Wednesday, June 21, 2017

The Vaccine Race: The Limits of Leonard Hayflick and the WI-38 Cell Line.

I have been anxiously awaiting the release of Meredith Wadman's book, The Vaccine Race: Science, Politics, and the Human Costs of Defeating Disease, which does a deep dive into the early history of cell culture and the development of vaccines for polio, rubella, and rabies. Wadman started researching this story for a piece in Nature in 2013 (I liked it so much that it is near the top of my list of great science blog posts.) Thus, the book is a well-researched and scientifically detailed account of how many lives have been saved and improved by these scientific advances. It also describes, with unflinching honesty, the sometimes questionable practices of researchers and doctors in the time before informed consent and clinical trial review boards. Like Rebecca Skloot's The Immortal Life of Henrietta Lacks, it is an important look at the history of medical ethics. The Vaccine Race also emphasizes why we need funding for basic research and details the beginning of intellectual property law in biomedical research.

While the book focuses on the scientific development of vaccines, Leonard Hayflick is still at the center of the action. Hayflick, an early practitioner of tissue culture, was interested in developing a normal diploid human cell line. Other cell lines in use at the time were either non-human or from cancer cells. The source of his cell lines were human fetuses obtained from legal abortions. In developing the normal diploid cell lines, he observed that the cells could not grow and replicate forever, but typically replicated only 40-60 times.This result contradicted the famous experiment of Alexis Carrel, whose normal chicken heart cells were continuously cultivated in the lab for 34 years. The Carrel result was considered dogma and so Hayflick's findings were initially met with skepticism and suggestions that Hayflick must not be culturing the cells properly. After increasingly careful repetitions with the same result, he performed a co-culture experiment using young cells from a female and old cells from a male, which showed that the cells died after 40-60 replications no matter when they were added to the culture. He finally convincing the scientific community of the existence of the Hayflick limit. Carrel's result was then called into question. Many years later, Carrel's former lab technician confirmed that the method of preparing the fresh culture media was constantly adding new, young cells to the supposedly elderly cells, allowing them to appear to grow immortally.

Images of WI-38 cells from ATCC; listed as diploid human fetal lung fibroblasts.
After 38 attempts, he found success with a cell line named WI-38 (WI for Wistar Institute), which could proliferate without problem and be infected with virus and continue to replicate. Once he had a reliable cell line, Hayflick then had to convince others of its efficacy and safety for vaccine development. The cell line was eventually used to develop vaccines for adenovirus, MMR, and chickenpox. (Of course, when the source of the cells became known, there was a backlash from religious groups who thought that there was no excuse for using fetal cells.)

The other major battle that Hayflick fought was with the NIH, who had given him a contract to distribute the cells to researchers working on certain NIH grants. When he left the Wistar Institute in 1968, he took the cells with him to his new lab at Stanford. Over the next 8 years, he distributed the WI-38 cells, first at prices similar to those of commercial distributors (like ATCC) and then at increasingly high prices, charging even more for young cells at low passage numbers. In 1974, Hayflick  interviewed for a position as director of the Institute of Aging; this spurred an investigation of his management and practices of the cell line. Eventually, Hayflick resigned from Stanford and hired a young lawyer whose expertise was intellectual copyright; this lawyer later represented many of Silicon Valley's biggest players, like Apple and Facebook. Hayflick was fighting a battle with the NIH that had little precedent. Wadman uses the WI-38 case to describe the advent of intellectual property and biological patent law and how these helped spurred the success of the biotech industry. These chapters were among the most interesting. 

Leonard Hayflick proudly displays his cell line.
The book includes some pretty harrowing details of clinical trials with early vaccines that were given to patients in mental wards, babies born in public hospitals where the majority of patients were African American, inmates in prisons whose release was tied to their participation, members of the US military, and terminal cancer patients all without informed consent. In most cases, the vaccines behaved as expected and side effects were minimal or nonexistent, but there were exceptions. These cases led Henry Beecher (an anesthesia specialist at Harvard Medical School) to publish an article in the New England Journal of Medicine entitled "Ethics and Clinical Research" which detailed the specifics of the questionable human experiments. After Beecher's article, the tide started to change.

Overall, The Vaccine Race is an excellent read, particularly for its coverage of the history of science and medical ethics. If I had one complaint it would be that it was not as detailed about the science of viruses and vaccines, but Wadman does end on a strong, scientific note as she describes the Nobel-prize winning discovery of the enzyme telomerase, which explains the mechanistic basis of the phenomenon observed by Leonard Hayflick. 

Wednesday, June 7, 2017

The impact of Nettie Stevens on genetics, chromosome theory, and the naming of cats in our house

We recently welcomed a new member to our family: a beautiful calico cat! We were struggling to find a suitable name for this feisty one. Because I have had so few female cats, I wasn't really sure where to start. As a woman of science, I decided to use female scientists for inspiration.

