Of Cells and Limits

“It is possible that the etiology of all age-associated diseases is in the molecular state that exists in old cells.”

But Hayflick noticed a consistent pattern: in all 25 normal fetal cell strains he created, after about 50 population doublings, the cells were metabolically active but had stopped dividing. Together with cytogeneticist Paul Moorhead, he designed a simple but clever experiment to test whether the cells’ limited capacity to replicate in culture was caused by microbial contamination or some unknown culture condition. It had recently been shown that female cells could be distinguished from male cells by the presence of Barr bodies formed from the inactive X chromosome in female cells. Hayflick mixed equal numbers of female cells that had divided 10 times with male cells that had doubled 40 times and found that after another 20 doublings of the mixed culture, only the female cells remained. “This showed that replication ceased not because of some extrinsic factor, but because of an intrinsic cell phenomenon—a revolutionary idea at the time.”

Hayflick’s study was published in Experimental Cell Research in 1961, after first being rejected by another prominent journal—The Journal of Experimental Medicine. The rejection letter came from Francis Peyton Rous who received the Nobel Prize a few years later for his discovery of chicken tumor viruses. “I can still quote from that letter: ‘Anyone who has worked with tissue culture knows that if the cells are provided with the proper milieu in vitro they will replicate indefinitely.’ He also called my suggestion that our observation suggests something about cellular senescence and aging ‘notably rash.’ ” The theory that all cells are generally immortal in culture was first postulated at the birth of cell culture in the early 1900s and was well publicized by Alexis Carrel of Rockefeller University in New York City who had developed a cell strain from chicken heart cells that he claimed had been growing for more than 40 years.

After showing that normal cells are mortal, Hayflick also reported, for the first time, that cancer cells were uniquely immortal—a claim that could not be made without first establishing that normal cells are mortal. He also discovered that his normal human fetal cells had a memory. When fetal cells frozen at different population doublings were thawed, the cells remembered the doubling at which they had been frozen and only divided until they reached a total of 50 divisions.

Hayflick’s work was criticized and he was ridiculed. It took about 10 years for a more general acceptance that normal cells have a limited life span in vitro, a phenomenon now known as the Hayflick limit. The Nobel Prize–winning discovery of telomere shortening and the expression of telomerase explained Hayflick’s observations.

Here, Hayflick talks about creating one of the most widely used cell strains, how he sued the federal government, and his discontent with the field of aging research.

Hayflick Heads Out

An explosive interest. Hayflick grew up in southwest Philadelphia and became interested in chemistry in elementary school, sparked by the gift of a Gilbert chemistry set. Hayflick made fireworks and rockets and experimented with heat and cold production. His mother encouraged him, even signing a permission statement that allowed Hayflick to purchase metallic sodium. With string, tin cans, water, and the sodium, he built rockets that traveled three to four stories into the air.

College-bound. After completing high school in 1945, Hayflick was accepted by the University of Pennsylvania, but first enlisted and served 18 months in the US Army so he could help pay for college through the GI Bill. “That was a fortunate decision. Had I not enlisted, I would have been drafted for the Korean War.”

Building confidence. After majoring in microbiology at Penn, Hayflick worked for the Philadelphia-based pharmaceutical company Merck, Sharp and Dohme and then returned to Penn as a graduate student. While earning a master’s degree, Hayflick identified the first field isolate of a mycoplasma as the source of an ear infection in the rat colony at the adjoining Wistar Institute.

A new type of culture. Hayflick studied the behavior of mycoplasmas in cell culture before it was known that these organisms are common, but often silent, contaminants of cell cultures. (See “Out, Damned Mycoplasma!,” The Scientist, December 2013.) “This was the first time that anyone purposefully put mycoplasmas into cell culture.”

Hayflick Hits His Stride

A popular cell strain. Hayflick cultured the famous WI-38 strain from fetal female lung tissue in 1962 and developed the technology to make poliovirus and other vaccines in this strain. By then, Hayflick was getting requests for his cells from all around the world. WI-38 cells have been used to produce most of the world’s human virus vaccines, including those against adenoviruses, measles, rabies, chicken pox, mumps, rubella, polio, and hepatitis A. Hayflick gave WI-38 gratis to the world’s human virus vaccine manufacturers because, at that time, living cells could not be patented. But while Hayflick promoted the use of his cells for vaccine production, there was resistance, mainly from the National Cancer Institute and the precursor of the Food and Drug Administration’s vaccine-control authority, which made unfounded statements that using Hayflick’s cells would be dangerous because of their alleged potential to mutate and become cancerous.

Mycoplasma expertise. As head of Wistar’s tissue-culture facility and an expert on mycoplasmas, colleagues would send Hayflick cell cultures to be tested for their presence. A visit from an NIH colleague got Hayflick working on “walking pneumonia,” known medically as primary atypical pneumonia, a common disease in young adults worldwide. Previously, researchers had been unsuccessful in isolating a virus as the etiological agent for this form of pneumonia. From an egg yolk sample thought to contain the causative virus, Hayflick isolated a mycoplasma that had not been described before and named it Mycoplasma pneumoniae. This was the first human disease shown to be caused by mycoplasmas, and it made the front page of The New York Times.

Aging clock. After moving to Stanford University in 1968, Hayflick’s lab continued to investigate how human cells in culture maintain a memory of the number of divisions they have made. In 1975, he and his graduate student, Woodring Wright, showed that this counter was located in the nucleus.

