Jordana Cepelewicz _ Interview with Leonard Hayflick _ 2016

The year was 1962, and it seemed like cell biologist Leonard Hayflick had made a mistake. After all, for 60 years it had been known that human cells were immortal, capable of dividing forever if they were cultivated in the right medium under the right conditions. But some of Hayflick’s cells were not dividing. He could have chalked it up to the usual suspects: Maybe his sample had been contaminated, or there was a problem with the way he’d prepared the cells. A few weeks passed, however, and not only did his finding remain the same, but a pattern emerged: The cells would double around 50 times, then stop dividing.

Today, we take it for granted that human cells multiply a finite number of times. They stop at the aptly-named Hayflick Limit, when their telomeres—the protective caps at the ends of our chromosomes—get too short. But at the time, the scientific community deemed Hayflick’s discoveries—that normal cells are mortal, that they have memory and an internal counting mechanism, that cancer cells are uniquely immortal—preposterous. It would take nearly a decade of criticism and skepticism before his ideas were accepted as fact.

Hayflick’s ideas have revolutionized the way we think about aging: as a process intrinsic to our cells, and not the result of outside stressors. He went on to a storied career, developing a cell strain used in most human virus vaccines, serving as an anatomy professor at the University of California, San Francisco, as president of the Gerontological Society of America, and as a co-founder of the National Institute on Aging.

Earlier this month, Hayflick spoke with Nautilus about his thoughts on the biological cause of aging, his frustration with current research in the field of gerontology, his skepticism toward claims that we’ll be able to use

science to extend the human lifespan, and his views on the relationship between research and commercial interest.

How did you first become interested in studying cells?

My initial interest as a young boy was in chemistry and, when a pre-teenager, I built a laboratory in the basement of my row house in Southwest Philadelphia. When I went to high school, my interest in chemistry was still maintained. Subsequently, I went to the University of Pennsylvania, where I majored in Medical Microbiology.

After graduating with a bachelor’s degree in that subject area I went to work for about a year and then returned to Penn to get my master’s and Ph.D. My boss, one of the professors, had just returned from the first course in the United States on cell culture, that is, the technology for growing both human, animal and plant cells in laboratory glassware or (now) plastic-ware. He was very enthusiastic, and convinced me that I should direct my dissertation to that subject area, which I did do. It was very easy to make the change because virtually every manipulation that I learned to do for bacteriology was identical to what you do for cell culture.

After graduating from Penn with my Ph.D., I spent two years at the University of Texas Medical Branch in Galveston, Texas as a post-doctoral student in the laboratory of one of the most famous cell culturists, Dr. Charles M. Pomerat. Later I was hired by the Wistar Institute in Philadelphia. This institute is the oldest scientific biological research institute in the United States. It was founded in the late 1800s and occupies an island in the midst of the University of Pennsylvania campus. It has no legal ties to the University of Pennsylvania. I was hired to organize and direct a cell culture laboratory that was to provide cell cultures to people who required them in other Wistar laboratories. I took that job knowing that once I had set it up, I would then be free to do my own research. More importantly this would relieve me from the onerous

task of seeking research funds because I knew that the surplus materials produced in my lab was all that I needed to do the research that interested me. That’s exactly what happened.

How did your work change our understanding of aging?

All of my work was done using cell cultures prepared from human donors. In order to understand the research I did it is necessary to understand the simple procedures used in this technology. I decided to do research on the possibility that some unknown virus might be causing some human cancers. The basis for my belief was that it was becoming known that viruses were the cause of some cancers in many lower animals. I decided to grow human cancer tissue and then expose normal human fetal cells to extracts from the cancer cells.

I arranged to obtain human fetal tissue from the University of Pennsylvania Hospital across the street from the Wistar Institute. Of course, the availability of this tissue occurred at random times. This fact was a key element in the discovery that I ultimately made. But, first I must describe the simple cell culture procedures that I used.

