environment. Viewed through a powerful microscope, the cells seemed to dance and glow as they grew in number, four becoming eight, then 16, then 32. On it went.

It was, in short, a breakthrough. The cells’ reproduction would go on to be the subject of two articles in Nature and to advance scientific thought on how blood diseases might someday be treated. But seeing something of this magnitude with their own eyes, as they did on an afternoon in July 2014, was not enough: the task of a pioneering researcher is not simply to trigger a phenomenon, but to then explain precisely how and why it occurred. They were, after all, on the path to assert that long-lasting blood stem cells could be grown outside of a human body. Even as their own doubts dissipated, they knew that questions would soon come from all sides.

Dr. Rafii’s lab, like so many cutting-edge research centers across the globe, is a landscape of hedged bets and fleeting euphoria. There are no bed-bound patients or pacing relatives begging for cures. Its warren of rooms sit largely quiet. Refrigerators hum; fluorescent lights glow; the hour of day is elusive. This, its greatest drama—cells bopping about on a monitor, an excited cluster of people pointing and talking—is rather muted, considering the potential clinical implications.

But imagine these landmark blood cells grown in Dr. Rafii’s lab bound for a cancer-stricken child—her bone marrow ravaged, her blood bereft of the T-cells central to life itself, her body wan and unable to help itself grow strong again, hope dimming. Globally, more than 300,000 children receive a cancer diagnosis each year and 80,000 die of the disease, according to the American Childhood Cancer Organization. And “a large number of patients who could be cured by a bone marrow transplant do not have a suitable donor,” says Dr. Joseph Scandura, scientific director of the Myeloproliferative Neoplasms Center, part of the Division of Hematology and Medical Oncology at Weill Cornell Medicine, an oncologist at NewYork-Presbyterian/Weill Cornell Medical Center, and a senior co-investigator in the project.

Here on Dr. Rafii’s screen was a possible new weapon against the carnage of cancer. The idea: extract a healthy cell from the patient themselves, multiply it in the lab, then send those cells back into the body to facilitate healing. “If we can do this,” Dr. Zev Rosenwaks, chief of reproductive medicine at Weill Cornell Medicine, recalls saying to Dr. Rafii, “then we have an unlimited source of stem cells for curing the patient. We can turn a single stem cell that is destined to become a blood vessel into blood.” Cracking the code of how a blood stem cell is triggered to repopulate, the scientists believed, could ultimately lead to a simple, bold statement that eludes many dedicated scientists like Dr. Rafii over an entire lifetime of research: this could someday form a new cure.

Colleagues underscore how much is at stake. Dr. Michel Sadelain, director of the Center for Cell Engineering at Memorial Sloan Kettering Cancer Center and an associate professor of immunology in medicine and in pediatrics at Weill Cornell Medicine, agrees. “The pursuit of hematopoietic stem cell expansion is one of the grand goals of biomedical research today,” he says. “If we were able to generate large numbers of unadulterated blood-forming stem cells, a number of diseases—from cancer to autoimmunity and more—could be better treated.”

Dr. Nancy Speck, an investigator at the Abramson Family Cancer Research Institute at the University of Pennsylvania’s Perelman School of Medicine, notes the all-or-nothing risk inherent in Dr. Rafii’s quest. “Shahin is trying to develop new technology—that could have potential clinical application,” she says. “It’s a risky proposition and requires a lot of courage to go down that path. However, the payoff could be enormous.”

Various researchers in this story have relationships with Angiocrine Bioscience that are independent of Weill Cornell Medicine.

This story first appeared in Weill Cornell Medicine, Vol. 17. No. 1

Reprogrammed blood stem cells (depicted in green and blue) expanding on top of their vascular niche (in red).

Reprogrammed blood stem cells (depicted in green and blue) expanding on top of their vascular niche (in red). Image: Provided

An All-Consuming Hypothesis

“Twenty years of my work!” Dr. Rafii says in his trademark mix of whimsy and astonishment, speaking in his office on an autumn afternoon between frequent visitors and dings of arriving e-mail. For 20 years, he held to the lonely notion that endothelial cells could be coaxed into generating human blood cells and in turn fight disease. The fate of his lab was tied to this belief, just as the fates of future patients would be tied to what unfolded there too.