Then I remembered the work of Nettie Stevens. I stumbled upon Dr. Stevens' work when I was researching Thomas Hunt Morgan for my two posts on the movie The Fly Room. Stevens was most famous for identifying the chromosomal basis of sex inheritance-she found that males are XY and females are XX. Calico cats are almost always female because the genes that control fur color are on the X chromosome, so we decided to name the cat Nettie. What I learned in my reading suggested that Nettie Stevens, like so many female scientists, was pretty amazing and definitely underappreciated for her impact on genetics, chromosome theory, and the use of model organisms in cell biology.

Nettie Stevens (1861-1912) was born in Vermont and grew up in Westford, MA as a fifth generation New Englander and the daughter of a carpenter. Her love of biology was likely spurred by summer science courses on Martha’s Vineyard. She taught high school zoology and physiology for many years, eventually saving up enough to attend Stanford (then Leland Stanford University) at the age of 35. Interestingly, Stanford was about 40% women at the time of Stevens’ matriculation in 1896. According to a Stanford newsletter about Nettie Stevens, they aren't really sure what attracted East Coasters and women in record numbers to their institution at that time. They speculate that it might have been the lower cost of tuition compared with the more established East Coast school.  

Stevens started her doctorate at Bryn Mawr in 1900; at that time the university was home to Thomas Hunt Morgan and Edmund Beecher Wilson, who were both leaders in the burgeoning field of cell biology (then called cytology). Stevens started her PhD in Morgan’s lab at a critical moment in the history of genetics: Gregor Mendel’s work had recently been rediscovered. The big question at that time: what is the biological basis for Mendel’s law of heredity? These ideas were causing excitement in parts of the scientific community. However, Thomas Hunt Morgan was initially not very interested in the Mendel revival. Instead, Morgan focused on the processes of regeneration (admittedly a very cool topic as I have covered in my post on axolotl), which is why Stevens started her PhD studying regeneration in various marine models, including the planaria.

Stevens received a fellowship to fund her travel to Germany to study in the lab of Theodor Boveri. At that time, Boveri was deeply involved in the Mendel revival. Using sea urchins, Boveri had shown that all the chromosomes had to be present for embryonic development to take place. He published these results in 1903 in parallel with Walter Sutton, who showed the same results in grasshoppers during his PhD work in the lab of E.B. Wilson. Wilson later termed this the Boveri-Sutton chromosome theory (notably Sutton, a derelict PhD student, was able to get the recognition that eluded Nettie Stevens.)   

Image courtesy of Rachel Ignotofsky 
Stevens returned from her time in the Boveri lab with a strong interest in the idea of inheritance through chromosomes. Morgan allowed her to work independently on this project, which was uniquely part of the culture in the Morgan lab; as the project progressed, Morgan became interested in the idea as well. For me, this story points out how Stevens was at the leading edge of the Mendel resurgence and brought this interest to the Morgan lab.

In 1903, Stevens completed her PhD at Bryn Mawr, but found it difficult to find a permanent research position. In her application to the Carnegie Institute requesting funding for her research, she stated, “College positions for women in Biology this year seem to be very few.” In her proposal, she planned to study the connection between chromosomes and sex determination, which was controversial at the time. While C. E. McClung had proposed that the “accessory chromosome” could be involved in sex determination, most researchers, including Morgan and Wilson, believed that sex was determined by environmental conditions.

To address this question, Stevens started looking at the full set of chromosomes of many different organisms. In 1905, while observing cell division in the mealworm Tenebrio molitor, she noted that females had 20 large chromosomes, while males had 19 large and 1 small chromosome. She suggested that the sperm determined the sex of the offspring, based on whether it carried the small or the large chromosome. This was in contrast to the results obtained by Wilson, who found that males had one fewer chromosome than females; in his case this was because the organism he studied was XO and XX. In the fall of 1905, Stevens published her work entitled "Studies in Spermatogenesis" just two months after Wilson's work was published. Her conclusions did not gain acceptance, but when Wilson published his next paper confirming the XX/XY inheritance model, the scientific community accepted the results as true. 

Stevens also examined the role of sex chromosomes in a variety of insects and marine organisms, which led her to conclude that the XX/XY inheritance was generalizable. (In contrast, Wilson’s XO males were relatively rare.) Thus, Nettie Stevens was at the vanguard of utilizing different organisms for research in cell biology. In her experimentation with different organisms, she even worked with the fruit fly, Drosophila melanogaster and introduced the model to Morgan's lab.
On July 7, 2016, a Google Doodle celebrated what would have been Dr. Stevens' 155th birthday. This brought her name out of obscurity. Like so many female scientists, perhaps most famously Rosalind Franklin, she had virtually been erased from scientific history. Why did she not get credit for her work? Was it the lack of a permanent position and lab of her own? Or was it a case of sexual discrimination? Some argue that Wilson was given credit not due to primacy of results, but the substance of his entire body of research. While it can't correct this historical oversight, we will acknowledge the contributions of Nettie Stevens in our house.

Further Reading

This post by Cristy Gelling is highly recommended; she details more about the sexism and Morgan's obituary of "Miss Stevens".

This podcast from Babes of Science was fun and filled with facts about Nettie Stevens that I hadn't read elsewhere.