Several hundred million cells can be obtained from a piece of tissue not much bigger than a match head. Those cells can then be introduced into a culture vessel along with a complex growth medium. The rectangular culture vessel is then rested on its side when placed in an incubator at body temperature. Thus, the cells grow on what was formerly the inside wall of the upright vessel. After a few hours, the cells begin to settle onto what is now called the floor of the vessel, and then within 24 hours or so, the cells begin to divide and soon carpet the entire floor of the vessel. When they touch each other they stop dividing. This is called a confluent culture. If you want more cells then techniques allow you to remove the cells from the first bottle and introduce, say, half of their numbers into two daughter vessels. This is called a one to two split or sub-cultivation.

What I discovered was that after making many subcultivations over several weeks, the cells in one of the cultures did not look right.

The cells were not dividing and looked different. I thought, as almost every culturist did at that time, that there must be some error in the way that the cells were subcultivated. For decades prior to my work some excuse would be invoked to explain why subcultivated cells had stopped dividing. It was always thought to be due to an unknown technical problem. After the passage of another few weeks, when I went to look at the cultures, another cell culture prepared from a different human fetus revealed lack of cell division as had appeared in the first culture in which this was observed.

I then looked at my laboratory records after seeing this event occur in more cultures. I discovered to my amazement that the cultures in which the cells had stopped dividing were those made eight or nine months ago. These were cultures made from human fetal tissues that had undergone the most subcultivations. That is, they were the oldest cultures in the series. Cultures made from human fetal tissue a few months or weeks ago were luxuriating in the same culture medium, in the same group of glassware, and cultivated by the same person on the same day.

These events elicited my curiosity. I did several other experiments to prove to myself that what was occurring was not attributable to some error. My discovery defied the 60-year-old dogma that was established from the beginning of cell culture procedures in the early 1900s. The dogma said that when you put cells in culture, they all have the capacity to divide forever, as long as you care for them properly. If they do stop dividing, then you made some mistake. The experiments that I designed disproved that dogma and proved the opposite. That is, that the cessation of division in normal human cells is determined by a mechanism inside the cell itself, or what we call an intracellular phenomenon.

I proposed that normal human cells have an internal counting mechanism and that they are mortal. This discovery allowed me to show, for the first time, that unlike normal cells, cancer cells are immortal.

I concluded also that these results were telling me something about human aging. This is the first time that evidence was found suggesting that aging might be caused by events occurring inside of cells. Until my discovery scientists thought that aging was caused by events outside of cells (extracellular events) like radiation, cosmic rays, stress, etc.

What I clearly had shown was that the cessation of cell division is a function of the number of times the cell divides or more exactly, that the DNA in the cell copies itself. DNA copies itself only a finite number of times in normal cells and that cancer cells must have a method to circumvent this mechanism. This discovery had an important impact in the field of aging because, prior to my discovery, people were looking for causes of aging as extracellular phenomena. Now the focus of the field was directed to intracellular events. I soon found that most of the several dozen normal human cell strains that I had developed had similar biological properties. In all subsequent studies I decided to focus my attention on the properties of one cell strain that I named WI-38. WI-38 was derived from human embryonic lung tissue and the cells were capable of doubling about 50 times over an eight- to 10-month period.

How did your cell strain, WI-38, come to be used throughout the world for the production of human virus vaccines?

I also suggested in the paper that described my work that, because my cultured normal human cells were free of any detectable viruses, they would be the solution to a serious problem then facing human virus vaccine manufacturers. At that time the polio vaccine was made by growing the virus in monkey kidney cells. They were soon found to contain several monkey viruses whose potential danger to humans was unknown. However, one virus called SV40 was then known to produce

cancers in some laboratory animals and, more importantly, to be capable of transforming my cultured normal cells into cancer cells. Many millions of people were accidentally fed or inoculated with polio vaccines containing live SV40 at that time.

I spent the next 10 years trying to convince the worlds’ vaccine manufacturers and the government authorities that controlled them to switch to my cell strain WI-38 to manufacture human virus vaccines that are free of contaminating viruses. I also showed that WI-38 was then the most sensitive cell strain then known that could grow all known human viruses. I also isolated a new common cold virus using these cells.