Dr. Rafii earned a bachelor’s degree from Cornell University in 1982 and a medical degree  from Albert Einstein College of Medicine four years later, then came to NewYork-Presbyterian/Weill Cornell Medical Center, where he completed his internship and residency in internal medicine and a fellowship as a hematologist and oncologist. After several years of taking care of patients, research in stem cell biology beckoned.

At the Ansary Institute, postdoctoral researchers came and went. Dr. Rafii, the institute’s director, would travel around the world for conferences he felt he could not miss, but otherwise stayed close to the lab. But since the promise of the multiplying cells so perpetually teased his mind, why leave? Mostly he continued—writing grants, revising papers, adding to a filing system of papers atop his desk and mulling over new ways to produce blood stem cells.

Dr. Rafii (left) and colleagues in the lab. Photo credit: Weill Cornell Medicine

Dr. Rafii (left) and colleagues in the lab. Photo credit: Weill Cornell Medicine

Uncovering Stem Cells’ Secrets

“Let’s go back 10, 15 years,” says Dr. Raphaël Lis. It’s late on a weeknight in November, and Dr. Lis—now an instructor in regenerative and reproductive medicine whose visibility has risen along with the lab’s recent work—is finally home at his apartment on the Upper East Side. “Cells that line blood vessels were seen as passive conduits for blood,” he says, summing up decades of conventional thinking. By contrast, Rafii theorized that these cells—known as endothelium—were more important. His thought, Dr. Lis says, “was that these endothelial cells, besides delivering nutrients and oxygen, assume a mastermind function.” Adds Dr. Lis: “Shahin had this idea, and he was alone. No one believed him.”

Dr. Raphaël Lis. Photo credit: John Abbott

It was 2008. Dr. Lis was on track to continue studying in his native France at the University of Paris-Sud when his mentor there, Dr. Jeremie Arash Rafii Tabrizi was lured away by Dr. Rafii (no relation) to establish a lab in Weill Cornell’s new location in Doha, Qatar. Dr. Lis, eager to continue work with Dr. Tabrizi researching the role that endothelial cells played in gynecological cancers, followed him. “The opening of the laboratories corresponded to everything I loved—adventure, out- of-the-box risk,” says Dr. Tabrizi, who continues work at Weill Cornell Medicine–Qatar as an associate professor of genetic medicine in obstetrics and gynecology. “But I would not have gone alone. I realized that Raphaël was probably one of the brightest students I ever had. I accepted the position when he told me that he would jump on the adventure, too.”

The Doha lab had no live specimens for research, so as Dr. Tabrizi and Dr. Lis pursued their own quest to understand how to generate true blood stem cells from embryonic-like pluripotent cells—a broad family of universal building blocks that could differentiate into a variety of cells, including hematopoietic stem cells—they collaborated with Dr. Rafii and his team in Manhattan. Once a year, when Dr. Lis came to work alongside Dr. Rafii in New York, they would inject human blood stem cell derivate from pluripotent cells into mice depleted of blood cells, then wait for something to happen. Under the right conditions, these cells eventually spur the creation of a multitude of healthy blood cells. Yet time and again they did not. “We were getting some blood stem cells in a dish,” Dr. Lis says. “Major disappointment. Literally nothing happened.” He continues: “When we transplanted these human cells, they looked like a true hematopoietic stem cell. But once you tried to assess their function, it turns out they don’t engraft”—the process whereby cells grow and make new blood cells—“in the way that true stem cells do.”

Hematopoietic stem cells (in red) expanding as a colony on the vascular niche (green). Image credit: Weill Cornell Medicine

Hematopoietic stem cells (in red) expanding as a colony on the vascular niche (green). Image credit: Weill Cornell Medicine

Hematopoietic stem cells reside in the safe haven of the vascular system, producing blood indefinitely. As innumerable cells of all types die, they work to replenish them. A body withers when illnesses like cancer disable hematopoietic cells and their crucial regenerative ability is lost. If Dr. Rafii was right about the endothelium’s power, his team was missing something in the relationship between it and stem cells—a conductor, a prompt. What triggered the hematopoietic cells to multiply and stay young? And what governed their differentiation?