A lawsuit. “My development of WI-38 was done without government or private support and because of the great demand for WI-38 cells, I was awarded an NIH contract only to ‘store and distribute’ the cells.” When Hayflick moved to Stanford the contract ended, but the demand for WI-38 cultures continued. Hayflick began to charge $25—the same amount charged by the American Type Culture Collection at the time—to ship his cells, and put the funds into a holding account until a decision was made about disposition of those funds. “This seemed to me to be reasonable, but some people at the NIH and the FDA interpreted this to be theft of government property, and that I was selling WI-38 for personal gain. They entered my lab in my absence and confiscated all of the frozen WI-38 ampoules.” Hayflick sued the federal government in 1975, claiming that the NIH had violated the Privacy Act of 1974 by releasing their findings before allowing him to reply and by illegally confiscating WI-38. “Who owns WI-38 or any other continuously dividing human cell culture is not easily settled. There are several stakeholders—the researchers who give the cells value, their institution, the individual from whom the cells are derived, and the organization that supports the research.”

The suit was ultimately settled outside of court, at the request of the Justice Department, who found itself in an impossible position for a number of reasons. First, living cells were now patentable. Second, an executive order by President Reagan and the Bayh-Dole Act made it legal to use tax payer–supported research materials for commercial exploitation. Third, the emerging biotechnology industry was founded on materials developed in federally funded academic research labs. If the government prevailed in Hayflick’s lawsuit, the biotech industry would be jeopardized, so industry lawyers prepared amicus briefs on Hayflick’s behalf should his case go to trial. Finally, the NIH itself was by now offering products made in their own laboratories for commercial use.

As part of the settlement, Hayflick received some of the confiscated ampoules of WI-38 as well as the funds from mailing the WI-38 cultures. All of these funds went to pay his legal expenses.

And Hayflick had supporters. After the settlement, a letter published in Science signed by 85 scientists stated that they objected to the behavior of the NIH and the FDA (215:240-42, 1982). According to Hayflick, his position that biologists, like other scientists, have intellectual property rights helped start a revolution in thinking, resulting in many biologists later becoming successful entrepreneurs.

Due credit. Hayflick has been credited with starting the field of cellular biogerontology, the study of the aging of cells. His work set a new direction for aging research. “The focus had been on extracellular causes of aging such as stress or radiation. I suggested that what I had found was teaching us something about the biology of aging, and the term senescence became widely used. My work refocused the field onto intracellular events as the etiology of aging,” he says.

Hayflick Has a Say

A matter of linguistics. “The term ‘research on aging’ has become muddled. Most scientists fail to understand that despite the fact that the ‘aging’ tag exists on their center, institute, or organization, most of the research is not on aging, but on age- associated diseases or longevity determinants. One problem that this linguistics issue creates is the diversion of more than 50 percent of the National Institute on Aging’s budget to Alzheimer’s disease research and less than 5 percent on the fundamental biology of aging. We should do Alzheimer’s research, but not at the expense of research on the fundamental biology of aging. For more than 50 years, aging research has meant research on age-associated diseases and this will tell us nothing about the cause of aging. It is remarkable to observe that a major medical concept that ‘the greatest risk factor for all age-associated diseases is aging’ has resulted in so little research on aging. It is possible that the etiology of all age-associated diseases is in the molecular state that exists in old cells or cells near the end of their lineage. I’ve been vocal about this for 40 years. And as a consequence of that you don’t make too many friends. But I am more interested in the truth than making friends.”

Longevity vs. aging. “Many confuse aging with longevity determination, which is indirectly driven by genes. There are no genes for aging. There are genes to maintain the biological functionality of an organism up to and including reproductive success. There is no mechanism that immediately causes death after reproductive maturation; it’s too costly. Longevity determination asks ‘Why do we live as long as we do?’ Aging research asks ‘Why does everything ultimately fail?’ and that is a huge difference.”

Just the facts. Rebecca Skloot, author of The Immortal Life of Henrietta Lacks, had contacted Hayflick during her research for the book as he knew the personalities involved, including those who had originally cultured the HeLa cells. “Unfortunately, much of what I told her was hyped by her editors, as I’ll give her the benefit of the doubt. There are a lot of inaccuracies in the book. One is the allegation that HeLa cells have saved lives and benefited the health of more people than any other cell population. That is false. WI-38 has benefited 2 billion people who have received vaccines made in these cells. Another is that HeLa cells were the first immortal cell population; that was L929 developed by Wilton Earle at the NIH nine years earlier. But this doesn’t take away from importance of the book. It raises the property rights and ownership issue of self-duplicating biological entities, which I have been addressing for 30 years.”

Retirement? Hayflick continues to mentor, consult, write, lecture, organize conferences on aging, and travel to meetings. “I have not stopped doing anything except running a wet lab. I’ve never retired because I don’t know a definition of that word that applies. I have an enormous correspondence network worldwide, and I spend a lot of time keeping up with the scientific literature. I am also writing my autobiography. My mother lived to be 106, so I am hopeful that I may have inherited some of her longevity determinants.” 

Greatest Hits

• Developed the first normal human diploid fibroblast cell strains such as WI-38 and the technology for producing vaccines against polio and other human viral diseases
• Discovered that normal human cells are mortal, have a memory located in the nucleus, and eventually become senescent. This finding is now called “the Hayflick limit” and is explained by telomere attrition.
• Discovered that cancer cells are immortal, now explained by the expression of the enzyme telomerase
• Discovered that “walking pneumonia” in humans is caused by a previously unknown species of mycoplasma
• Invented powdered cell-culture media and the first inverted microscope for viewing cell cultures
• Changed the thinking of biologists about their intellectual property rights

Further Reading
“Medical Research: Cell Division” (Nature, 498:422-26, 2013)
“Hayflick, his limit, and cellular ageing” (Nat Rev Mol Cell Biol, 1:72-76, 2000)
“A Novel Technique for transforming the theft of mortal human cells into praiseworthy federal policy” (Exp Gerontol, 33: 191-207)
Web of Stories 2012 interview with Leonard Hayflick