There was considerable resistance to my suggestion to use WI-38 to prepare human virus vaccines for political reasons and also because, it was argued, WI-38 might contain dangerous viruses that could not be detected. I argued that this fear was equally tenable for the monkey kidney cells then used and for any other cell population. After several deaths had occurred in people working with monkey kidney cells, eventually the detractors’ fears were overcome because no one has ever found a contaminating virus in WI-38 during the 50 years that it has been studied worldwide. My efforts, and those of others who shared my views, resulted in the next decades to benefit more than 2 billion people from human virus vaccines made in WI-38. These included vaccines against polio, measles, rubella, chicken pox, rabies, hepatitis A, mumps, and shingles.

I also discovered that the cells composing a cell strain like WI-38 could be stored indefinitely at sub-zero temperatures in liquid nitrogen. What was remarkable was my finding that the cells had a memory! When stored at many different population doubling levels from two to their maximum of 50, the cells remembered the level at which they were stored and, when reconstituted, continued to divide until the total of 50 doublings was reached. The cells clearly had a memory! Their memory has remained

accurate even after 54 years in the frozen state. I concluded that the cells must have a mechanism that counts each time they divide. Our later experiments revealed that the putative mechanism was located in the nucleus of the cells.

What did it take to change a 60-year-old dogma in science?

I engaged my cytogeneticist colleague Paul Moorhead to determine that the cells I had worked with were chromosomally normal which I felt was a critical determination to distinguish them from the abnormal chromosomes found in the many known cancer cell lines available at that time. In 1961 Paul Moorhead, and I wrote the paper describing our results and our interpretation of those results. We sent it to one of the most prestigious scientific journals in the field at that time called The Journal of Experimental Medicine.

To our great disappointment the paper was rejected by a scientist, who wrote that, “Anyone working in tissue culture knows that given the right milieu in vitro normal cells are immortal.” That is exactly what we believed we had proven to be absolutely wrong.

To suggest, as I did in this paper, that my proof that normal cells are mortal and that this might be telling us something about aging, the author of the rejection letter wrote, that concept is “notably rash”. (Later the author of the rejection letter won the Nobel Prize in Medicine or Physiology.)

We sent the paper to another journal called Experimental Cell Research. It was published immediately without change, and as of today, it has been cited by other scientists over the years about 10,000 times, which is a huge number. Most papers in science are not cited more than three or four times. Initially, acceptance of my work by the scientific community was minimal and indeed, in the first few years, even ridiculed. This reaction is not uncommon in science. The bigger the concept that you

torpedo, the greater ridicule and suspicion you can expect. That’s indeed what happened. For 10 years, my work was barely accepted despite the fact that people had cultured the same cells that I did and made the same observations. Many scientists remained skeptical, until a series of unusual of events occurred about 10 years later. When scientists working in disparate fields meet and exchange information it is sometimes results in important discoveries.

In this case a “scientific grandson” of mine by the name of Calvin Harley was working at McMaster University in Canada when the girlfriend of another scientist working there arrived for a visit. Her name was Carol Greider. Carol was a scientist, at that time working at John Hopkins University in Baltimore. She had been studying a single cell organism and structures within them called telomeres. These structures are found at the ends of chromosomes. Little was known about them at that time. As a visiting scientist to see her boyfriend, she was invited to sit in on the discussions that were taking place that day where department scientists were discussing their research results.

Carol met Calvin Harley, who was working with my cell system. The two of them, after some discussion, decided to see what happened to the telomeres in the cultured normal human cells from the start and until they reached the limit of their division capacity. What they discovered was profound. The telomeres got shorter and shorter as the cells approached their replicative limit. One can imagine the two chromosome ends shortening each time the cell divides. The discovery of that molecular mechanism which explained my phenomenological discovery was widely received and pretty much vindicated the validity of my findings. Because I had postulated that this event might be telling us something about aging the shortening of telomeres then became a popular area of aging research.

What does aging mean?