Labs elsewhere were taking different approaches to probing the relationship between cells and their incubating, encasing vascular niche. As the cells went about creating various organs and tissues, something inside them was dictating the multiplication and specialization. Some researchers focused on mesenchymal fibroblasts—cells common in animals and the essence of connective tissue—believing they might be key. Others looked at forms of pluripotent cells— and while the potential of these types of stem cells is tantalizing, efforts to coax them into working blood stem cells had long posed daunting hurdles. But if, as Dr. Rafii held, the endothelium itself inside the vascular niche were essentially governing a blood stem cell’s reproduction and future role in an organism (“a kind of bar code,” Dr. Lis says), then the researchers’ failed attempts to grow cells proved that this process was not simply happening on its own. “It took us several years to come to the realization that maybe the actual experimental pluripotent model we were working on was skewed toward failure,” Dr. Lis says. “We were about to say it’s impossible, but we went back to the drawing board to look at endothelial cells and teach them to turn into stem cells. That was the beginning of the first Nature paper.”

Dr. Lis, his doctorate now in hand, moved to New York to work alongside Dr. Rafii. There he joined forces with Drs. Rosenwaks and Scandura, Dr. Jason Butler, an assistant professor of regenerative medicine, and Dr. Vladislav Sandler, then an instructor in genetic medicine, as they focused their work on a number of micro-environmental cues secreted by a vascular niche. By now they knew that simply introducing human endothelium into this environment was not enough to cause the creation of blood cells. The key, they theorized, was figuring out which blood-specific transcription factors—essentially on and off switches that regulate gene expression—triggered it to interact.

In contrast to more circuitous pathways that could prompt the endothelium to produce blood cells, “this approach makes the most sense,” says Dr. Speck, the University of Pennsylvania researcher, because “it starts with what is developmentally the closest relative of the hematopoietic stem cell. So, theoretically, less manipulation will be required to tease these cells into adopting a new fate.” The team added 27 known transcription factors directly onto the adult derived endothelial cells that were carefully mixed with the nurturing vascular niche, where the human endothelium sat ready to interact with the stem cell. Then, they closed the lab incubator and waited.

“Boom! It was like magic,” Dr. Lis says. “Within 20 days, the adult endothelial cells were receiving instructions from the transcription factors to turn into hematopoietic stem cells. And the newly born stem cells were getting the proper signals from the vascular niche to divide, stay healthy and prepare to form many types of blood cells.” The ensuing Nature paper, published in 2014, trumpeted the group’s significant advance: direct conversion of adult endothelial cells into hematopoietic cells could be achieved. Then came the knocks. “The major criticism about the first paper was, one, we could not show that the blood cells we created could form T cells [which guide immune response to disease],” Dr. Lis recalls. “And that’s how the second Nature paper started.” Dr. Butler, his fellow researcher, recalls wondering whether they could “show this more carefully—and figure out how the stem cells are created.” Could these cells truly rebuild the immune system?

Hematopoietic stem cells (in red) expanding as a colony on the vascular niche (green).

Hematopoietic stem cells (in red) expanding as a colony on the vascular niche (green). Image credit: Weill Cornell Medicine

Understanding Their Discovery

The next task was to untangle this unprecedented switching process. Which of the transcription factors turned an endothelial cell into a potentially life-saving magic bullet? All 27? One? Eight? While the lab’s first scientific article had established that hematopoietic stem cells could be created in a bold new way, it did not sufficiently show how to quell doubts that the process could be reliably replicated. To find out, “we had to run an n-minus-one experiment,” Dr. Lis says, in which factors were subtracted, one by one, to identify the genetic instigator. “This part of the work was tedious,” he says, describing a process that spanned from late 2012 into 2014. “It was purely experimentally driven. Here, we really wanted to just find the key players that could elicit this kind of answer. If you put in 26 and you don’t see the phenomenon happening, you knew that the one you removed was actually driving something. The most difficult thing was to figure out that part.” Yet it was crucial to seeing how it all might one day help to cure disease.

Coming as it did after Nature heralded the lab’s first big advance, the plodding work to expand on their discovery was even more perilous. Further research could reveal their approach required several refinements, for one. And competitors, never far behind, were now tipped to the fact that Dr. Rafii’s lab was on to something. “There’s the frustration created by the nature of the work,” Dr. Lis says. “You can’t control everything, you know you’re competing with someone else, and if their article comes first, your career is not going to advance. And on top of that, you worry about the experiment working—and whether it can be replicated.”

The researchers pressed on—adding one factor, subtracting another. Twelve-hour days were standard; toward the end, Dr. Lis recalls being at the lab until 3 a.m. “There’s kind of two teams: the early birds and the late-nighters,” he said. “Shahin belongs to both.”

Eventually the team narrowed the transcription factors critical for generation down from 27 to four. But with the pathway identified, an additional challenge remained: how to prove the cells being created by the process were in fact blood stem cells, not simply cells that looked potent but would prove less useful when reintroduced into a future patient. “So we used a mouse in which all the blood stem cells are genetically painted green, but endothelial cells are not green,” Dr. Scandura says. “So if an endothelial cell becomes a hematopoietic stem cell, they become green.”

As Dr. Lis says of Dr. Rafii, “There’s kind of two teams: the early birds and the late-nighters. Shahin belongs to both.”

As Dr. Lis says of Dr. Rafii, “There’s kind of two teams: the early birds and the late-nighters. Shahin belongs to both.”

The Cells ‘Last forever’

In his office, Dr. Rafii watches as the microscopic movie of the process plays out on his computer monitor for a visitor. What unfolds is undeniable and easy to see: flat endothelial cells blossom into green, round blood stem cells. “Look, no fake cells here—round and green!” he says. “Nobody has done that in tissue culture before. That one newly born blood stem cell became multiple cells. And now you can get each of these, transplant them into mice, and the mouse’s whole bone marrow comes back.”

Four months later—a critical scientific measuring point—the cells were still alive. And they were seen to last just as long inside another mouse through a second so-called serial transplantation. “If it was a progenitor cell, the supply would exhaust,” says Dr. Butler, the team’s transplant expert. Adds Dr. Rafii: “If they are really stem cells, then they should last forever, right?” He pauses. “They last forever.”

After multiple iterations and funding from numerous agencies, including the New York State Stem Cell Initiative, the team’s second paper was submitted to Nature on April 4, 2016, and accepted nearly a year later. While the birth of new hematopoietic stem cells with the potential of forming full immune cells was a major finding of this paper, another major benchmark was less emphasized: “The newly formed blood stem cells were generating more of themselves, ‘self-renewing’ over and over again,” Dr. Scandura marvels. “The key was the vascular niche we engineered, which mimicked the environment from which they grow and multiply.”

The road ahead, and the implications it may bring, is clearer than ever before. The Rafii team’s discoveries “will likely lead to multiple first-in-human clinical trials,” says Dr. Isabelle Rivière, director of Memorial Sloan Kettering’s Cell Therapy and Cell Engineering Facility. The work, she adds, is “potentially transformative,” with Penn’s Dr. Speck adding that it could ultimately cure patients who need a bone marrow transplant but cannot find a suitable donor. Dr. Lis agrees—but cautions that the burst of recent breakthroughs belies the long road ahead. “People aren’t going to hear from us for a while.”

For the multitude of academic implications that come from producing blood stem cells in the way Dr. Rafii has pioneered, patients and their families obviously care about only one: will it work? That is why the Dr. Rafii team’s breakthrough discovery, like so many in the field, answered one big question yet raised so many others. From a clinical perspective, the process created inside the Ansary Stem Cell Institute and Division of Regenerative Medicine would simply take too long to help an ailing patient in the everyday world. The odds would be long to survive a nearly month-long wait for blood cells to be regenerated outside of your body and then sent back inside to heal. “That’s the question we are trying to solve right now—how scale-able this process is—and that’s why we’re switching to a larger model,” says Dr. Lis, pointing to trials being prepared in collaboration with Dr. Hans-Peter Kiem, a noted stem cell biologist at Fred Hutchinson Cancer Research Center in Seattle. “That’s the entire idea of the trial we’re doing now: safety and scale-ability.” Next, Dr. Rafii says, “We want to decrease the time to form the stem cells, from 28 days to maybe 10. We’ll make the niche more efficient—and most importantly, we will watch the mysteries of stem cell self-renewal unfold in front of our eyes in real time.”