The term aging has an enormous number of meanings depending on the person who hears or reads the word. Even expert biologists in the field (biogerontologists), cannot agree on the definition of fundamental words like “aging” itself. The word “aging” has been used to describe human experiences in virtually every field of human endeavor. Aging plays a role in architecture, economics, politics, science, medicine, biology, religion, literature, and all other human, animal, and plant activities. In fact, aging occurs in inanimate objects as well. Many years ago I tried to bring experts together in the field of biological aging in an effort to agree on the definition of key terms in the field. I told them, “If we didn’t agree, then they will understand that what we are now doing, and have been doing, is talking and writing over each other’s head. And that is exactly what happened because this group of 20 or 30 experts in the field could not agree even on the definition of the terms “age” or “aging.”

What causes aging?

Over the past 55 years that I’ve been in this field, I have given this subject a great deal of thought. Along with a few others we have reached the conclusion that the cause of aging is a function of the Second Law of Thermodynamics, which is not difficult to understand. The Second Law says that energy tends to spread out or dissipate unless it’s constrained. At the molecular level, where the fundamental action is, the chemical bonds are the constraints. They hold complex biological molecules together and tend to break because the molecular components are in constant motion and the energy that holds the components together tends to dissipate. As the bonds break and the molecular structures change, the molecules become inactive or dysfunctional. When repair mechanisms, also composed of complex molecules, fail the accumulation of dysfunctional molecules produce changes at higher levels of organization (cell, tissue, organ) until they become manifest at the clinical level. We now recognize a person or animal whom we recognize as aging.

For complex reasons there is very little research done on the fundamental cause of biological aging. In varying time frames everything in the universe ages or changes form. For example, the source of age changes is identical in your automobile and in you as described above. Your automobile is brilliant because it knows how to age without any instructions in the blueprints or elsewhere. The car ages because of the Second Law. That law applies to all animate and inanimate entities. This is the current speculation because we cannot raise either awareness or research money to study this fundamental question.

Should we try to slow the aging process?

When looking into the future, I can say with a good deal of confidence that because little, or no, research is done on the fundamental cause of age changes there is little likelihood that tampering with the aging process will ever be possible. However, the question that few people give serious thought to is what kind of a world would we be living in if we were able to tamper with the aging process.

For example, if middle-aged parents decided to slow their aging process and their children did not, the children would eventually reach the biological age of their parents. To slow, or even arrest, the aging process in humans is fraught with serious problems in the relationships of humans to each other and to all of our institutions. By allowing asocial people, tyrants, dictators, mass murderers, and people who cause wars to have their longevity increased should be undesirable. Yet, that would be one outcome of being able to tamper with the aging process. At first it might be very expensive to do, as most medical breakthroughs originally prove to be. If the cost is very high a pill that slows aging would become available, first, to the rich and powerful. Are they the first, perhaps, the only people to have their lives lengthened? I can think of no scenario that would argue for the ability to interfere with the fundamental aging process. My personal belief is that I would rather experience the aging

process as it occurs and death when it occurs in order to avoid allowing the people who I just described to live longer.

What is the difference between aging and longevity?

This is another area of very great confusion because many people including the so-called experts in the field do not understand the concept of longevity determination. Aging is the result of the accumulation of unrepaired changes or losses in molecules (a catabolic process). Research on aging tries to answer the question: “Why do things ultimately go wrong?

Longevity determinants are all of life’s biological pathways. All of these processes lead to answering the question, “Why do we live as long as we do?” Many of the studies in the field of aging, as I indicated earlier, focus on age-associated diseases and another huge segment focuses on longevity determinants misunderstood to be studying aging. A good example is the huge enterprise using worms and flies, as simple biological material with very short lifespans, to study the aging phenomenon.

Virtually all of the experiments are designed to compare a treated and untreated group of animals. The purpose is to see how many members of the treated group live longer than the members of the untreated group. When it is found that members of the treated group live longer, the conclusion is reached that the aging process was affected. That is an indefensible conclusion, because the cause of death in the treated animals could be for many reasons other than aging. For example, all old humans do not die because of the aging process. We die from a cause that is specified on a death certificate (even though it may be erroneous, it is not stated as aging. Those who conclude that their experimental animals have lived longer because the aging process was affected have really observed “all-cause mortality”. That is the cause of death could be from many causes other than aging.

How should we be studying aging?

We should be studying aging in the ways in which it is not now being studied because it is not being studied at the fundamental level. Most studies are either descriptive, studies on longevity determinants, or studies on age-associated diseases. None of this research will reveal information about the fundamental biology of aging. Less than 3 percent of the budget of the National Institute on Aging in the past decade or more has been spent on research on the fundamental biology of aging.

I am one of the original founders of the Council of the National Institute on Aging, (and Chairman of its’ Executive Committee) so I am reasonably informed about their activities. Most of the money that is spent by the National Institute on Aging and, I might add, other NIH institutes, is spent on research on age-associated diseases like cardiovascular disease, cancer, stroke, and Alzheimer’s disease. In fact, about one half of the budget, of the National Institute on Aging is spent on Alzheimer’s disease research.

The resolution of Alzheimer’s disease as a cause of death will add about 19 days onto human life expectancy. I have suggested that the name of the institute be changed to the National Institute on Alzheimer’s Disease. Not that I support ending research on Alzheimer’s disease, I do not, but the study of Alzheimer’s Disease and even its resolution will tell us nothing about the fundamental biology of aging.

Curing cancer, cardiovascular disease, or stroke as a cause of death also will tell us nothing about the fundamental biology of aging. People who have had cardiovascular disease or cancer in their mid-years, let’s say in their 40s, and then recover, find that the aging process continues. Aging is not arrested or accelerated by those ailments. When we resolved causes of death in infants like iron deficiency anemia, Wilms tumors, poliomyelitis, and other ailments of young people, the cures revealed no information about childhood development.

These disease causes have nothing to do with childhood development just as age-associated diseases have nothing to do with aging other than that they increase vulnerability to those diseases. This should suggest to even a first-year biology student that it should be a top priority to study the fundamental biology of aging. It strongly suggests that there may be something unique about the biology of an old cell that provides the milieu, or the environment, in which the leading causes of death more often occur. This concept is almost completely ignored by the National Institute on Aging and also by many other organizations that claim to study aging. There are very few, if any, organizations in this country, including centers, institutes, and departments that have the word aging in their name, that actually study the fundamental biology of aging at the molecular level.

I have written about this to a large extent. The title of one of my papers is, “The One Billion Dollar Misunderstanding”, because that’s precisely what it is.

If aging is the true cause of death in most cases, why don’t we just say that on our birth certificates?

It is illegal in this country, and in all developed countries, to die of “old age” or “natural causes.” You cannot legally write that on a death certificate so that when you read or see that in a newspaper, or hear someone saying that, it is illegal.

One of the most important events that has occurred in respect to efforts to answer the true causes of death is that over the past 40 or 50 years there has been a sharp decline in autopsies. Autopsies are now usually done only for forensic reasons. It is fair to say that the cause of death in individuals over the age, arbitrarily, of 75 or 80 is unknown, despite what is written on the death certificate.

What is written on the death certificate is merely a legal requirement for which you must use a term that’s on a list of acceptable legal causes. The problem is that almost all older people have multiple pathologies and to determine which one can be the scientifically proven cause of death is rarely determined. The cause of death on the death certificates of older people is usually arbitrary, even if the cause of the death might be caused by being struck by a bus. The cause of death in that case may not be the bus, but it may be the individual whose eyesight or hearing may have been impaired by old age or some pathology.

Determining the cause of death in older people is not being addressed because the cost of doing autopsies is high. Furthermore, loved ones may not wish to pay for an autopsy because of religious or ethical reasons. In the three or four cases in which a large number of autopsies have been done in an effort to understand the cause of death all have revealed that about half of the causes of death written on the death certificates were wrong. Relying on causes of death on death certificates is very risky. Yet, it governs decision makers who, not only decide where to put research funds but make crucial decision s about public health.

How can we generate more interest in the study of aging?

The three most important ways to increase interest in doing research on the fundamental biology of aging are “education, education and education”. That education must include an understanding that the massive amount of research funds spent on studying the leading causes of death will not advance our understanding of the basic biology of aging. It also must include an understanding that the study of longevity determinants (anabolic processes) will not reveal information about the basic biology of aging (catabolic processes). Finally, we need to educate scientists and the public, to support research on the differences between young cells and old cells that make the latter more vulnerable to age-associated diseases.

There is a mantra said and written about many thousands of times by geriatricians, biogerontologists, and researchers on the leading causes of death. It is that “Aging is the greatest risk factor for each of these leading causes.” It does not take a great leap of intellect to then ask: “So, why are we not doing research on the fundamental biology of aging?”

Have scientists taken unfair advantage of misconceptions about the aging process?

We have known about alleged unproven methods to stop, slow, or interrupt aging since recorded human history. Those who advocate these spurious methods are members of the first, second, or both of the oldest professions.

Many people have discovered through the years that they can make huge amounts of money by selling the public on so-called means of preventing, slowing, or stopping the process entirely. There has always been an enormous lunatic fringe around this field. There are many people who have become extremely wealthy as a result of their ability to convince other people that one particular diet, nostrum, or lifestyle will increase their longevity. I, and dozens of my colleagues, have published that: “We know of no lifestyle, nostrum, or any other treatment that will interfere in any way with the human aging process.” Extending longevity or life expectancy by modifying the effects of disease or pathology does not affect the aging process.

Of course you can cover up many of the so-called signs of aging. The huge industry that manufactures cosmetics, or other cover-ups, thrives on that belief.

Unfortunately, there are a substantial number of scientists in this field who have become very wealthy as a result of pushing various products and lifestyle changes. I and many other colleagues have become very disappointed because it is easier for the public to be convinced of the

alleged effect of a particular nostrum or treatment by a professional in the field than it is by a non-professional. There has always been a great temptation for some people in this field to exploit their position by making unsupported claims about the use of various devices, chemicals or diets to treat aging.

How has the relationship between science and commercial interest changed over the past 50 years?

Unlike the circumstances when I became a scientist 55 years ago, today if a scientist does not have some commercial interest, they are considered to be a failure. Fifty-five years ago a respected scientist rarely thought about profiting from their work. That scientific attitude has changed 180 degrees since the time I became a scientist. My belief at that time was to do research for the benefit of humanity. I learned within a matter of 10 or 15 years that that belief was no longer tenable. I slowly learned that I was living in a pool of sharks.

Until recently there was a time in the 1950s or the 1960s when, by moving from an academic institution to an industrial organization, or pharmaceutical company, your academic career ended. In later years, this has changed. Now there is a free traffic between profit-making organizations and academic institutions. What has happened is that the academic institutions have become more like commercial organizations and the reciprocal. For example, when I first went to university, there was no such thing as lawyers employed by deans or even presidents of universities. In subsequent years they all have not only one lawyer, but usually batteries of lawyers who specialize in various areas. In addition to that, major universities, especially so-called research universities, have departments or units with patent attorneys, and also with people whose major job is to search for products produced by faculty members that could be financial exploited by the university.

Several years ago I said and wrote about my belief that the only distinction between major research universities and pharmaceutical companies, for example, is in the eyes of the IRS. That is still substantially true. The amount of educational activities done by some good biotechnology companies is equivalent to that done by many universities. There are courses given, lectures held, and mentors assigned to young scientists, so that the elements of education are used by commercial organizations who realize the good by engaging in those kinds of activities.

This dramatic change in the merging of academic and industrial values has not impaired research but it has compromised some ethical values long sought to be the provenance of only universities. However, the merger has helped research because many academic scientists have benefitted from commercial support of their university departments. Or, scientists have moved to industrial environments because doing so makes it easier to have research support in a commercial environment. Of course, the commercial entity expects something in return—namely, a product. In that sense the marriage between the two institutions has benefited society.

In an academic environment you must spend enormous amounts of time writing grant applications to fund your research. Many academic researchers now spend more time writing and re-writing grant applications than they spend time doing actual research.

What would you be if you weren’t a scientist?

I would probably be a writer. I do a lot of writing and I enjoy it.

I would write about the things I’ve been writing about above and also about biology in general. I have some other ideas about biological matters and I think I could do a fair job explaining them. I’ve been told by several people that I do a pretty good job explaining complex biological and other

scientific concepts to make them understood by lay people. I would probably try to exploit that.